HIGH THROUGHPUT QUANTIFICATION OF THE FUNCTIONAL GENES ASSOCIATED WITH RDX DEGRADATION USING THE SMARTCHIP PLATFORM By Jennifer Collier A THESIS Submitted to Michigan State University i n partial fulfillment of the requirements f or the degree of Environmental Engineering Master of Science 2018 ABSTRACT HIGH THROUGHPUT QUANTIFICATION OF THE FUNCTIONAL GENES ASSOCIATED WITH RDX DEGRADATION USING THE SMARTCHIP PLATFORM By Jennifer Collier Hexahydro - 1,3,5 - trinitro - 1,3,5 - triazine (RDX) is a contaminant of concern at many military sites in the US. One approach that may be used to clean up these sites is bioremediation, and six functional genes, diaA , nfsI , pnrB , xenA , xenB , and xplA , have been linked to RDX degradation to date. Quantitative polymerase chain reaction (qPCR) is typically used to detect these genes in environmental samples, but the primer sets previously used do not have good theoretical coverage of the known gene sequences, so th ey may yield false negative results. To address this, new primer sets were designed with the EcoFunPrimer tool based on sequences collected by the Functional Gene Pipeline and Repository and verified based on residues and motifs. These primer sets were the n used to quantify the RDX functional genes in RDX - contaminated groundwater before and after biostimulation, RDX - contaminated sediment and uncontaminated samples (Red Cedar River water and agricultural soils) using the SmartChip Real - Time PCR System. The n ewly designed primer sets improved upon the theoretical coverage of the previously published ones, and this corresponded to more detections in the environmental samples. All genes except diaA , were detected in the environmental samples, with xenA and xenB being the most predominant. In the sediment samples, nfsI was the only gene detected. The primer sets designed in this study may be used for more reliable detection of the RDX functional genes at contaminated sites. However, additional work with high throu ghput sequencing is required to confirm the specificity of these assays. iii Dedicated to my family and friends iv ACKNOWLEDGEMENTS I am very grateful to my research advisor, Dr. Alison Cupples, for providing me the opportunity to work on this project. In addition, I would like to thank her and my other committee members, Dr. Syed Hashsham and Dr. James Cole, for the support and guidance they provided throughout this work. Dr. Robert Ste lab provided invaluable guidance and assistance in teaching me many of the methods used in Chapter 2. I am particularly indebted to Tiffany for providing the Red Cedar River water samples us ed in Chapter 2. Moreover, I am appreciative of the assistance provided by Benli Chai and developed and helped troubleshoot problems I came across. I would also like to thank the US Army Corps of Engineers (USACE) and Naval Facilities Engineering Command (NAVFAC) for providing the RDX - contaminated groundwater and sediment samples used in Chapter 2. Specifically, Mandy Michalsen, Aaron King and Jeffrey Weiss from USACE and Malcolm Gander from NAVFAC. Additionally, I am grateful to Michigan State University for funding this work through the Interdisciplinary Team Building Initiative grant and Academic Achievement Graduate Assistantship. In addition to all those listed ab ove, many other people provided me with support throughout this project. I would like to acknowledge my lab group members Jean - Rene Thelusmond, Vidhya Ramalingam, Hongyu Dang, Emily Kawka, and Yogendra Kanitkar as well as Lori Larner. Also, I am extremely grateful to my family and Dean Keffer for their encouragement and support. v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ........................ vii LIST OF FIGURES ................................ ................................ ................................ ....................... xi KEY TO ABBREVIATIONS ................................ ................................ ................................ ...... xiii 1.0 IN TRODUCTION ................................ ................................ ................................ ............... 1 1.1 General ................................ ................................ ................................ ............................. 1 1.2 Biodegradation of RDX ................................ ................................ ................................ ... 1 1.3 Quantitative Polymerase Chain Reaction ................................ ................................ ......... 7 1.4 qPCR Analysis ................................ ................................ ................................ ................. 8 1.5 Study objectives ................................ ................................ ................................ ............... 9 2.0 HIGH THROU GHPUT QUANTIFICATION OF RDX FUNCTIONAL GENES USING THE SMARTCHIP PLATFORM ................................ ................................ ................................ 11 2.1 Introduction ................................ ................................ ................................ .................... 11 2.2 Methods ................................ ................................ ................................ .......................... 14 2.2.1 FunGene and Primer Design Sequence Sets ................................ ........................... 14 2.2.2 Primer Design ................................ ................................ ................................ ......... 14 2.2.3 Published Primer Sets ................................ ................................ ............................. 15 2.2.4 Environmental Sample Collection and DNA Extraction ................................ ........ 16 2.2.5 Quantitative PCR ................................ ................................ ................................ .... 17 2.2.6 qPCR Analysis ................................ ................................ ................................ ........ 19 2.3 Results and Discussion ................................ ................................ ................................ ... 20 2.3.1 FunGene and Primer Design Sequence Sets ................................ ........................... 20 2.3.2 Primer Design ................................ ................................ ................................ ......... 20 2.3.3 Published Primer Sets ................................ ................................ ............................. 22 2.3.4 DNA Extraction ................................ ................................ ................................ ...... 23 2.3.5 Quantitative PCR ................................ ................................ ................................ .... 24 3.0 R ECOMMENDATIONS AND FUTURE WORK ................................ ........................... 33 3.1 Absolute Quantification ................................ ................................ ................................ . 33 3.1.1 Standard Curves ................................ ................................ ................................ ...... 33 3.1.2 Linear Regression of Efficiency Method ................................ ................................ 34 3.2 Relative Quantification ................................ ................................ ................................ .. 34 3.3 Future Work ................................ ................................ ................................ ................... 35 3.3.1 High throughput sequencing ................................ ................................ ................... 35 3.3.2 Proteomics ................................ ................................ ................................ ............... 36 APPENDICES ................................ ................................ ................................ .............................. 37 APPENDIX A : Sequence Sets ................................ ................................ ................................ ... 38 APPENDIX B : Assay Validation ................................ ................................ ............................ 121 vi APPENDIX C : Primer and Plasmid Sequence Information ................................ .................... 126 APPENDIX D : SmartChip Data and Standard Curves ................................ ............................ 133 APPENDIX E : Groundwater Pre - and Post - Biostimulation Results ................................ ....... 176 APPENDIX F : Statistical Analysis ................................ ................................ .......................... 181 REFERENCES ................................ ................................ ................................ ........................... 188 vii LIST OF TABLES Table 1. Sequence set size for identifying FunGene and ................................ .................... 2 0 Table 2. Designed Suite of Assays. ................................ ................................ .............................. 21 Table 3. Purity and concentration of replicate DNA extracts from groundwater and sediment samples from an RDX - contaminated site ................................ ................................ ..................... 24 Table 4. Purity a nd concentration of replicate DNA extracts from uncontaminated soil and water samples ................................ ................................ ................................ ................................ .......... 24 Table 5. Summary of SmartChip Standard Curve Characteristics ................................ ............... 30 Table 6. Recommended assays and plasmids for a single SmartChip run. ................................ ... 33 Table A.1. Search terms used to gather protein sequences ................................ ........................... 39 Table A.2. Identified residues and motifs of functional genes. The positions refer to the reference ................................ ................................ ................................ ......... 41 Table A.3. FunGene and primer design sequences for diaA ................................ ......................... 44 Table A.4. FunGene and primer design sequences for nfsI ................................ .......................... 51 Table A.5. FunGene and primer design sequences for pnrB ................................ ........................ 80 Table A.6. FunGene and primer design sequences for xenA ................................ ........................ 83 Table A.7. FunGene and primer design sequences for xenB ................................ ........................ 97 Table A.8. FunGene and primer design sequences for xplA ................................ ....................... 119 assay did not perform well enough to generate a standard curve. ................................ .............. 124 Table C.1. Designed assays for diaA ................................ ................................ .......................... 127 Table C.2. Designed assays for nfsI ................................ ................................ ............................ 128 Table C.3. Designed assays for pnrB ................................ ................................ .......................... 128 viii Table C.4. Designed assays for xenA ................................ ................................ .......................... 129 Table C.5. Designed assays for xenB ................................ ................................ .......................... 130 Table C.6. Designed assays for xplA ................................ ................................ .......................... 131 Table C.7. qPCR Primer Sets from Literature. The primer set(s) with the highest theoretic al coverage of the relevant primer design sequence set is marked with an asterisk (*). ................ 131 Table C.8. Plasmid sequence insert in formation ................................ ................................ ........ 132 Table D.1. SmartChip Ct values for diaA assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cycle threshold of 28 and a zero value indicates no amplification. ................................ ...................... 134 Table D.2. SmartChip Ct values for nfsI assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the stand ard curve or is above the cycle threshold of 28 and a zero value indicates no amplification ................................ ....................... 135 Table D.3. SmartChip Ct values for pnrB assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cycle threshold of 28 and a zero value indicates no amplification ................................ ....................... 137 Table D.4. SmartChip Ct values for xenA assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cycle threshold of 28 and a zero value indicates no amplification. ................................ ...................... 139 Table D.5. SmartChip Ct values for xenB assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the stand ard curve or is above the cycle threshold of 28 and a zero value indicates no amplification. ................................ ...................... 142 Table D.6. SmartChip Ct values for xplA assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cycle threshold of 28 and a zero value indicates no amplification ................................ ....................... 145 Table D.7. SmartChip Ct values for the 16S rRNA assay. These results correspond to the 3 chip runs performed: 1) xenB and pnrB , 2) xplA and nfsI , and 3) xenA and diaA. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cycle threshold of 28 and a zero value indicates no amplification ........... 147 Table D.8. SmartChip standard curve characteristics ................................ ................................ . 149 Table D.9. Gene copies per mL or g of starting material for nfsI amplification or false positive amplification. ................................ ................................ ............. 151 ix Table D.10. Gene copies per mL or g of starting material for pnrB no amplification or false positive amplification. ................................ ................................ ........ 152 Table D.11. Gene copies per mL or g of starting material for xenA no amplification or false positive amplification. ................................ ................................ ........ 154 Table D.12. Gene copies per mL or g of star ting material for xenB no amplification or false positive amplification. ................................ ................................ ........ 157 Table D.13. Gene copi es per mL or g of starting material for xplA no amplification or false positive amplification. ................................ ................................ ........ 161 either no amplification or false positive amplification ................................ ............................... 162 Table D.15. Gene copies per 16S rRNA gene copy for nfsI amplification or false positive amplification. ................................ ................................ ............. 164 Table D.16. Gene copies per 16S rRNA gene copy for pnrB amplification or false positive amplification. ................................ ................................ ............. 165 Table D.17. Gene copies per 16S rRNA gene copy for xenA amplification or false positive amplification. ................................ ................................ ............. 167 Table D.18. Gene copies per 16S rRNA gene copy for xenB amplification or false positive amplification. ................................ ................................ ............. 170 Table D.19. Gene copies per 16S rRNA gene copy for xplA amplification or false positive amplification. ................................ ................................ ............. 174 Table F.1. P - indicates the test was not appropriate and p - 0.05). ................................ ................................ ................................ ................................ ........... 183 Table F.2. P - indicates the test was not appropriate and p - 0.05). ................................ ................................ ................................ ................................ ........... 183 Table F.3. P - indicates the test was not appropriate and p - values in bold indicate 0.05). ................................ ................................ ................................ ................................ ........... 184 Table F.4. P - indicates the test was not ap propriate and p - 0.05). ................................ ................................ ................................ ................................ ........... 184 x Table F.5. P - values for statistical tests with the MW64 gene copies per mL resul indicates the test was not appropriate and p - 0.05). ................................ ................................ ................................ ................................ ........... 185 Table F.6. P - values for statistical tests indicates the test was not appropriate and p - 0.05). ................................ ................................ ................................ ................................ ........... 185 Tabl e F.7. P - indicates the test was not appropriate and p - 0.05). ................................ ................................ ................................ ................................ ........... 186 Table F.8. P - values for statistical tests with the shallow zone aquifer gene copies per mL results. - values in bold indicate a significant difference (p 0.05). ................................ ................................ ................................ ................... 186 Table F.9. P - values for statistical tests with the perched zone aquifer gene copies per mL results. - values in bold indicate a significant difference (p 0.05). ................................ ................................ ................................ ................... 187 xi LIST OF FIGURES Figure 1. Pathways of RDX metabolism under anaerobic and aerobic conditions (reprinted from Fuller et al. (11) with permission from American Society for Microbiology) ............................... 3 Figure 2. Propose d pathway of diaphorase catalyzed RDX transformation based on stoichiometry of metabolites recovered and NADH consumed (reprinted from Bhushan et al. (17) with permission from Elsevier). Primary reactions involve RDX reduction to RDX anion radical I that u ndergoes denitrohydrogenation to produce III. Secondary reactions involve ring cleavage and spontaneous decomposition in water. Intermediates shown inside brackets were not detected. .... 6 Figure 3. Breakdown of assay coverage. The coverage for xplA is only based on the 27 flavodoxin sequences. ................................ ................................ ................................ ................... 22 Figure 4. Theoretical Coverage of Published and Designed Assays. The theoretical coverage shown is for the assay with the highest coverage for each gene. For xplA , this coverage is only based on the 27 sequences that include the flavodoxin domain as this is the portion of the gene that the assay targeted. ................................ ................................ ................................ .................. 23 Figure 5. Heatmap of groundwater (M W) and Red Cedar River (RC) log gene copies per mL of groundwater or river water, respectively. Black cells indicate either no amplification or false positive amplification. The 16S_rRNA results correspond to the 3 chip runs performed: 1) xenB and pnrB , 2) xplA and nfsI , and 3) xenA and diaA indicate whether they are pre - or post - biostimulation, respectively. ................................ ............ 28 Figure 6. Heatmap of sediment (MW) and soil log gene copies per g of sediment or soil, respectively. Black cells indicate either no amplification or false positive amplification. The 16S_rRNA results correspond to the 3 chip ru ns performed: 1) xenB and pnrB , 2) xplA and nfsI , and 3) xenA and diaA . ................................ ................................ ................................ ................... 29 Figure 7. Boxplots of RDX genes in contaminated groun dwater and uncontaminated river water. respectively. Results for the Red Cedar River (n=2) are provided for comparison to uncontaminated water. The number of detections for each assay are noted on plots (a) and (b). 31 Figure 8. Boxplots of RDX genes in contaminated sediment (n=2) and uncontaminated soil to the 16S rRNA gene, respectively. The number of dete ctions for each assay are noted on plot (a). ................................ ................................ ................................ ................................ ................. 32 xii Figure E.1. Average log gene copies per mL of groundwater in shallow zone aquifer well MW62. Error bars represent the standard deviation and an asterisk (*) denotes a statistically significant difference (p<0.05) between the pre - and post - biostimulation levels. ...................... 177 Figure E.2. Average log gene copies per mL of groundwater in shallow zone aquifer well MW62. Error bars represent the standard deviation and an asterisk (*) denotes a statistically significant difference (p<0.05) between the pre - and post - biostimulation levels. ...................... 178 Figure E.3. Average log gene copies per mL of groundwater in perched zone aquifer well MW48. Error bars represent the standard deviati on. ................................ ................................ .. 178 Figure E.4. Average log gene copies per mL of groundwater in perched zone aquifer well MW60R. Error bars represent the standard deviation. ................................ ............................... 179 Figure E.5. Average log gene copies per mL of groundwater in perched zone aquifer well MW64. Error bars represent the standard deviation. ................................ ................................ .. 179 Figure E.6. Average log gene copies per mL of groundwater in perched zone aquifer well MW66. Error bars represent the standard deviation. ................................ ................................ .. 180 Figure E.7. Average log gene copies per mL of groundwater in perched zone aquifer well MW67. Error bars represent the standard deviation. ................................ ................................ .. 180 xiii KEY TO ABBREVIATIONS aa amino acid BLAST Basic Local Alignment Search Tool BLASTP protein - protein BLAST bp base pair(s) DNX 1,3 - dinitroso - 5 - nitro - 1,3,5 - hexahydrotriazine EPA Environmental Protection A gency FunGene Functional Gene Pipeline and Repository HMM HMMER Hidden Markov Model HPCC High Performance Computing Center KBS Kellogg Biological Station LRE Linear Regression of Efficiency MNX 1 - nitroso - 3,5 - dinitro - 1,3,5 - hexahydrotriazine NCBI Natio nal Center for Biotechnology Information NDAB 4 - nitro - 2,4 - diazabutanal nt nucleotide(s) PS p rimer s et PDB Protein Data Bank qPCR quantitative polymerase chain reaction RDX royal demolition explosive, hexahydro - 1,3,5 - trinitro - 1,3,5 - triazine rxn r eaction TNX 1,3,5 - trinitroso - 1,3,5 - hexahydrotriazine 1 1.0 INTRODUCTION 1.1 General Hexahydro - 1,3,5 - trinitro - 1,3,5 - triazine (RDX) is a xenobiotic compound that enters the environment through the production, use and disposal of explosives (1) . It has contaminated many military sites across the US (2) and is a cause for concern because RDX has a low soil sorption coefficient, which means it is mobile through soil and may contaminate groundwater (3) . Through a lite rature review, the US Environmental Protection Agency (EPA) has found RDX to have toxic effects and identified it as a possible human carcinogen based on evidence of adverse nervous system effects in humans and liver and lung tumors observed in animal expe riments (4) . While RDX does not have a federal drinking water standard currently, the US EPA set a lifetime health advisory guidance level at 2 µg/L for drinking water (5) . 1.2 Biodegradation of RDX Between the 1970s and 1990s, it was discovered that RDX was susceptible to biodegradation, both aerobically and anaerobically. In 1981, McCormick, Cornell and Kaplan investigated the biological degradation of RDX and the products generated under both aerobic and anaerobic conditions, using activated and anaerobic s ewage sludge, respectively, as the inoculum. While the authors did not observe any degradation for the aerobic case, they did find rapid degradation under anaerobic conditions with the production of formaldehyde and nitroso derivatives (6) . About a decade later, Binks, Nicklin and Bruce cultured Stenotrophomonas maltophilia PB1 from soil samples from an RDX contaminated site. This microorganism utilized RDX as its sole nitrogen source under aerobic and nitrogen - limiting conditions (1) . Later work identified additional conditions that RDX may be degraded under, including nitrate - reducing, acetogenic 2 and methanogenic, and bacteria capable of the degradation (7, 8) . These studies confirmed the potential for using biodegradation to remediate RDX contaminated sit es. Along with the discovery of bacteria capable of degrading RDX, the genes responsible for this degradation were investigated. To date, six functional genes have been linked to RDX degradation: diaA , nfsI , pnrB , xenA , xenB , and xplA (9, 10) . These genes may be used as biomarkers to detect the presence and monitor the level of bacteria capable of degrading RDX at a contaminated site. Also, the genes present at a site will indicate the end products likely to be produced from biodegradation because the enzy mes encoded by these functional genes attack the RDX compound in different ways. While Crocker et al. (7) identified seven possible degradation pathways, Fuller et al. (11) noted that there are three main ones: 1) anaerobic nitro - reduction, 2) anaerobic ri ng cleavage, and 3) aerobic denitration ( Figure 1 ). The known RDX functional genes have all been connected to one of these three pathways. The most well researched of the RDX functional genes is xplA . This gene encodes a cytochrome P450 - like enzyme that also contains a flavodoxin domain on the n - terminus (12) . It has been identified in multiple aerobic bacteria genera, including Gordonia , Wil liamsia and Rhodococcus , and it works with the xplB gene, a flavodoxin reductase (10) . Fournier et al. (13) investigated the metabolites of aerobic RDX degradation using Rhodococcus sp. strain DN22 and hypothesized a degradation pathway based on these. Dur ing RDX degradation, these authors measured the production of nitrite (N ), nitrous oxide (N 2 O), formaldehyde (HCHO), carbon dioxide (CO 2 ) , ammonia (NH 3 ), and 4 - nitro - 2,4 - diazabutanal (NDAB, C 2 H 5 N 3 O 3 ) ; both nitrite and formaldehyde were found to be trans ient intermediates. The authors hypothesized that the early 3 Figure 1 . Pathways of RDX metabolism under anaerobic and aerobic conditions (reprinted from Fuller et al. (11) with permission from American Society for Microbiology) production of nitrite indicated that denitration was the initial mechanism used to breakdown RDX, and this denitration would be catalyzed by the xplA enzyme. Seth - Smith et al. (12) supported these findings when they investigated the RDX degradation pathw ay of Rhodococcus 4 rhodochrous strain 11Y under aerobic conditions. These authors also observed early production of nitrite, and noted that the other products formed, formate and formaldehyde in this case, would also be explained by a denitration mechanism since denitration would destab ilize the molecule for spontaneous ring cleavage to occur. Fuller, Perreault and Hawari (14) determined that xplA may also operate under microaerophilic conditions, although the degradation rates are much slower than for aerobic conditions, and NDAB was me asured as a breakdown product, which is expected for the aerobic denitration pathway. Therefore, the current evidence indicates that the xplA enzyme catalyzes aerobic denitration of RDX (Pathway C in Figure 1). Another two genes found in aerobic bacteria are xenA and xenB . Both genes are xenobiotic reductases that have been identified in the Pseudomonas genus to date, and the enzymes contain a flavin mononucleotide - containing oxidoreductase domain (10) . Even though the bacteria are aerobic, Fuller et al. ( 15) found RDX degradation to occur under anoxic and anaerobic conditions, and it was severely inhibited under aerobic conditions for Pseudomonas putida II - B ( xenA ) and Pseudomonas fluorescens I - C ( xenB ) . The authors hypothesized that at least for xenB , the inhibition may be due to oxygen competing with RDX for the active site. Regarding the breakdown products, formaldehyde and small levels of methylenedinitramine (MEDINA), nitrous oxide, nitrite, and ammonia were measured, although it was inconclusive if th e ammonia might have been nitramide or some other product. Based on this, the authors hypothesized that the degradation pathway was the anaerobic ring cleavage route. A later study by Fuller et al. (11) used the same bacteria strains and supported this hyp othesis as they did not measure any 1 - nitroso - 3,5 - dinitro - 1,3,5 - hexahydrotriazine (MNX), 1,3 - dinitroso - 5 - nitro - 1,3,5 - hexahydrotriazine (DNX) or 1,3,5 - trinitroso - 1,3,5 - hexahydrotriazine (TNX) intermediates during 5 RDX degradation. The presence of these inter mediates would have indicated that these enzymes catalyzed the anaerobic nitro - reduction pathway. While the mechanism of attack is currently not known for xenA and xenB , these authors hypothesized that it may involve an initial denitration reaction based o n the production of nitrite during RDX degradation. This corresponds to Pathway B2 (Figure 1), which includes a denitration step prior to ring cleavage. Contrary to the previous genes , diaA has been isolated from an anaerobic bacterium. It is a diaphorase found among the Clostridium genus, and it contains a flavin reductase - like domain with a rubredoxin - like domain on the n - terminus (16) investigations into the degradation mechanism of diaA have been done with whole cell assa ys . Bhushan et al. (17) investigated the degradation route for RDX using the purified diaphorase enzyme from Clostridium kluyveri . They found that diaA functioned under anaerobic conditions and was inhibited by oxygen, and they measured the production of n itrite, ammonium, nitrous oxide, formaldehyde, and MEDINA, with MEDINA being a transient intermediate, during RDX degradation. From this, they concluded the degradation pathway involved an initial denitration step followed by ring cleavage. The diaA enzyme is expected to catalyze the transformation of RDX to ( species I) through a transfer of one redox equivalent ( Figure 2 ). Spontaneous denitration, ring cleavage and decomposition in water completes the degradation pathway. This proposed pathway agrees with the more general anaerobic ring cleavage pathway with an initial denitration step (Pathway B2 in Figure 1 ). 6 Figure 2 . P roposed pathway of diaphorase catalyzed RDX transformation based on stoic hiometry of metabolites recovered and NADH consumed (reprinted from Bhushan et al. (17) with permission from Elsevier) . Primary reactions involve RDX reduction to RDX anion radical I that undergoes denitrohydrogenation to produce III. Secondary reactions i nvolve ring cleavage and spontaneous decomposition in water. Intermediates shown inside brackets were not detected. The remaining two genes, nfsI and pnrB , are both type I oxygen - insensitive nitroreductases. N fsI has been isolated from the Enterobacter and Morganella genera, which are both facultative anaerobes , and pnrB from the Pseudomonas and Stenotrophomonas genera, which are both aerob ic . A study on the nfsI gene by Kitts, Cunningham and Unkefe r (18) investigated the degradation pathway and measured MNX, DNX and TNX as intermediate products produced by M. morganii strain B2 under O 2 - depleted conditions. These intermediates suggest that nfsI functions according to the anaerobic nitro - reduction pathway and may catalyze the reduction of the nitro groups on RDX, which would eventually lead to spontaneous ring cleavage. These authors also measured the production of CO 2 , which supported the thought that the ring in the RDX structure had been broken. It is as sumed that pnrB follows the same pathway as nfsI , but not much work has been done to elucidate the products generated by this enzyme. Lee et al. (19) 7 investigated the degradation of RDX under aerobic conditions with Pseudomonas sp. HK - 6 and a later study u sed Stenotrophomonas maltophilia OK - 5 (20) , but both only noted that metabolic intermediates were not detected. While these enzymes are not sensitive to oxygen, Kitts et al. (21) discovered that only the purified nfsI enzyme will degrade RDX under aerobic conditions, while whole cell assays with Enterobacter cloacae and Morganella morganii strain B2 only degraded RDX under anaerobic conditions. For pnrB , it is unknown if a similar situation exists because work has only been done with this gene under aerobic conditions. 1.3 Quantitative Polymerase Chain Reaction Quantitative polymerase chain reaction (qPCR) has been used extensively to detect and quantify genes of interest in environmental samples. The conventional approach uses a thermocycler that can run eith er 96 or 384 reactions simultaneously. Reactions include a DNA - binding dye, deoxyribonucleotides (dNTPs), DNA polymerase, MgCl 2 , stabilizers, primers, and DNA template. Primers include a forward and reverse primer designed to specifically target the gene o f interest, and the DNA template is DNA extracted from the environmental samples to be assayed. Many studies exist that used this method to detect and quantify RDX functional genes (22 - 24) . Conventional qPCR, however, is limited by the number of reaction s that can be run, the sample When there are multiple genes of interest for a set of samples, it will quickly become necessary to perform multiple runs with th e thermal cycler. The number of runs that may be performed, however, is constrained by the available sample volume. While it may be feasible to test around 8 20 genes, depending on the original sample volume, DNA concentration and technical replicates desire d, this method is impossible for studies investigating hundreds of genes. Lastly, the primer sets designed to be used with conventional qPCR are historically designed based on only one gene sequence, or possibly a consensus sequence. In the case where ther e is high genetic variability known to exist among a gene, this method may not be capable of capturing the genetic diversity as multiple primer sets would be required. Therefore, it is necessary to consider high throughput options. High throughput qPCR us es the same methodology as conventional qPCR, but it runs significantly more reactions with smaller volumes in parallel. The SmartChip Real - Time PCR System (SmartChip) is one platform that is capable of high throughput qPCR. With this technology, 5,184 reactions may be run simultaneously with a reaction volume of only 100 nL. This allows multiple genes and/or multiple primer sets for a single gene to be tested against m ultiple samples using a much smaller sample volume as compared to conventional qPCR. This platform has been used for a wide range of applications including detecting disease biomarkers and antibiotic resistance genes (25, 26) ere are no studies to date detecting or quantifying RDX genes using a high throughput qPCR platform. 1.4 qPCR Analysis Both absolute and relative quantification techniques may be used with high throughput qPCR. The common absolute quantification method requires running reactions with known quantities of a standard simultaneously with the samples. The C t values from the standard reactions are plotted against the logarithm of the known starting quantity to generate a standard curve, which can be 9 described by the equation of a line: . The gene copies in the sample reactions may then be calculated using this equation. An alternative absolute quantification approach is the linear regression of efficiency (LRE) method, wh ich bases quantification off of fluorescence readings and an optical calibration factor (27) . This optical calibration factor is determined from a few reactions with a known standard, and Rutledge and Stewart (27) recommend using lambda gDNA as the univers al standard. For relative quantification, the method is typically used for determining fold changes in the gene levels of environmental samples. In this method, C T is the difference in C T values between the target sample and reference sample, w here C T is the difference in C t values between the gene of interest and a reference gene (28) . Another relative quantification method uses the standard curve equation for absolute quantification to estimate gene copy numbers and then normalizes the result for the target gene to the 16S rRNA gene. However, a standard curve is not actually generated in this case, instead the amplification efficiency is assumed to be 100% and the y - intercept is set at the threshold cycle (29) . While relative quantification do es not require sample space to be dedicated to standard curve reactions and therefore permits the maximum number of genes and samples to be tested at one time, it is desirable to be able to perform absolute quantification if needed. 1.5 Study objectives 1. To de sign a new suite of primer sets for each RDX degrading functional gene , to ensure a high coverage of all gene sequences currently available. 2. To ensure the primer sets work efficiently with the SmartChip Real - Time PCR System, which is a massive - parallel sin gleplex PCR platform. 10 3. To quantify RDX functiona l genes 1) in groundwater at a contaminated site before and after biostimulation, 2) in contaminated sediment and 3) in uncontaminated water and soils. 11 2.0 HIGH THROUGHPUT QUANTIFICATION OF RDX FUNCTIONAL GENES USING THE SMARTCHIP PLATFORM 2.1 Introduction Hexahydro - 1,3,5 - trin itro - 1,3,5 - triazine (RDX) has contaminated many military sites across the US (2) . It is released to the environment through its production, use and disposal (1) , and typically ends up in groundwater due to its high mobility through soil, as determined by i ts low soil sorption coefficient (3) . RDX is not regulated in drinking water, but the US Environmental Protection Agency (EPA) has set a lifetime health advisory guidance level of 2 µg/L since the compound has been identified as a possible human carcinogen (5) . Bioremediation is one feasible treatment approach for RDX - contaminated sites. Bacteria from many different genera, including Clostridium , Enterobacter , Gordonia , Pseudomonas , and Rhodococcus , have been found capable of degrading RDX under a wide ran ge of conditions, such as aerobic, anaerobic, nitrate - reducing, and methanogenic (7, 8) . Six of the functional genes responsible for this degradation have also been identified: diaA , nfsI , pnrB , xenA , xenB , and xplA (9, 10) . These functional genes may be u sed as biomarkers for detecting the presence of bacteria capable of RDX degradation in environmental samples. Besides detecting RDX - degrading bacteria, the identification of the functional genes present at a contaminated site can also indicate the product s expected to be formed during RDX biodegradation. The three primary degradation pathways identified for RDX are aerobic denitration, anaerobic ring cleavage and anaerobic nitro - reduction (11) . Three of the RDX genes, diaA , xenA and xenB , are expected to d egrade RDX through the a naerobic ring cleavage route, 12 which produces methylenedinitramine (MEDINA), formaldehyde and nitrous oxide (15, 17) . NfsI and pnrB , on the other hand, are thought to follow the anaerobic nitro - reduction pathway and produce 1 - nitroso - 3,5 - dinitro - 1,3,5 - hexahydrotriazine (MNX), 1,3 - dinitroso - 5 - nitro - 1,3,5 - hexahydrotriazine (DNX) or 1,3,5 - trinitroso - 1,3,5 - hexahydrotriazine (TNX) as interm ediates before ring cleavage yields carbon dioxide, methanol, nitrous oxide, and formaldehyde (18, 19) . Lastly, the xplA gene is expected to catalyze the aerobic denitration pathway with carbon dioxide, ammonia, and 4 - nitro - 2,4 - diazabutanal (NDAB ) being p roduced (13) . Knowing the expected intermediates and end products provides a second line of evidence that may be used to confirm biodegradation is occurring. Quantitative PCR (qPCR) has been widely used for detecting and quantifying RDX functional genes i n environmental samples. Andeer et al. (22) investigated RDX degradation in contaminated soil and detected the presence of xplA in 11 out of 16 soil samples tested. Michalsen et al. (24) monitored xplA gene copy levels while testing the feasibility of bios timulation and bioaugmentation for a contaminated aquifer, and Wilson and Cupples (30) quantified x enA , xenB and xplA in contaminated groundwater and sediment samples. However, Fuller et al. (23) tested groundwater from two RDX - contaminated sites and did n ot detect xenA , xenB nor xplA . They identified multiple potential reasons for this, with one being that the primers used could not amplify the gene variants present. This highlights a concern with conventional qPCR, which is that one primer set may not be capable of amplifying all of the gene variants. been used to detect and quantify RDX functional genes to date. Conventional qPCR utilizes a 13 thermal cycler that c an only run either 96 or 384 reactions simultaneously, which limits the number of samples and assays included in each run. This can make the methodology expensive and time consuming when there are a lot of samples and assays to test, and sample volume may also become a limiting factor in this situation. The use of a single primer set for each gene also potentially restricts the ability of the gene to be detected, especially for genes that have high sequence diversity that cannot be captured by a single prim er set. This could result in false negative results and indicate the need for bioaugmentation when it is not actually necessary. These limitations of conventional qPCR are addressed with high throughput qPCR platforms. One high throughput qPCR platform is the SmartChip Real - Time PCR System (SmartChip) which can run 5,184 reactions simultaneously with only 100 nL reaction volumes. This system provides multiple configurations for testing a large number of assays and samples, and it has been used for many app lications, including detecting antibiotic resistance genes in environmental samples (26) . The purpose of this study was to design a suite of assays for each gene to use with the SmartChip platform for detecting RDX functional genes in environmental samples . These suites of assays were designed with the goal of maximizing the theoretical coverage of known gene sequences. This will provide a more efficient method for quantifying all six RDX functional genes and a higher probability that the genes will be dete cted if they are present at RDX - contaminated sites. 14 2.2 Methods 2.2.1 FunGene and Primer Design Se quence Set s A preliminary set of sequences for each of the six RDX genes was collected from the National Center for Biotechnology Information (NCBI), Protein Data Bank (PDB), and UniProt databases. These sequences were subjected to strict criteria concerning the length, identity and residues and motifs to ensure they were relevant to the gene. Those that passed were identified as the FunGene se quence set, which was used to develop the HMMER Hidden Markov Model (HMM) search program for the Michigan State University RDP Functional Gene Pipeline and Repository (FunGene) (31) . With this HMM model, FunGene was capable of performing a more thorough search of the NCBI non - r edundant protein database. The sequences gathered by FunGene were subjected to the same criteria before being included in the final primer design sequence set. Additional detail regarding the identification of these sequence sets is provided in Appendix A. 2.2.2 Primer Design New primer sets were designed using the EcoFunPrimer tool (32) (https://github.com/rdpstaff). This tool designed complementary primer sets for each gene to maximize the overall theoretical coverage of the input primer design sequence set . Input parameters were set for the tool to limit de generacy among each primer and mismatches allowed between a primer and target sequence to two, melting temperature to between 59°C and 61°C, and primer length to between 18 and 21 nt. However, the primer length for diaA was expanded to between 15 and 25 nt in order to meet the T m requirement. Regions for the forward and reverse primers were also specified to ensure all primer sets for a single gene were amplifying the same region, that the region amplified contained some of the identified residues and motif s, and to 15 control the length of the amplicon generated. Multiple runs were performed with different primer regions and subsets of the initial sequence set to identify the optimum suite of primers. The final suite of assays for each gene were run through RD (32) tools to determine the theoretical coverage. Primer sets were also validated on a C1000 Touch thermal cycler with CFX96 real - time platform (Bio - Rad, Hercules , C A) before proceeding with running them on the SmartChip. Ad ditional detail regarding this validation is provided in Appendix B. This test was to confirm the primer Primer sets were removed if the linear dynamic range did not include at least 4 orders of magnitude, if multiple dilutions in this range had scattered replicates, if the range did not extend to 10 3 copies per reaction or fewer, and if the amplification efficiency was less than 70% or greater than 120%. In the case that removing assa ys would greatly reduce the theoretical coverage of the assay suite, - BLAST tool to replace them. 2.2.3 Published Primer Sets Literature was searched to generate a list of currently published quantitative PCR (qPCR) p rimer sets for each gene. These primer sets were then run against the appropriate primer design coverage and enable a comparison between these and the designed assays . They were also validated like the designed assays on a C1000 Touch thermal cycler with CFX96 real - time platform before running them on the SmartChip (Appendix B). 16 2.2.4 Environmental Sample Collection and DNA Extraction Contaminated and uncontaminated water and soil samples were collected for analysis. From an RDX - contaminated naval site, groundwater samples were collected before and after biostimulation with fructose and sediment samples before biostimulation . The groundwater included two wells from a shallow z one aquifer and five from a perched zone aquifer . For the shallow zone aquifer, samples were collected in November 2017 and June 2018 and for the perched zone aquifer, samples were collected in January 2018 and April 2018. Biostimulation was performed in M arch 2018 using injection wells and an injection trench for the shallow and perched zone aquifers, respectively. The water was collected in 1 L amber glass bottles, shipped overnight on ice, and stored in the dark at 4°C prior to filtration . The sediment s ample was collected in a Z iplock bag, shipped overnight and stored at 4°C prior to DNA extraction. For the uncontaminated samples, water was collected from the Red Cedar River in Michigan and a gricultural soils were taken from MSU cal Station (KBS). The river water was collected in 1 L plastic bottles and frozen at - 20 ° C until filtration. The agricultural soil samples were collected in Ziplock bag s and immediately stored at 4°C until DNA extraction. For the groundwater and river water samples, DNA was extracted with the DNeasy PowerWater . Approximately 950 mL of ground water or 250 mL of rive r water was filtered on a 0.47 µm diameter 0.22 µm filter ( GSWG047S6, Millipore) using a vacuum pump and then the filter was stored in the PowerBead tube at - 20°C until proceeding with the rest of the extraction protocol . Triplicate extractions were done f or the groundwater from each well before biostimulation and from the shallow zone aquifer wells after biostimulation . Duplicate extractions were done for the groundwater from the 17 perched zone aquifer wells after biostimulation and from the Red Cedar River water. DNA was eluted in 50 µ L and stored at - 20°C. For the sediment and soil samples, DNA was extracted with the DNeasy PowerSoil kit (Qiagen) . Approximately 1 g of sediment or soil was used for each extraction and extr actions were done in triplicate for each sample . With the contaminated sediment sample, 40 mg of Carnation instant nonfat dry milk (Nestlé, Rosslyn, VA) per g of sediment was added at the beginning of the extraction to improve the DNA yield (33) . DNA was e luted in 50 µ L and stored at - 20°C. The A260/A280 value w as measured for each sample with a NanoDrop ND - 1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) and the DNA concentration was determined using the appropriate Qubit dsDNA BR or H S assay kits with a Qubit fluorometer (Invitrogen, Eugene, Oregon) 2.2.5 Quantitative PCR RDX genes present in environmental samples were quantified on the SmartChip using the primer sets that passed all of the above tests. T hese runs included serial plasmid dilutio ns to generate standard curves and DNA from the environmental samples. All primers were manufactured by Integrated DNA Technologies (IDT, Coralville, IA) and tested against plasmids containing a partial gene sequence (GenScript, Piscataway, NJ). Gene 18 copy number for the plasmids was calculated as described by Ritalahti et al. (34) . Primer and plasmid sequence information is provided in Appendix C. Samples and assays were dispensed into the 72x72 nanowell chip with the Multisample Nanodispenser. Individual reactions on the chip consisted of a total volume of 100 nL with 1X Lig htCycler 480 SYBR Green I Master (Roche Applied Sciences, Indianapolis, IN), 0.5 µM each of the forward and reverse primers (2.5 µM each in the case of two forward and/or reverse primers), DNA, and balance PCR grade water. Extracted DNA from the environmen tal samples w as diluted to 20 ng/µ L for a total of 0.2 ng in the reaction or run at concentration if the s ample concentration was be low 20 ng/µ L . Cycling was performed following the SmartChip protocol specified by Wang et al. (35) . The 24 assays by 216 samples configuration was used for each SmartChip run . Each chip included the designed and published primer sets for two genes along with the universal bacteria primer set, which targets the 16S rRNA gene (36) . Triplicate 10 - fold serial plasmid dilutio ns (10 1 - 10 7 copies/reaction) were included for each plasmid required to generate the standard curves for the primer sets being used . For the 16S rRNA primer set, the plasmid dilution series also included reactions with 10 8 copies . All environmental samples were run on each chip as biological duplicates or triplicates. A reaction blank and triplicate negative controls using a plasmid with an aceA gene insert ( 10 3 copies/reaction ) were also included . 19 2.2.6 qPCR Analysis Reactions that did not amplify or were ide ntified as false positives were considered as missing data for all analyses. False positives were reactions that amplified later than the threshold cycle, which was set at 28 as recommended for the SmartChip (37) , reactions with C t values outside of the st andard curve range, or reactions that were the only replicate to amplify. The copy number of each gene per reaction was calculated based on the standard curve for the corresponding primer set and plasmid. This was then analyzed in two ways: 1) copy numbe r per reaction was converted to per mL or per g of starting material, as appropriate, and 2) copy number per reaction was normalized to the 16S rRNA gene copy number per reaction as described previously (29) . Heatmaps were generated for the copy numbers pe r mL or per g of starting material using the gplots v3.0.1 package in R v3.5.1 (38, 39) , and bar charts were created in Excel to display the groundwater pre - and post - biostimulation results for each well. Additionally, boxplots were generated from the resu lts of both analysis approaches using the ggplot2 v3.0.0 package (40) . For the groundwater results, only assays that had detections in both the pre - and post - biostimulation samples were plotted, and for the sediment and soil results, the assays that had de tections in at least one of the samples were plotted. Statistical analysis of the difference between the pre - and post - biostimulation samples for the gene copy number per mL of groundwater values was performed in R using the car v3.0 - 2 and matrixTests v0 .1.0 packages (41, 42) - sample t - - test or Mann - Whitney U test was used for each primer set as appropriate. A detailed explanation of the statistical analysis is provided in Appendix F. 20 2.3 Results a nd Discussion 2.3.1 Fun Gene and Primer Design Sequence Sets Table 1 summarizes the number of sequences at the beginning and final stages of identifying the FunGene and primer design sequence sets. The elimination criteria specified in Appendix A greatly reduced the number of sequences included in these sequence sets. Howe ver, the use of FunGene did increase the number of sequences used for primer design. The accession numbers for the nucleotide and protein sequences included in the final FunGene and primer design sequence sets are reported in Tables A.3 - A.8 (Appendix A). Table 1 HMM mod el. Functional Gene Initial Sequences FunGene Se quence Set FunGene Sequences Primer Design Sequence Set diaA 1,051 96 123,691 16 1 nfsI 2,926 23 58,706 723 pnrB 1,178 32 61,601 56 xenA 1,852 14 199,903 309 xenB 4,435 58 110,413 425 xplA 7 7 189,222 49 2 1 Initial criteria yielded 123 sequences, but these were further filtered to only include Clostridium . 2 Partial sequences were allowed for this gene 27 sequences included the flavodoxin domain and 30 included the cytochrome P450 domain. 2.3.2 Primer Design A suite of assays was successfully designed for each gene to have at least 80% coverage of the primer design sequence set according to the EcoFunPrimer Tool, except for diaA ( Table 2 ). For diaA , the EcoFunPrimer tool was unable to find many regions in the sequences to design primers that met the T m requirements even with a larger allowable primer length and more variability permitted in the amplicon lengths. This relaxation of the design parameters did yield assays that covered a majority of the diaA sequences though. All assays amplify at least one residue and 21 Table 2 . Designed Suite of Assays. Functional Gene Number of Assays Total Coverage (%) Amplicon Length (bp) Residues Amplified Motifs Amplified diaA 3 62.4 84 - 136 2 1 nfsI 8 92.5 181 - 200 4 1 pnrB 5 83.9 156 - 189 10 1 xenA 17 81.6 190 - 200 4 1 xenB 19 90.4 129 - 134 1 1 xplA 3 96.3 1 157 - 165 1 2 1 The coverage for xplA is only based on the 27 flavodoxin sequences motif, and these may be used in future work to confirm the amplicons generated in qPCR are from the expected gene. All primer sequences are reported in Appendix C. Results from the qPCR runs on the Bio - Rad thermal cycler to validate the assays are provided in Appendix B. One, five and two assays were removed from the xenA , xenB and xplA gene su ites, respectively, due to poor performance on the Bio - Rad thermal cycler. The two assays for xplA - BLAST tool, and the new primer sets had similar coverages to the original ones and performed better on the Bio - Rad thermal cycler (Appendix C). For xenA and xenB , h owever , these primer sets were not redesigned because their removal did not change the theoretical coverage of the full assay suites. A breakdown of the coverage for each assay as determined by the EcoFunPrimer t ool is presented in Figure 3 . The first assay has the highest coverage for each gene and the subsequent assays generally decrease in coverage. Therefore, only the first, or a select few of the assays, could be used for evaluating environmental samples and still maintain a good covera ge of the gene of interest. 22 Figure 3 . Breakdown of assay coverage. The coverage for xplA is only based on the 27 flavodoxin sequences . T he theoretical coverage determined with the SeqFilters and ProbeMatch tools was higher th an that from the EcoFunPrimer tool (Tables C.1 - C.6 in Appendix C). This occurred because these tools allowed certain mismatches that the EcoFunPrimer tool does not. Along with increasing the coverage of the assays, this analysis also revealed that a single sequence may be amplified by additive, and the full suite should only be used to ensure a high probability that the gene will be detected if it is present in the sample. The copy numbers present may then be estimated based on the assay with the highest initial copy number. 2.3.3 Published Primer Sets Primer sets already published in literature and used for qPCR were identified for all genes and are listed in Table C.7 (Appendix C). Only diaA was found to have no previously published qPCR primer sets. The theoretical coverage of the complete suite of designed assays is consistently higher than the published assays, which supports that the designed assays improve 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% diaA nfsI pnrB xenA xenB xplA Coverage (%) Functional Gene PS1 PS2 PS3 All Other Primer Sets Not Hit 23 upon tho se currently used ( Figure 4 ). Only xplA had a small amount of improvement with the designed assays, and this was due to a high similarity among most of the xplA sequen ces so one primer set was able to capture most of the sequences. Figure 4 . Theoretical Coverage of Published and Designed Assays. The theoretical coverage shown is for the assay with the highest coverage for each gene . For xplA , this coverage is only based on the 27 sequences that include the flavodoxin domain as this is the portion of the gene that the assay targeted. 2.3.4 DNA Extraction The purity and concentration values for each DNA extraction are reported in Tables 3 and 4 for the RDX - contaminated and uncontaminated samples, respectively. The purity values were generally around 1.80 as desired for DNA, except for cases where DNA yield was very low, or less than 10 ng/µL. In most cases for the groundwater samples, the ones fr om after biostimulation yielded more DNA than those before. The RDX - contaminated sediment sample, MW71, had very low DNA yields even with the addition of dry milk during the extraction process. This is assumed to be partially due to the fact that the sedim ent appeared very clay - like, and clay would strongly bind DNA (33) . 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% diaA nfsI pnrB xenA xenB xplA Theoretical Coverage (%) Functional Gene Published Assays Designed Assays 24 Table 3 . Purity and concentration of replicate DNA extracts from groundwater and sediment samples from an RDX - contaminated site Source Sample ID Biostimulation Stat us A260/A280 Concentration (ng/µ L ) 1 2 3 1 2 3 Shallow Aquifer MW32 Pre 1.74 1.94 2.14 11.8 9.78 15.2 Post 1.86 1.93 1.86 43.7 109 142 MW62 Pre 1.91 1.96 1.93 101 90 133 Post 1.70 1.89 1.84 19.4 119 68.0 Perched Aquifer MW48 Pre 1.48 1.74 1.52 0.079 6.06 13.9 Post 1.85 1.85 N/A 405 465 N/A MW60R Pre 2.48 1.76 1.29 3.42 0.129 0.098 Post 1.98 1.74 N/A 48.9 38.2 N/A MW64 Pre 2.02 - 0.80 1.23 4.11 1.14 2.70 Post 1.86 1.89 N/A 249 352 N/A MW66 Pre 2.15 1.94 1.70 19.8 18.1 12.4 Post 2.07 1.7 N/A 8.07 43.5 N/A MW67 Pre 2.53 6.91 1.83 4.10 4.92 5.75 Post 2.16 1.64 N/A 0.086 0.102 N/A Sediment MW71 Pre 1.04 1.00 1.05 2.31 2.75 2.29 Table 4 . Purity and concentration of replicate DNA extracts from uncontaminated soil and water samples S ource Sample ID A260/A280 Concentration (ng/µ L ) 1 2 3 1 2 3 KBS Agricultural Soil T1 1.89 1.69 1.85 31.0 27.9 47.5 T2 1.65 1.96 1.71 24.4 35.2 32.0 T3 1.76 1.92 1.90 24.7 27.0 41.7 T4 1.71 1.89 1.87 37.5 43.8 46.5 MSU Agricultural Soil Soil E 1.88 1.67 1.87 31.7 17.3 16.3 Soil F 1.83 1.81 1.57 27.8 14.4 13.8 Red Cedar River RC_B 1.86 1.95 N/A 26.3 28.1 N/A 2.3.5 Quantitative PCR The SmartChip C t values, standard curve characteristics, gene copies per mL or g, and gene copies per 16S rRNA gene for each assay are reported in Appendix D. All samples amplified consistently with the 16S rRNA assay, except one replicate of MW66_Pre on the first chip, c onfirming that DNA extraction was successful and that qPCR was not inhibited ( Figure 5 and Figure 6 ). The heatmaps also show that the designed assays resulted in more detections of each gene than the published ones, which suggests that the greater theoretical coverage of these assays did translate into better detection. In most cas es, the standard curves were also better in terms of 25 amplification efficiency and R 2 values. However, pnrB and xplA did have a few designed assays with low amplification efficiencies ( Table 5 ). For pnrB , this was only PS2 that had a low amplification efficiency, and the other designed assays were comparable to the published ones with regard to standard curve characteristics. For x plA , on the other hand, Pub1, Pub2 and Pub4 performed much better than the designed assays, and this suggests that the results for these assays may be more reliable. RDX functional genes were detected in most of the samples, and almost every gene was det ected. The sediment samples (MW71) were the only ones to yield detection of the RDX functional genes by only one primer set, but this may have been due to the low DNA concentration of these samples. This is supported by the lack of gene detection in MW48_P re.1, which also had very low DNA yield. Regarding the genes detected, xenA and xenB were the most commonly detected. NfsI , pnrB and xplA were also detected in at least a few of the samples, and diaA was the only gene not detected in any of the samples. The perched zone aquifer had positive detection of every gene except diaA , while the shallow zone aquifer only had detections of pnrB , xenA , and xenB . The assays that had detections at both time points yielded gene copies in the range of 10 2 to 10 5 per mL of groundwater, and this value was generally consistent at both time points for each assay, whether looking at the wells individually (Appendix E) or combined for each aquifer ( Figure 7 a - b). This agrees with the results of Michalsen et al. (24) who found xplA gene copies to be present at 10 3 to 10 5 copies per mL in an RDX contaminated aquifer before biostimulation and experience no change after 26 biostimul ation. There were a few assays, xenB PS1, PS5 and PS7 for the shallow zone aquifer and pnrB PS2 and xenB PS1 for the perched zone aquifer, that did have a statistically significant difference (p<0.05) between the pre - and post - biostimulation levels (Append ix F). For the shallow zone aquifer, this was a decrease in the post - biostimulation level compared to pre - biostimulation, while for the perched zone aquifer, it was an increase ( Figure 7 a - b). Further sampling would be required to confirm either of these trends. Considering the 16S rRNA normalized results for the groundwater samples, there is a clear decrease in the relative a bundance of the genes detected in the post - biostimulation samples ( Figure 7 c - d). This implies that biostimulation may have initially promoted the growt h of a wide range of bacteria, and a later time point may show the RDX degraders gaining dominance. Interestingly, when the genes were also detected in the Red Cedar River samples, they were present at similar levels and relative abundance to those in the contaminated groundwater ( Figure 7 ). For the sediment and soil samples, most of the detections were with the agricultural soils. All of the genes we re detected in the KBS soils, except for diaA , and all of the genes, except diaA and pnrB , were detected in the MSU soils at levels ranging from 10 4 to 10 8 copies per g of soil ( Figure 8 a). Only one assay, PS7 from nfsI , yielded positive detection in the contaminated sediment samples. With this assay, nfsI was detected at levels around 10 5 copies per g of contaminated sediment ( Figure 8 a). This is similar to the findings of Wilson and Cupples (30) who quantified xenA , xenB and xplA in RDX - contaminated sediment at levels around 10 5 copies per g; they d id not test for nfsI . The uncontaminated KBS soils had a higher copy number, approximately 10 6 copies per g, and the MSU soils had no detection ( Figure 8 a). Despite the KBS soils having a higher copy number, the contaminated sediment had a higher relative 27 abundance ( Figure 8 b). This is as expected because the microorganisms capable of RDX degradation should be able to compete better in the RDX contaminated sediment than uncontaminated soils and therefore be more dominant. 28 Figure 5 . Heatmap of groundwater (MW) an d Red Cedar River (RC) log gene copies per mL of groundwater or river water, respectively. Black cells indicate either no amplification or false positive amplification. The 16S_rRNA results correspond to the 3 chip runs performed: 1) xenB and pnrB , 2) xplA and nfsI , and 3) xenA and diaA . The - or post - biostimulation, respectively. 29 Figure 6 . Heatmap of sediment (MW) and soil log gene copies per g of sediment or soil, respectively. Black cells indicate either no amplification or false positive amplification. The 16S_rRNA results correspond to the 3 chip runs performed: 1) xenB and pnrB , 2) xp lA and nfsI , and 3) xenA and diaA . 30 Table 5 . Summary of SmartChip Standard Curve Characteristics Functional Gene Assays Log( Linear Range) Efficiency (%) R 2 Min Max Min Max Min Max diaA Designed 2 to 7 1 to 7 78.3 102.6 0.986 0.998 nfsI Designed 2 to 7 1 to 7 76.1 87 0.983 0.995 Published 2 to 7 2 to 7 76.2 91.9 0.918 0.935 pnrB Designed 3 to 7 1 to 7 60.1 91.5 0.987 0.997 Published 2 to 7 2 to 7 89.9 91 0.968 0.98 0 xenA Designed 4 to 7 1 to 7 81.5 97.3 0.989 0.998 Published 3 to 6 3 to 6 74.8 74.8 0.989 0.989 xenB Designed 4 to 7 1 to 7 68.1 93 0.986 0.996 Published 3 to 7 3 to 7 68.5 68.5 0.99 0 0.99 0 xplA Designed 4 to 7 2 to 7 40.2 75.4 0.93 0 0.993 Published 3 to 7 1 to 7 61.8 97.3 0.984 0.995 16S rRNA Published 2 to 8 1 to 8 93 107 0.994 0.998 31 Figure 7 . Boxplots of RDX genes in contaminated groundwater and uncontaminated river water. Plots (a) and (c) for the shallow zone aq (d) fo . Results for the Red Cedar River (n=2) are provided for comparison to uncontaminated water. The number of detections for each assa y are noted on plots (a) and (b). a) b ) c ) d ) Shallow Zone Aquifer Perched Zone Aquifer 3 2 3 3 3 3 6 6 3 3 3 6 0 5 3 6 6 6 6 6 3 5 6 5 6 6 3 5 3 0 2 2 2 2 2 2 2 0 0 0 0 0 0 0 0 0 0 0 0 13 11 14 11 13 12 11 14 3 9 4 2 8 6 6 2 4 2 4 2 8 2 0 0 8 7 10 7 7 7 11 12 4 6 6 6 2 4 6 8 2 2 2 2 2 2 2 2 2 2 32 Figure 8 The number of detections for each assay are n oted on plot (a). a) b ) 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 3 10 7 3 4 2 2 10 8 8 6 12 11 10 2 2 11 12 5 6 8 12 12 12 5 2 0 0 0 0 0 0 3 4 6 2 5 0 5 2 0 2 6 6 0 0 0 0 2 2 5 33 3.0 RECOMMENDATIONS AND FUTURE WORK 3.1 Absolute Quantification 3.1.1 Standard Curves To test all of the designed and published assays (as described in Chapter 2), it is recommended to run 3 chips, with 2 RDX functional genes each, in the 24 as says by 216 samples format. Pairing diaA with xenA , nfsI with xplA and pnrB with xenB maximizes the use of each chip. These arrangements permit 64 samples to be tested with each chip, assuming the functional gene plasmids include 7 10 - fold dilutions in tri plicate, the 16S rRNA plasmid include 8 10 - fold dilutions in triplicate and there is one no template control and one negative control. This permits 32 samples to be tested in duplicate or 21 samples to be tested in triplicate. However, if funds are limited , and only chip can be analyzed, a subset of these assays can be used (Table 6). These SmartChip and the need for only one plasmid per gene. In this case, the 12 ass ays by 384 samples layout would be used and this would enable 232 samples to be tested, which equates to 116 samples in duplicate or 77 samples in triplicate. The proposed one chip format does increase the potential for false negatives because the total th eoretical coverage is less than the three chip s approach. Additional assay and sample configurations could be designed depending on the requirements of the project. Table 6 . Recommended assays and plasmids for a single SmartChip ru n. Functional Gene Assays Plasmids Total Theoretical Coverage (%) diaA PS1 CP012395 62.5 nfsI PS 1 CP001918 99.9 pnrB PS1 and PS5 CP010979 98.2 xenA PS1, PS4, PS12, and PS16 CP004045 81.9 xenB PS2 and PS19 CP014205 86.6 xplA Pub1 AF449421 85.2 16S rRNA 16S_rRNA KX858536 N/A 34 3.1.2 Linear Regression of Efficiency Method An alternative absolute quantification method that could be used with these assays is the linear regression of efficiency (LRE) method. This method was developed by Rutledge and Stewart (27) , and it uses fluorescence readings with an optical calibration fa ctor to calculate the number of target molecules present in the reaction. For the optical calibration factor, a few reactions with lambda gDNA and the CAL1 primer set (43) would need to be included on the chip, but substantial space would be saved for runn ing samples since the plasmid standards would not be required. Rutledge and Stewart (27) found absolute accuracies of 25% using this method, and Boyle, Dallaire and MacKay (44) determined that the number of target molecules calculated from LRE and average standard curves were highly correlated. This method does require high optical precision to be accurate, however (27) . Therefore, it will be necessary to test the precision achieved with the SmartChip platform and ensure the results are reliable before sol ely relying on this approach. 3.2 Relative Quantification Other approaches that can maximize the number of samples run on each chip are relative quantification methods. These include the (28) method and the relative gene copies calculation described by Looft et al. (29) . The method normalizes the target gene to a reference gene and calibrator, and it assumes that the amplification efficiencies of the target and reference genes are the same (28) . These two normalizations yield the fold change of the target gene relative to the calibrator. This calibrator could be the sample before biostimulation, and in this case the method would yield the resulting fold change in the target gene from biostimulation. The relative gene copies calculation described by Looft et al. (29) uses the s ame formula as the 35 standard curve absolute quantification method, but assumes an efficiency of one and sets the y - intercept to the threshold cycle. The result is normalized to the 16S rRNA gene for a measure of the relative abundance. These methods are app ropriate in situations where only the fold changes in gene levels or relative abundances are required, such as for bioremediation projects that want to check if biostimulation or bioaugmentation was successful at increasing the gene copies present. Care is recommended in the interpretation of such results, however, as these methods could be useful for comparing results from a specific primer set among all the samples tested, but comparisons between primer sets would not be recommended due to the inherent ef ficiency assumptions. 3.3 Future Work 3.3.1 High throughput sequencing Additional work is needed to confirm the spe cificity of the assays designed in the current research. High throughput sequencing of the gDNA used in qPCR as well as of the amplicons generated t hrough qPCR will aid in this. Sequencing of the gDNA can be used to confirm that the genera expected based on the genes detected are actually present. However, all of the genera that contain these functional genes are not known. This means that cases where the expected genera are not detected will remain inconclusive regarding the accuracy of the qPCR results. Sequencing the qPCR amplicons will help resolve this because the amplicon sequences can be compared against the sequence sets identified here to dete rmine if they are of the expected length and contain the identified residues and motifs. These two efforts together have the potential to both confirm the specificity of the assays and identify new genera containing these RDX functional genes. 36 3.3.2 Proteomics T he results from high throughput qPCR may also be supported by proteomics. In proteomics, the enzymes associated with functional genes are used as the quantitative biomarker. This enables both the detection of RDX - degraders and confirmation of enzymatic act ivity. This technique has already been used to detect reductive dehalogenase enzymes from organohalide - respiring bacteria (45) . However, it should be noted that no detection through proteomics may only mean degradation is not actively occurring, and not th at the bacteria capable of degradation are not present. 37 APPENDICES 38 APPENDIX A Sequence Sets 39 NCBI, PDB, and UniProt databases were all searched for relevant protein sequences using the search terms in Table A. 1 . From the initial search results, the expected protein length was determined, and a minimum length criterion of 90% was set to avoid the inclusion of partial sequences. Sequences retrieved fr om the UniProt database were also used to run individual Basic Local Alignment Search Tool (BLAST) searches within this database. The automatic BLAST search settings were used for this with the exception that the number of hits was increased to 1,000. A su bset of the sequences obtained from UniProt were also used to perform a single protein - protein BLAST (BLASTP) search in the NCBI database. R esults from these searches were subject to the criteria of 90% coverage and Table A. 1 . Search terms used to gather protein sequences Functional Gene Search terms diaA diaA diaA nfsI nfsI pnrB pnrB xenA xenA xenB xenB xplA xplA Clustering tool (32) and aligned using MAFFT v7.215 (46) through the Michigan State University High Performance Computing Center (HPCC). After dereplication, c onfirm ation of these sequences was done based on common protein motifs and residues identified in literature ( Table A. 2 ). Jalview (47) was used to view the aligned sequences, mark the residues and motifs as sequence features, and remove sequences that did not match the identified features. At this stage for diaA , it w as observed that most sequences had lengths less than 270 aa, another set of sequences had lengths greater than 400 aa and a few sequences had gaps between the two motifs. Therefore, two additional criteria were set for the diaA FunGene sequence set: 1) ma ximum 40 length of 270 aa and 2) no gap between the motifs when aligned. The remaining sequences were used as the seed set, or training sequences, for the FunGene HMM model. In FunGene, the HMM model pulled sequences from the NCBI non - redundant protein datab ase. These sequences were first filtered by setting the minimum score to 90% of the max . The results were reviewed to ensure all of the seed set sequences were present and that the minimum length was only 90% of the max. The exception to this was xplA , whi ch was determined to require the inclusion of partial sequences due to many reported sequences only covering one of the two domains present in this gene, so the minimum score was set at 200. In most cases, a few of the seed set sequences were not present in the list from FunGene because they were from a different database. These sequences were manually added to the others, and a ll sequences were then vetted based on the previously identified residues and motifs. For diaA , the sequences remaining had a hig h diversity in both the organisms included and the aligned sequences. Therefore, this sequence set was restricted to only the Clostridium genus, because it is the only genus that has been linked to diaA to date. The final primer design sequence list s are r eported in Tables A.3 - A.8 . 41 Table A. 2 . Identified residues and motifs of functional genes . The positions refer to the reference noted in the Gene (reference) Residue/Motif Position Reference Residue/Motif Position Reference diaA (16) Y/F/W 180 (48) W 213 (48) C - x - y - C - G - Y 1 182 - 187 (48) C - P - y - C - G - z 2 215 - 220 (48) C 182 (16) C 215 (16) C 185 (16) C 218 (16) Y 189 (48) K 222 (48) F 206 (48) F 225 (48) nfsI (49, 50) R 10 (49) F 124 (51, 52) K 14 (51 - 53) Y 144 (51) S[PA]SSXNSQPW [HK]FIV 37 - 46 (49, 50) L 145 (51) SPSS 37 - 40 (53) GXGXXG 3 153 - 158 (54) S 39 (49, 51, 53) PIEGF 163 - 167 (49, 53) S 40 (51, 52) P 163 (51, 53) T 41 (51) I 164 (51) Y 68 (51) E 165 (51) F 70 (51, 52) G 166 (49, 51) N 71 (51) G 192 (49) K 74 (51, 52) HH[SP]XE D[FY]NA [TGS]LPKSR 193 - 207 (49, 50) Y 123 (51) pnrB (55) M 1 (55) Q 117 (55) L 6 (55) R 121 (55) R 10 (55) R 122 (55) T 12 (55) Y 124 (55) K 14 (55) V 125 (55) A 15 (55) H 128 (55) P 23 (55) D 134 (55) Q 24 (55) W 138 (55) S 4 25 (55) M 139 (55) L 30 (55) Q 142 (55) L 31 (55) YLALG 144 - 148 (55) L 34 (55) L 151 (55) SPSSVNSQPWHF 37 - 48 (55) GAA 153 - 155 (55) V 50 (55) G 158 (55) A 51 (55) DATP 160 - 163 (55) pnrB (55) E 55 (55) EGFD 165 - 168 (55) A 58 (55) D 173 (55) A 61 (55) A 174 (55) R 4 67 (55) LGLRERG 176 - 182 (55) N 71 (55) T 184 (55) 42 Gene (reference) Residue/Motif Position Reference Residue/Motif Position Reference pnrB (55) ASHVI 78 - 82 (55) S 185 (55) R 87 (55) V 187 (55) M 90 (55) SLG 190 - 192 (55) H 94 (55) A 4 195 (55) L 95 (55) T 4 197 (55) L 99 (55) DFNA 198 - 201 (55) E 102 (55) L 203 (55) DGRF 105 - 108 (55) KSRL 205 - 208 (55) A 115 (55) FTFL 214 - 217 (55) V 4 116 (55) xenA (56) PMC[MQ]Y 5 23 - 27 (57) R 231 (56, 58) M 24 (57, 58) W 302 (58, 59) C 25 (57 - 62) F 304 (56) Y 27 (57 - 59) S 323 (56) A 57 (56) G 325 (56) GRI[TS]X(4)GIW 5 64 - 74 (57) R 326 (56) Q 99 (56, 58) A 330 (58) H 178 (56, 58, 60) P 354 (58) H 181 (56, 58, 60) H 357 (58) Y 183 (58, 59) W 358 (58, 59, 61) [TS]NXRTDX YGGSX[DE]NR [SA]R[FL]LLE 5 193 - 213 (56) xenB (56) T 25 (56) [TS]NXRTDX YGGSX[DE]NR [SA]R[FL]LLE 6 188 - 208 (56) A 56 (56) YGGS 195 - 198 (63) Q 98 (56) H 225 (56) HGA 173 - 175 (63) F 288 (56) H 173 (56) A 306 (56) N 176 (56) G 308 (56) xplA 7 (64) T 10 (64) Y 97 (64) E 11 (64) P 132 (64) T 12 (64) P 136 (64) N 14 (64) V 391 (64) STYGEG E LPA 57 - 66 (64) M 394 (64, 65) Y 59 (64) A 395 (64, 65) F 90 (64) Q 438 (65) DS T Y DT YNN 94 - 102 (64) 1 Sieker et al. (48) reports the last amino acid as being maintained as Y by the rubredoxin sequences analyzed. However, the sequence reported by Chakraborty et al. (16) has F in this position so this residue was not used to eliminate sequences. 43 2 Sieker et al. (48) reports th e second amino acid as being an invariant P by the rubredoxin sequences analyzed. The sequence reported by Chakraborty et al. (16) agrees with this so this residue was used to eliminate sequences. 3 Bryant et al. (54) reports that nfsI conserves this motif which is found among flavoproteins. However, Zenno et al. (50) notes that this motif is not conserved among other members of this protein family so this was not used to eliminate sequences. 4 Amino acids identified by Kahng et al. (55) as unique to pnrB . Th ese were not used to eliminate sequences from the FunGene or primer design sequence sets. 5 Sequences for the FunGene sequence set were not eliminated based on positions in this motif that could be more than one amino acid, but these positions were used as inclusion criteria for the primer design sequence set. 6 For determining the FunGene sequence set, this motif was kept strictly to TNQRTDNYGGSLENRARLLLE, which is the specific sequence reported for xenB rather than the general motif observed for the OYE family. 7 For xplA , not all of the residues and motifs were used eliminate sequences. The ones indicated in bold were permitted to have some variability, otherwise sequences that had been identified in the ir respective databases as cytochrome p450 would have been removed. 44 Table A. 3 . FunGene and primer design sequences for diaA Protein Accession No. Nucleotide Accession No. Organism Definition Size FunGene Sequence Set FunGene Sequence ID (Database) ABC76945 CP000252 Syntrophus aciditrophicus SB ferric - chelate reductase / Rubredoxin 236 X 2 Q2LS83 (UniProt) ACV63492 CP001720 Desulfotomaculum acetoxidans DSM 771 flavin reductase domain protein FMN - binding 227 X 2 ACV63492 (NCBI) ADG82833 CP002028 Thermincola potens JR flavin reductase domain protein FMN - binding protein 234 X 2 ADG82833 (NCBI) ADK15010 CP001666 Clostridium ljungdahlii DSM 13528 putative flavin reductase - like protein with rubredoxin domain 229 AEA33387 CP002606 Hippea maritima DSM 10411 flavin reductase domain protein FMN - binding protein 234 X 2 F2LTY8 (UniProt) AEG16589 CP002770 Desulfotomaculum kuznetsovii DSM 6115 flavin reductase domain protein FMN - binding protein 228 X 2 F6CLH1 (UniProt) AGL02744 CP003273 Desulfotomaculum gibsoniae DSM 7213 conserved protein of DIM6/NTAB family 228 X 2 R4KJE5 (UniProt) AGY74262 CP006763 Clostridium autoethanogenum DSM 10061 Rubredoxin domain containing protein 229 AHF96662 CP007051 Desulfurella acetivorans A63 High molecular weight rubredoxin 223 X 2 WP_025391426 (NCBI) AKI97034 CP011232 Kosmotoga pacifica High molecular weight rubredoxin 229 X 2 WP_047754169 (NCBI) AKN31180 CP011803 Clostridium carboxidivorans P7 High molecular weight rubredoxin 232 X AKN31180 (NCBI) ALU34453 CP012395 Clostridium autoethanogenum DSM 10061 Rubredoxin domain - containing protein 229 APM40069 CP018335 Clostridium kluyveri High molecular weight rubredoxin 229 X WP_073539679 (NCBI) BAH07534 AP009049 Clostridium kluyveri NBRC 12016 hypothetical protein 229 CBX27933 FR695868 uncultured Desulfobacterium sp. High molecular weight rubredoxin 230 X 2 CBX27933 (NCBI) CEO88390 CDRZ01000090 Syntrophaceticus schinkii High molecular weight rubredoxin 227 X 2 WP_044664557 (NCBI) 45 Protein Accession No. Nucleotide Accession No. Organism Definition Size FunGene Sequence Set FunGene Sequence ID (Database) EDK34804 CP000673 Clostridium kluyveri DSM 555 Hrb 229 X EDK34804 (NCBI) EET85839 ACVI01000074 Clostridium carboxidivorans P7 flavin reductase domain protein FMN - binding 232 GAG55455 BART01002025 marine sediment metagenome Marine sediment metagenome DNA, contig: S01H4_C02025, whole genome shotgun sequence 213 X 2 X0YHV6 (UniProt) GAG71827 BART01000399 marine sediment metagenome Marine sediment metagenome DNA, contig: S01H4_C00399, whole genome shotgun sequence 233 X 2 X1AR62 (UniProt) GAH02136 BART01022901 marine sediment metagenome Marine sediment metagenome DNA, contig: S01H4_S07771, whole genome shotgun sequence 227 X 2 X1C206 (UniProt) GAH71630 BARU01029844 marine sediment metagenome Marine sediment metagenome DNA, contig: S03H2_S13408, whole genome shotgun sequence 223 X 2 X1IQV1 (UniProt) GAI21343 BARV01015951 marine sediment metagenome Marine sediment metagenome DNA, contig: S06H3_S01427, whole genome shotgun sequence 227 X 2 X1LQG4 (UniProt) GAI31013 BARV01019462 marine sediment metagenome Marine sediment metagenome DNA, contig: S06H3_S04938, whole genome shotgun sequence 218 X 2 X1MIC9 (UniProt) KFI37218 JPEC01000073 Peptococcaceae bacterium SCADC1_2_3 High molecular weight rubredoxin 232 X 2 KFI37218 (NCBI) KFO68279 JQDQ01000086 Smithella sp. SCADC High molecular weight rubredoxin 229 X 2 KFO68279 (NCBI) KJS03435 LADN01000009 Peptococcaceae bacterium BRH_c4a High molecular weight rubredoxin 227 X 2 KJS03435 (NCBI) KJS11230 LADP01000035 Peptococcaceae bacterium BRH_c8a High molecular weight rubredoxin 224 X 2 KJS11230 (NCBI) KJS76399 JUED01000041 Desulfotomaculum sp. BICA1 - 6 High molecular weight rubredoxin 224 X 2 KJS76399 (NCBI) KNZ69687 LGTE01000010 Thermincola ferriacetica flavin reductase domain - containing FMN - binding protein 239 X 2 KNZ69687 (NCBI) 46 Protein Accession No. Nucleotide Accession No. Organism Definition Size FunGene Sequence Set FunGene Sequence ID (Database) KPJ49086 LIZT01000076 candidate division TA06 bacterium DG_26 High molecular weight rubredoxin 235 X 2 KPJ49086 (NCBI) KPJ60922 LJNC01000006 Latescibacteria bacterium DG_63 High molecular weight rubredoxin 236 X 2 KPJ60922 (NCBI) KPJ66142 LIZW01000040 Coxiella sp. DG_40 High molecular weight rubredoxin 236 X 2 KPJ66142 (NCBI) KPL01198 LJUX01000004 candidate division Zixibacteria bacterium SM23_73_3 High molecular weight rubredoxin 231 X 2 KPL01198 (NCBI) KPL09964 LJVA01000046 candidate division TA06 bacterium SM1_40 High molecular weight rubredoxin 242 X 2 KPL09964 (NCBI) KPL14439 LJVE01000050 candidate division WOR_3 bacterium SM1_77 High molecular weight rubredoxin 232 X 2 KPL14439 (NCBI) KPL19139 LJUY01000010 candidate division Zixibacteria bacterium SM23_81 High molecular weight rubredoxin 253 X 2 KPL19139 (NCBI) KPL20732 LJUZ01000178 Anaerolineae bacterium SM23_84 High molecular weight rubredoxin 236 X 2 KPL20732 (NCBI) KPU45454 LKET01000021 Oxobacter pfennigii high molecular weight rubredoxin 230 X 2 WP_054873804 (NCBI) KQC07238 LKUC01000307 Smithella sp. SDB High molecular weight rubredoxin 229 X 2 KQC07238 (NCBI) KUG22969 LNQE01000929 hydrocarbon metagenome rubredoxin 229 X 2 A0A0W8FQ88 (UniProt) KUK51921 LGFZ01000128 Desulfotomaculum sp. 46_296 Rubredoxin 229 X 2 KUK51921 (NCBI) KUK87408 LGGX01000005 candidate division TA06 bacterium 34_109 flavin reductase - like, FMN - binding 233 X 2 A0A101I288 (UniProt) KUO39544 LQMQ01000061 Hadesarchaea archaeon YNP_45 High molecular weight rubredoxin 228 X 2 A0A147JSV1 (UniProt) KYK20996 LSSE01000053 Thermoplasmatales archaeon SG8 - 52 - 2 High molecular weight rubredoxin 230 X 2 KYK20996 (NCBI) KYK23145 LSSI01000040 Thermoplasmatales archaeon SM1 - 50 High molecular weight rubredoxin 233 X 2 A0A151E3C7 (UniProt) 47 Protein Accession No. Nucleotide Accession No. Organism Definition Size FunGene Sequence Set FunGene Sequence ID (Database) KYK24869 LSSF01000008 Thermoplasmatales archaeon SG8 - 52 - 4 High molecular weight rubredoxin 231 X 2 A0A151E8W7 (UniProt) KYK27312 LSSD01000056 Thermoplasmatales archaeon SG8 - 52 - 1 High molecular weight rubredoxin 231 X 2 A0A151EFD3 (UniProt) KYK33978 LSSG01000019 Thermoplasmatales archaeon SG8 - 52 - 3 High molecular weight rubredoxin 230 X 2 KYK33978 (NCBI) KZL91355 LWAE01000003 Clostridium magnum DSM 2767 high molecular weight rubredoxin 246 X KZL91355 (NCBI) OAA21971 JGCK01000033 Kosmotoga sp. DU53 High molecular weight rubredoxin 229 X 2 WP_063727853 (NCBI) OAA31561 JFHK01000003 Kosmotoga arenicorallina S304 high molecular weight rubredoxin 228 X 2 WP_068345860 (NCBI) OAA87671 LITS01000008 Clostridium ljungdahlii DSM 13528 High molecular weight rubredoxin 229 X OAA87671 (NCBI) OAA90537 LITT01000011 Clostridium ljungdahlii High molecular weight rubredoxin 229 X WP_063554973 (NCBI) OAA95145 LITQ01000001 Clostridium coskatii High molecular weight rubredoxin 229 OBR89701 LROS01000079 Clostridium ragsdalei P11 high molecular weight rubredoxin 229 X WP_065079974 (NCBI) OBR97507 LROR01000022 Clostridium coskatii high molecular weight rubredoxin 229 X OBR97507 (NCBI) OFY91363 MEPH01000049 Bacteroidetes bacterium RIFOXYA2_FULL_33_7 High molecular weight rubredoxin 233 X 2 OFY91363 (NCBI) OGC05759 METP01000034 candidate division WOR - 1 bacterium RIFCSPLOWO2_02_FUL L_46_20 High molecular weight rubredoxin 224 X 2 OGC05759 (NCBI) OGD15969 MEYH01000043 Candidatus Atribacteria bacterium RBG_19FT_COMBO_35_ 14 High molecular weight rubredoxin 238 X 2 OGD15969 (NCBI) OGN89297 MGML01000095 Chloroflexi bacterium RBG_13_46_14 High molecular weight rubredoxin 227 X 2 OGN89297 (NCBI) OGN89489 MGMM01000043 Chloroflexi bacterium RBG_13_46_9 High molecular weight rubredoxin 228 X 2 OGN89489 (NCBI) 48 Protein Accession No. Nucleotide Accession No. Organism Definition Size FunGene Sequence Set FunGene Sequence ID (Database) OGN93299 MGMP01000067 Chloroflexi bacterium RBG_13_50_10 High molecular weight rubredoxin 229 X 2 OGN93299 (NCBI) OGN97618 MGMS01000010 Chloroflexi bacterium RBG_13_51_36 High molecular weight rubredoxin 230 X 2 OGN97618 (NCBI) OGO02426 MGMW01000067 Chloroflexi bacterium RBG_13_53_26 High molecular weight rubredoxin 237 X 2 OGO02426 (NCBI) OGO02536 MGMX01000156 Chloroflexi bacterium RBG_13_54_8 High molecular weight rubredoxin 229 X 2 OGO02536 (NCBI) OGO60596 MGOH01000046 Chloroflexi bacterium RBG_19FT_COMBO_48_ 23 High molecular weight rubredoxin 229 X 2 OGO60596 (NCBI) OGP54962 MGQF01000035 Deltaproteobacteria bacterium RBG_13_52_11 High molecular weight rubredoxin 230 X 2 OGP54962 (NCBI) OGP85639 MGQS01000023 Deltaproteobacteria bacterium RBG_16_54_11 High molecular weight rubredoxin 231 X 2 OGP85639 (NCBI) OGP89539 MGQZ01000066 Deltaproteobacteria bacterium RBG_19FT_COMBO_43_ 11 High molecular weight rubredoxin 231 X 2 OGP89539 (NCBI) OGP91237 MGQZ01000015 Deltaproteobacteria bacterium RBG_19FT_COMBO_43_ 11 High molecular weight rubredoxin 231 X 2 OGP91237 (NCBI) OGP94917 MGQT01000009 Deltaproteobacteria bacterium RBG_16_54_18 High molecular weight rubredoxin 234 X 2 OGP94917 (NCBI) OGS20528 MGVC01000068 Elusimicrobia bacterium RIFOXYA2_FULL_39_19 High molecular weight rubredoxin 234 X 2 OGS20528 (NCBI) OGS21815 MGVD01000012 Elusimicrobia bacterium RIFOXYA2_FULL_40_6 High molecular weight rubredoxin 236 X 2 OGS21815 (NCBI) OGS24501 MGVO01000041 Elusimicrobia bacterium RIFOXYB2_FULL_50_12 High molecular weight rubredoxin 234 X 2 OGS24501 (NCBI) OGS31001 MGVL01000010 Elusimicrobia bacterium RIFOXYB2_FULL_46_23 High molecular weight rubredoxin 228 X 2 OGS31001 (NCBI) OGS34140 MGVN01000219 Elusimicrobia bacterium RIFOXYB2_FULL_49_7 High molecular weight rubredoxin 238 X 2 OGS34140 (NCBI) 49 Protein Accession No. Nucleotide Accession No. Organism Definition Size FunGene Sequence Set FunGene Sequence ID (Database) OGS36449 MGVN01000083 Elusimicrobia bacterium RIFOXYB2_FULL_49_7 High molecular weight rubredoxin 238 X 2 OGS36449 (NCBI) OGS40611 MGVV01000075 Euryarchaeota archaeon RBG_13_31_8 High molecular weight rubredoxin 234 X 2 A0A1F9YLW4 (UniProt) OHB81972 MHYO01000240 Planctomycetes bacterium RBG_19FT_COMBO_48_ 8 High molecular weight rubredoxin 237 X 2 OHB81972 (NCBI) OHD44682 MIAW01000022 Spirochaetes bacterium GWD1_27_9 High molecular weight rubredoxin 234 X 2 OHD44682 (NCBI) OHE16697 MICJ01000099 Syntrophobacterales bacterium GWC2_56_13 High molecular weight rubredoxin 233 X 2 OHE16697 (NCBI) OHE20549 MICK01000095 Syntrophobacterales bacterium GWF2_56_9 High molecular weight rubredoxin 233 X 2 OHE20549 (NCBI) OIO01048 MNUP01000068 Candidatus Desantisbacteria bacterium CG1_02_49_89 High molecular weight rubredoxin 232 X 2 OIO01048 (NCBI) OIO87023 MNXI01000071 Actinobacteria bacterium CG2_30_50_142 High molecular weight rubredoxin 233 X 2 OIO87023 (NCBI) OIP25752 MNYE01000136 Dehalococcoidia bacterium CG2_30_46_9 High molecular weight rubredoxin 228 X 2 OIP25752 (NCBI) OIP68335 MNYY01000124 Candidatus Atribacteria bacterium CG2_30_33_13 High molecular weight rubredoxin 236 X 2 OIP68335 (NCBI) OIP93149 MNZQ01000033 Syntrophaceae bacterium CG2_30_58_14 High molecular weight rubredoxin 233 X 2 OIP93149 (NCBI) OPL11334 LQBH01000070 delta proteobacterium MLS_D High molecular weight rubredoxin 234 X 2 OPL11334 (NCBI) OPL19420 LQBI01000006 Candidatus Aegiribacteria bacterium MLS_C High molecular weight rubredoxin 235 X 2 OPL19420 (NCBI) OPX18296 MUKB01000025 Candidatus Omnitrophica bacterium 4484_100 High molecular weight rubredoxin 233 X 2 OPX18296 (NCBI) OPX28692 MVCX01000136 Gemmatimonadaceae bacter ium 4484_173 High molecular weight rubredoxin 236 X 2 OPX28692 (NCBI) SEM25682 FOBS01000008 Syntrophus gentianae NADH - FMN oxidoreductase RutF, flavin reductase (DIM6/NTAB) family 239 X 2 SEM25682 (NCBI) 50 Protein Accession No. Nucleotide Accession No. Organism Definition Size FunGene Sequence Set FunGene Sequence ID (Database) SFH18630 FOOX01000019 Desulfotomaculum arcticum NADH - FMN oxidoreductase RutF, flavin reductase (DIM6/NTAB) family 227 X 2 SFH18630 (NCBI) SHH39297 FQXL01000005 Clostridium magnum DSM 2767 NADH - FMN oxidoreductase RutF, flavin reductase (DIM6/NTAB) family 246 WP_027718493 1 NZ_AUBR01000039.1: c22180 - 21491 Desulfovirgula thermocuniculi high molecular weight rubredoxin 229 X 2 WP_027718493 (NCB I) WP_028894048 1 NZ_KI867150.1: 928490 - 929185 Syntrophorhabdus aromaticivorans high molecular weight rubredoxin 231 X 2 WP_028894048 (NCBI) WP_031515655 1 NZ_JONT01000011.1: c2640 - 1936 Desulfotomaculum alkaliphilum high molecular weight rubredoxin 234 X 2 WP_031515655 (NCBI) WP_035588975 1 NZ_JQLX01000014.1: 152368 - 153078 Hippea jasoniae high molecular weight rubredoxin 236 X 2 WP_035588975 (NCBI) WP_041584747 1 NC_007759.1: c1091004 - 1090300 Syntrophus aciditrophicus high molecular weight rubredoxin 234 X 2 WP_041584747 (NCBI) WP_051965572 1 NZ_JQKL01000008.1: 84143 - 84793 Clostridiales bacterium DRI - 13 high molecular weight rubredoxin 216 X 2 WP_051965572 (NCBI) WP_052217953 1 NZ_LGTE01000010.1: 87345 - 88067 Thermincola ferriacetica high molecular weight rubredoxin 240 X 2 WP_052217953 (NCBI) WP_066625043 1 NZ_FQXL01000005.1: c30610 - 29912 Clostridium magnum high molecular weight rubredoxin 232 X WP_066625043 (NCBI) 1 These sequences were not on FunGene so were added back in to the primer design sequence set manually . 2 Th ese sequence s w ere not included in the final primer design sequence set because they did not meet the stricter criteria used in the final review. 51 Table A. 4 . FunGene and primer design sequences for nfsI Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AAA62801 M63808 Enterobacter cloacae oxygen - insensitive NAD(P)H nitroreductase 217 X Q01234 (UniProt) ADF62693 CP001918 Enterobacter cloacae subsp. cloacae ATCC 13047 retro - nitroreductase 217 AEN63793 CP003026 Enterobacter asburiae LF7a nitroreductase 217 X G2SB42 (UniProt) AFM58872 CP003678 Enterobacter cloacae subsp. dissolvens SDM dihydropteridine reductase 217 X A0A0M1R1B2 (UniProt) AFP69024 CP003737 Enterobacter cloacae subsp. cloacae ENHKU01 dihydropteridine reductase 217 AHW95293 CP007546 Enterobacter asburiae L1 dihydropteridine reductase 217 AIN21788 CP008897 Enterobacter cloacae ECNIH3 dihydropteridine reductase 217 AIN27131 CP008905 Enterobacter cloacae ECR091 dihydropteridine reductase 217 AIV28758 CP009756 Enterobacter cloacae dihydropteridine reductase 217 AIX55855 CP009850 Enterobacter cloacae dihydropteridine reductase 217 AIX58255 CP009854 Enterobacter cloacae dihydropteridine reductase 217 AJB61696 CP010376 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 AJB70122 CP010377 Enterobacter hormaechei subsp. hormaechei dihydropteridine reductase 217 AJB80828 CP010384 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 AKL01059 CP011591 Enterobacter asburiae dihydropteridine reductase 217 AKZ83319 CP012167 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 ALA01283 CP012165 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 ALL16580 CP012999 Enterobacter sp. E20 dihydropteridine reductase 217 AMA02343 CP011863 Enterobacter asburiae dihydropteridine reductase 217 AMJ71713 CP014280 Enterobacter cloacae NAD(P)H - dependent oxidoreductase 217 X A0A126PS74 (UniProt) AMX06383 CP014993 Enterobacter asburiae oxygen - insensitive NAD(P)H nitroreductase 217 AMY66853 CP010512 Enterobacter cloacae dihydropteridine reductase 217 AMZ77222 CP015227 Enterobacter sp. ODB01 NAD(P)H nitroreductase 217 AOE94628 CP016906 Enterobacter cloacae NAD(P)H nitroreductase 217 52 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AOL14985 CP017087 Enterobacter sp. HK169 Oxygen - insensitive NAD(P)H nitroreductase 217 AOP77117 CP017179 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 AOP81553 CP017180 Enterobacter hormaechei subsp. oharae NAD(P)H nitroreductase 217 AOP85852 CP017181 Enterobacter kobei NAD(P)H nitroreductase 217 AOP90393 CP017183 Enterobacter xiangfangensis NAD(P)H nitroreductase 217 AOP94610 CP017184 Enterobacter cloacae complex 'Hoffmann cluster IV' NAD(P)H nitroreductase 217 AOP99046 CP017186 Enterobacter cloacae complex 'Hoffmann cluster III' NAD(P)H nitroreductase 217 APR42534 CP018814 Enterobacter cloacae NAD(P)H nitroreductase 217 AQT90310 CP019839 Enterobacter cloacae NAD(P)H - dependent oxidoreductase 217 ARA26060 CP020053 Enterobacter cloacae NAD(P)H nitroreductase 217 ARZ77846 CP021749 Enterobacter cloacae NAD(P)H nitroreductase 217 ASA06611 CP021776 Enterobacter cloacae NAD(P)H nitroreductase 217 ASB74171 CP021896 Enterobacter cloacae NAD(P)H nitroreductase 217 ASD58149 CP017990 Enterobacter cloacae complex sp. ECNIH7 NAD(P)H nitroreductase 217 ASG39592 CP022148 Enterobacter cloacae NAD(P)H - dependent oxidoreductase 217 ASO98863 CP022532 Enterobacter cloacae retro - nitroreductase 217 ASQ16953 CP017475 Enterobacter cloacae Oxygen - insensitive NAD(P)H nitroreductase 217 ASQ75974 CP019889 Enterobacter cloacae NAD(P)H nitroreductase 217 ATW94206 CP024812 Enterobacter sp. CRENT - 193 oxygen - insensitive NAD(P)H nitroreductase 217 AUJ82597 CP025225 Enterobacter cancerogenus oxygen - insensitive NAD(P)H nitroreductase 217 AUM02628 CP020817 Enterobacter sp. Crenshaw NAD(P)H - dependent oxidoreductase 217 CBK85782 FP929040 Enterobacter cloacae subsp. cloacae NCTC 9394 dihydropteridine reductase 217 CQR78470 CTRQ01000008 Enterobacter cloacae Oxygen - insensitive NAD(P)H nitroreductase 217 53 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) CUJ19923 CYTA01000035 Achromobacter sp. ATCC13047 Oxygen - insensitive NAD(P)H nitroreductase 217 CZU19544 FJXF01000001 Enterobacter cloacae nitroreductase 217 CZU20024 FJXK01000001 Enterobacter cloacae nitroreductase 217 CZU23511 FJXT01000001 Enterobacter cloacae nitroreductase 217 CZU24251 FJWG01000001 Enterobacter cloacae nitroreductase 217 CZU26231 FJXH01000001 Enterobacter cloacae nitroreductase 217 CZU28052 FJWM01000001 Enterobacter cloacae nitroreductase 217 CZU33565 FJWK01000001 Enterobacter cloacae nitroreductase 217 CZU35296 FJXQ01000001 Enterobacter cloacae nitroreductase 217 CZU37660 FJXE01000001 Enterobacter cloacae nitroreductase 217 CZU38649 FJWX01000001 Enterobacter cloacae nitroreductase 217 CZU41411 FJXL01000001 Enterobacter cloacae nitroreductase 217 CZU48432 FJWW01000001 Enterobacter cloacae nitroreductase 217 CZU50099 FJXV01000001 Enterobacter cloacae nitroreductase 217 CZU50140 FJXN01000002 Enterobacter cloacae nitroreductase 217 CZU50741 FJWV01000001 Enterobacter cloacae nitroreductase 217 CZU54269 FJWN01000005 Enterobacter cloacae nitroreductase 217 CZU57345 FJXP01000001 Enterobacter cloacae nitroreductase 217 CZU60949 FJWQ01000004 Enterobacter cloacae nitroreductase 217 CZU61229 FJXC01000002 Enterobacter cloacae nitroreductase 217 CZU65748 FJWJ01000001 Enterobacter cloacae nitroreductase 217 CZU66880 FJWT01000003 Enterobacter cloacae nitroreductase 217 CZU68610 FJWY01000004 Enterobacter cloacae nitroreductase 217 CZU74858 FJWR01000006 Enterobacter cloacae nitroreductase 217 CZU76805 FJWS01000002 Enterobacter cloacae nitroreductase 217 CZU83876 FJWO01000002 Enterobacter cloacae nitroreductase 217 CZU84775 FJWC01000005 Enterobacter cloacae nitroreductase 217 CZU86851 FJWP01000002 Enterobacter cloacae nitroreductase 217 CZU87995 FJXA01000007 Enterobacter cloacae nitroreductase 217 CZU94381 FJWI01000007 Enterobacter cloacae nitroreductase 217 CZV12637 FJWF01000004 Enterobacter cloacae nitroreductase 217 CZV18957 FJXB01000006 Enterobacter cloacae nitroreductase 217 CZV35823 FJXJ01000020 Enterobacter cloacae nitroreductase 217 CZV39330 FJXO01000009 Enterobacter cloacae nitroreductase 217 54 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) CZV40446 FJXU01000001 Enterobacter cloacae nitroreductase 217 CZV58614 FJXM01000015 Enterobacter cloacae nitroreductase 217 CZV80438 FJWL01000019 Enterobacter cloacae nitroreductase 217 CZV87735 FJXD01000009 Enterobacter cloacae nitroreductase 217 CZV88602 FJXI01000007 Enterobacter cloacae nitroreductase 217 CZV93255 FJXY01000002 Enterobacter cloacae nitroreductase 217 CZV99032 FJYF01000001 Enterobacter cloacae nitroreductase 217 CZW04811 FJXR01000027 Enterobacter cloacae nitroreductase 217 X A0A157GLI8 (UniProt) CZW07158 FJYC01000004 Enterobacter cloacae nitroreductase 217 CZW17832 FJYB01000001 Enterobacter cloacae nitroreductase 217 X A0A0P8J201 (UniProt) CZW26425 FJYM01000001 Enterobacter cloacae nitroreductase 217 CZW28558 FJYJ01000002 Enterobacter cloacae nitroreductase 217 CZW29068 FJYT01000001 Enterobacter cloacae nitroreductase 217 CZW29237 FJYH01000002 Enterobacter cloacae nitroreductase 217 CZW32992 FJYN01000001 Enterobacter cloacae nitroreductase 217 CZW36114 FJXS01000032 Enterobacter cloacae nitroreductase 217 CZW50797 FJYE01000005 Enterobacter cloacae nitroreductase 217 CZW62010 FJXZ01000014 Enterobacter cloacae nitroreductase 217 CZW65348 FJYY01000001 Enterobacter cloacae nitroreductase 217 CZW72685 FJYK01000005 Enterobacter cloacae nitroreductase 217 CZW74276 FJYU01000001 Enterobacter cloacae nitroreductase 217 CZW74715 FJYA01000007 Enterobacter cloacae nitroreductase 217 CZW78701 FJZB01000001 Enterobacter cloacae nitroreductase 217 CZW80294 FJZC01000001 Enterobacter cloacae nitroreductase 217 CZW80321 FJYQ01000004 Enterobacter cloacae nitroreductase 217 CZW83490 FJYS01000002 Enterobacter cloacae nitroreductase 217 CZW86349 FJYV01000002 Enterobacter cloacae nitroreductase 217 CZW89600 FJYR01000001 Enterobacter cloacae nitroreductase 217 CZW91698 FJYW01000002 Enterobacter cloacae nitroreductase 217 CZW95394 FJYG01000005 Enterobacter cloacae nitroreductase 217 CZW96222 FJZH01000001 Enterobacter cloacae nitroreductase 217 CZX00489 FJZD01000001 Enterobacter cloacae nitroreductase 217 CZX05303 FJXX01000014 Enterobacter cloacae nitroreductase 217 55 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) CZX11286 FJZG01000001 Enterobacter cloacae nitroreductase 217 CZX33936 FJZI01000002 Enterobacter cloacae nitroreductase 217 CZX34071 FJZK01000002 Enterobacter cloacae nitroreductase 217 CZX39460 FJYL01000007 Enterobacter cloacae nitroreductase 217 CZX48956 FJZJ01000002 Enterobacter cloacae nitroreductase 217 CZX51289 FJZF01000006 Enterobacter cloacae nitroreductase 217 CZX54413 FJZM01000004 Enterobacter cloacae nitroreductase 217 CZX68213 FJYX01000022 Enterobacter cloacae nitroreductase 217 CZX70772 FJZP01000006 Enterobacter cloacae nitroreductase 217 CZX71404 FJZE01000006 Enterobacter cloacae nitroreductase 217 CZX71419 FJYZ01000030 Enterobacter cloacae nitroreductase 217 CZX82876 FJZR01000007 Enterobacter cloacae nitroreductase 217 CZX90662 FJZT01000002 Enterobacter cloacae nitroreductase 217 CZY00734 FKAA01000001 Enterobacter cloacae nitroreductase 217 CZY03920 FJZZ01000001 Enterobacter cloacae nitroreductase 217 CZY15676 FJZQ01000005 Enterobacter cloacae nitroreductase 217 CZY20210 FJZX01000002 Enterobacter cloacae nitroreductase 217 CZY26570 FJZN01000007 Enterobacter cloacae nitroreductase 217 CZY37120 FJZO01000010 Enterobacter cloacae nitroreductase 217 CZY38711 FKAG01000001 Enterobacter cloacae nitroreductase 217 CZY39159 FJZY01000002 Enterobacter cloacae nitroreductase 217 CZY40875 FKAB01000002 Enterobacter cloacae nitroreductase 217 CZY56648 FKAC01000001 Enterobacter cloacae nitroreductase 217 CZY58696 FKAD01000002 Enterobacter cloacae nitroreductase 217 CZY59519 FKAM01000001 Enterobacter cloacae nitroreductase 217 CZY61426 FKAE01000003 Enterobacter cloacae nitroreductase 217 CZY66124 FJZL01000009 Enterobacter cloacae nitroreductase 217 CZY72955 FJZS01000005 Enterobacter cloacae nitroreductase 217 CZY74135 FKAR01000001 Enterobacter cloacae nitroreductase 217 CZY78281 FKAU01000001 Enterobacter cloacae nitroreductase 217 CZY85360 FKAP01000002 Enterobacter cloacae nitroreductase 217 CZY85613 FKAW01000001 Enterobacter cloacae nitroreductase 217 CZY94616 FKAZ01000001 Enterobacter cloacae nitroreductase 217 CZZ00442 FKAF01000009 Enterobacter cloacae nitroreductase 217 CZZ04384 FKAN01000002 Enterobacter cloacae nitroreductase 217 56 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) CZZ05290 FKBC01000001 Enterobacter cloacae nitroreductase 217 CZZ10435 FKBA01000001 Enterobacter cloacae nitroreductase 217 CZZ10793 FKAV01000002 Enterobacter cloacae nitroreductase 217 CZZ15592 FKBD01000001 Enterobacter cloacae nitroreductase 217 CZZ17546 FKBE01000001 Enterobacter cloacae nitroreductase 217 CZZ19399 FKAT01000002 Enterobacter cloacae nitroreductase 217 CZZ35379 FKBH01000001 Enterobacter cloacae nitroreductase 217 CZZ47430 FKAX01000011 Enterobacter cloacae nitroreductase 217 CZZ49686 FKBF01000003 Enterobacter cloacae nitroreductase 217 CZZ67135 FKAJ01000005 Enterobacter cloacae nitroreductase 217 CZZ75660 FKBL01000001 Enterobacter cloacae nitroreductase 217 CZZ80303 FKAQ01000006 Enterobacter cloacae nitroreductase 217 CZZ87099 FKAS01000007 Enterobacter cloacae nitroreductase 217 CZZ90710 FKAH01000010 [Enterobacter] aerogenes nitroreductase 217 EFC55379 ABWM02000020 Enterobacter cancerogenus ATCC 35316 oxygen - insensitive NAD(P)H nitroreductase 217 X D2ZGD5 (UniProt) EGK62528 AFHR01000020 Enterobacter hormaechei ATCC 49162 oxygen - insensitive NAD(P)H nitroreductase 217 X F5RUG0 (UniProt) EIM37332 AJXP01000003 Enterobacter cloacae subsp. cloacae GS1 dihydropteridine reductase 217 EJO48778 ALNS01000001 Enterobacter sp. SST3 Oxygen - insensitive NAD(P)H nitroreductase 217 EPY96152 ATHX01000006 Enterobacter cloacae EC_38VIM1 dihydropteridine reductase 217 ERP02085 AXLJ01000001 Enterobacter sp. MGH 8 oxygen - insensitive NAD(P)H nitroreductase 217 ESL78320 AYIE01000007 Enterobacter cloacae UCICRE 11 oxygen - insensitive NAD(P)H nitroreductase 217 ESL83262 AYID01000001 Enterobacter cloacae UCICRE 12 oxygen - insensitive NAD(P)H nitroreductase 217 X V3E2X6 (UniProt) ESL91992 AYIG01000007 Enterobacter cloacae UCICRE 9 oxygen - insensitive NAD(P)H nitroreductase 217 ESM19499 AYIK01000004 Enterobacter cloacae UCICRE 5 oxygen - insensitive NAD(P)H nitroreductase 217 ESM22487 AYIM01000001 Enterobacter cloacae UCICRE 3 oxygen - insensitive NAD(P)H nitroreductase 217 57 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) ESM32927 AYIP01000009 Enterobacter cloacae BWH 31 oxygen - insensitive NAD(P)H nitroreductase 217 ESM44307 AYIR01000011 Enterobacter cloacae BWH 29 oxygen - insensitive NAD(P)H nitroreductase 217 ESM79363 AYIY01000008 Enterobacter sp. MGH 38 oxygen - insensitive NAD(P)H nitroreductase 217 ESM83780 AYJA01000005 Enterobacter cloacae 'Hoffmann cluster IV' MGH 34 oxygen - insensitive NAD(P)H nitroreductase 217 ESN11078 AYJE01000005 Enterobacter sp. MGH 26 oxygen - insensitive NAD(P)H nitroreductase 217 ESN17841 AYJF01000018 Enterobacter sp. MGH 25 oxygen - insensitive NAD(P)H nitroreductase 217 ESN26004 AYJH01000005 Enterobacter sp. MGH 23 oxygen - insensitive NAD(P)H nitroreductase 217 ESN28155 AYJI01000001 Enterobacter sp. MGH 22 oxygen - insensitive NAD(P)H nitroreductase 217 ESN53371 AYJO01000001 Enterobacter sp. MGH 16 oxygen - insensitive NAD(P)H nitroreductase 217 EUL38804 JCKK01000002 Enterobacter cloacae UCI 50 oxygen - insensitive NAD(P)H nitroreductase 217 EUL64446 JCKQ01000001 Enterobacter cloacae UCI 39 oxygen - insensitive NAD(P)H nitroreductase 217 EUL67820 JCKR01000005 Enterobacter cloacae UCI 36 oxygen - insensitive NAD(P)H nitroreductase 217 EUL73477 JCKS01000001 Enterobacter cloacae UCI 35 oxygen - insensitive NAD(P)H nitroreductase 217 EUL78189 JCKT01000002 Enterobacter cloacae UCI 30 oxygen - insensitive NAD(P)H nitroreductase 217 EUL88446 JCKW01000004 Enterobacter cloacae UCI 24 oxygen - insensitive NAD(P)H nitroreductase 217 EUL91820 JCKX01000009 Enterobacter cloacae UCI 23 oxygen - insensitive NAD(P)H nitroreductase 217 EUM08906 JCLB01000007 Enterobacter sp. BIDMC 29 oxygen - insensitive NAD(P)H nitroreductase 217 58 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) EUM15526 JCLC01000009 Enterobacter sp. BIDMC 28 oxygen - insensitive NAD(P)H nitroreductase 217 EUM21943 JCLD01000002 Enterobacter sp. BIDMC 27 oxygen - insensitive NAD(P)H nitroreductase 217 EUM34474 JCLG01000014 Enterobacter sp. BWH 39 oxygen - insensitive NAD(P)H nitroreductase 217 EUM49863 JCLI01000001 Enterobacter sp. MGH 37 oxygen - insensitive NAD(P)H nitroreductase 217 EUM53867 JCLJ01000002 Enterobacter sp. MGH 33 oxygen - insensitive NAD(P)H nitroreductase 217 EUM57100 JCLK01000001 Enterobacter sp. MGH 15 oxygen - insensitive NAD(P)H nitroreductase 217 EUM63475 JCLM01000004 Enterobacter sp. MGH 12 oxygen - insensitive NAD(P)H nitroreductase 217 EUM65749 JCLN01000008 Enterobacter sp. MGH 11 oxygen - insensitive NAD(P)H nitroreductase 217 EUM75320 JCLP01000007 Enterobacter sp. MGH 9 oxygen - insensitive NAD(P)H nitroreductase 217 EUM82347 JCLQ01000006 Enterobacter sp. MGH 7 oxygen - insensitive NAD(P)H nitroreductase 216 EUM94544 JCLS01000005 Enterobacter sp. MGH 5 oxygen - insensitive NAD(P)H nitroreductase 217 EUN03671 JCLV01000014 Enterobacter sp. MGH 2 oxygen - insensitive NAD(P)H nitroreductase 217 EWG65056 AZUA01000013 Enterobacter sp. DC1 Oxygen - insensitive NAD(P)H nitroreductase 217 EWG72594 AZUB01000007 Enterobacter sp. DC4 Oxygen - insensitive NAD(P)H nitroreductase 217 X W7P1G3 (UniProt) EWG78545 AZXZ01000004 Enterobacter sp. DC3 Oxygen - insensitive NAD(P)H nitroreductase 217 EZR15844 JJNL01000005 Enterobacter sp. BWH 27 oxygen - insensitive NAD(P)H nitroreductase 217 KDF43196 JMUQ01000011 Enterobacter cloacae BIDMC 67 oxygen - insensitive NAD(P)H nitroreductase 217 59 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KDF50040 JMUR01000002 Enterobacter cloacae BWH 43 oxygen - insensitive NAD(P)H nitroreductase 217 KDF51086 JMUS01000021 Enterobacter cloacae CHS 79 oxygen - insensitive NAD(P)H nitroreductase 217 KDF59885 JMUT01000007 Enterobacter cloacae MGH 53 oxygen - insensitive NAD(P)H nitroreductase 217 KDF60961 JMUU01000004 Enterobacter cloacae MGH 54 oxygen - insensitive NAD(P)H nitroreductase 217 KDF77739 JMUV01000001 Enterobacter cloacae UCI 29 oxygen - insensitive NAD(P)H nitroreductase 217 KDM55519 JMZV01000009 Lelliottia amnigena CHS 78 oxygen - insensitive NAD(P)H nitroreductase 217 KGB03739 JPPR01000041 Enterobacter cloacae oxygen - insensitive NAD(P)H nitroreductase 217 KGI65104 JRJC01000008 Enterobacter sp. UCD - UG_FMILLET dihydropteridine reductase 217 KGY63834 JTBF01000001 Enterobacter cloacae dihydropteridine reductase 217 KGZ08118 JTBO01000001 Enterobacter cloacae dihydropteridine reductase 217 KHG49224 JTEP01000015 Enterobacter hormaechei subsp. oharae oxygen - insensitive NAD(P)H nitroreductase 217 KHK40536 JRUR01000037 Enterobacter sp. E20 dihydropteridine reductase 217 KHM15979 JUHY01000016 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KHO34010 JSZC01000013 Enterobacter sp. FB dihydropteridine reductase 217 KHQ43105 JWRQ01000001 Enterobacter cloacae dihydropteridine reductase 217 KHQ47705 JWRR01000001 Enterobacter cloacae dihydropteridine reductase 217 KIF96196 JXAE01000012 Enterobacter cloacae dihydropteridine reductase 217 KJC02823 JYGA02000001 Enterobacter cloacae dihydropteridine reductase 217 X A0A0J0GYZ6 (UniProt) KJF33504 JZKC01000002 Enterobacter cloacae BIDMC 33A oxygen - insensitive NAD(P)H nitroreductase 217 KJI52102 JYME01000018 Enterobacter cloacae dihydropteridine reductase 217 KJI63937 JYMG01000012 Enterobacter cloacae dihydropteridine reductase 217 KJI67088 JYMF01000003 Enterobacter cloacae dihydropteridine reductase 217 KJI77864 JYMJ01000026 Enterobacter cloacae dihydropteridine reductase 217 KJI80093 JYMM01000070 Enterobacter cloacae dihydropteridine reductase 217 60 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KJL53990 JZXS01000010 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJL58524 JZXR01000012 Enterobacter kobei dihydropteridine reductase 217 KJL62788 JZXQ01000004 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJL65872 JZXV01000014 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJL71098 JZXT01000012 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJL82019 JZXW01000022 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reduct ase 217 KJL91614 JZXX01000006 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJL96458 JZYB01000016 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJM25698 JZYE01000004 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJM31332 JZYF01000019 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJM39857 JZYG01000008 Enterobacter cloacae subsp. cloacae dihydropteridine reductase 217 X A0A0F0TWZ9 (UniProt) KJM40163 JZYH01000001 Enterobacter kobei dihydropteridine reductase 217 KJM53393 JZYK01000004 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJM60000 JZYM01000008 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJM69539 JZYN01000005 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 215 KJM73897 JZYP01000010 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJM83489 JZYQ01000002 Enterobacter hormaechei dihydropteridine reductase 217 KJM84692 JZYS01000051 Enterobacter kobei dihydropteridine reductase 217 KJM96715 JZYT01000006 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJN06334 JZYW01000015 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJN09901 JZYU01000001 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJN15129 JZYY01000015 Enterobacter hormaechei dihydropteridine reductase 217 KJN22542 JZYZ01000004 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJN24874 JZYX01000036 Enterobacter cloacae complex sp. 35699 dihydropteridine reductase 217 X A0A0F1AUY8 (UniProt) KJN28736 JZZA01000011 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 61 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KJN33593 JZZB01000014 Enterobacter cloacae complex sp. 35669 dihydropteridine reductase 217 KJN40016 JZZC01000012 Enterobacter hormaechei subsp. hormaechei dihydropteridine reductase 217 KJN48312 JZZD01000001 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJN63831 JZZJ01000015 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJN64553 JZZH01000008 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJN78680 JZZK01000012 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 215 KJN82211 JZZL01000003 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJN90460 JZZM01000002 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJN94148 JZZO01000007 Enterobacter hormaechei dihydropteridine reductase 217 KJN96050 JZZN01000005 Enterobacter hormaechei dihydropteridine reductase 217 KJO04448 JZZP01000007 Enterobacter hormaechei subsp. hormaechei dihydropteridine reductase 217 KJO07497 JZZQ01000006 Enterobacter hormaechei dihydropteridine reductase 217 KJO18773 JZZT01000007 Enterobacter hormaechei dihydropteridine reductase 217 KJO22169 JZZU01000006 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJO22423 JZZS01000004 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJO28651 JZZW01000013 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJO56047 LAAE01000079 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJO61267 LAAD01000004 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJO70875 LAAH01000091 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJO72059 LAAG01000015 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJO74471 LAAF01000011 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJO82549 LAAJ01000019 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJO95282 LAAK01000013 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJO97513 LAAL01000028 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJO99505 LAAN01000046 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 62 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KJP17220 LAAP01000023 Enterobacter asburiae dihydropteridine reductase 217 KJP20970 LAAQ01000012 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJP25065 LAAS01000087 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJP28956 LAAR01000009 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJP37881 LAAT01000003 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJP47639 LAAW01000021 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJP50767 LAAV01000011 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJP54695 LAAY01000039 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJP68624 LAAZ01000006 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJP71642 LABB01000027 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJP72531 LABA01000005 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJP80366 LABC01000008 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJP95304 JZCX01000045 Enterobacter cloacae dihydropteridine reductase 217 KJQ14588 JZDA01000017 Enterobacter cloacae dihydropteridine reductase 217 KJQ40098 JZDF01000006 Enterobacter cloacae dihydropteridine reductase 217 KJW76773 JZKP01000096 Enterobacter asburiae dihydropteridine reductase 217 KJW92826 JZKN01000015 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJX00074 JZKO01000105 Enterobacter asburiae dihydropteridine reductase 217 X A0A0F3WPM3 (UniProt) KJX08594 JZKU01000031 Enterobacter cloacae subsp. cloacae dihydropteridine reductase 217 KJX15256 JZLE01000091 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJX20650 JZKM01000005 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KJX34743 JZKY01000001 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KJX47824 JZLB01000004 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KKA58171 JYMH01000005 Enterobacter cloacae dihydropteridine reductase 217 KKJ29292 LBLX01000021 Enterobacter hormaechei dihydropteridine reductase 217 KKY76395 JSWY01000043 Enterobacter cloacae dihydropteridine reductase 217 KLF79275 LDCC01000090 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLF80514 LDCD01000036 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 63 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KLF88527 LDCE01000036 Enterobacter asburiae dihydropteridine reductase 217 KLF93227 LDCB01000006 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLF95206 LDCF01000038 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLG05662 LDCG01000016 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KLG07420 LDCI01000028 Enterobacter cloacae subsp. cloacae dihydropteridine reductase 217 KLG12786 LDCH01000009 Enterobacter asburiae dihydropteridine reductase 217 KLG16574 LDCL01000065 Enterobacter kobei dihydropteridine reductase 217 KLG23926 LDCJ01000025 Enterobacter kobei dihydropteridine reductase 217 KLP27586 LEEQ01000011 Enterobacter kobei dihydropteridine reductase 217 KLP43733 LECX01000031 Enterobacter asburiae dihydropteridine reductase 217 KLP47377 LECY01000030 Enterobacter kobei dihydropteridine reductase 217 KLP59854 LEDA01000066 Enterobacter asburiae dihydropteridine reductase 217 KLP62170 LEDB01000033 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLP75032 LEDE01000031 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLP75103 LEDC01000017 Enterobacter kobei dihydropteridine reductase 217 KLP81665 LEDD01000008 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLP83346 LEDG01000028 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLP86873 LEDH01000068 Enterobacter asburiae dihydropteridine reductase 217 KLP92362 LEDF01000028 Enterobacter hormaechei dihydropteridine reductase 217 KLP99732 LEDK01000036 Enterobacter hormaechei subsp. oharae dihydropteridine reductase 217 KLQ16646 LEDL01000009 Enterobacter cloacae subsp. cloacae dihydropteridine reductase 217 KLQ26057 LEDP01000064 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLQ27743 LEDO01000027 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLQ30737 LEDQ01000030 Enterobacter cloacae complex sp. GN02548 dihydropteridine reductase 217 KLQ38423 LEDR01000024 Enterobacter cloacae subsp. dissolvens dihydropteridine reductase 217 KLQ46072 LEDS01000040 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 64 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KLQ48645 LEDU01000057 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLQ67113 LEDY01000059 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLQ72829 LEDZ01000057 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLQ76728 LEEA01000031 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLQ85237 LEEB01000032 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLQ90636 LEEC01000026 Enterobacter kobei dihydropteridine reductase 217 KLQ96663 LEEF01000057 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLQ97280 LEEE01000022 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLR02651 LEEG01000062 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLR07252 LEEH01000061 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLR11692 LEEI01000036 Enterobacter hormaechei subsp. hormaechei dihydropteridine reductase 217 KLR21134 LEEK01000108 Enterobacter kobei dihydropteridine reductase 217 KLR25454 LEED01000008 Enterobacter hormaechei dihydropteridine reductase 217 KLR26621 LEEJ01000006 Enterobacter cloacae complex sp. GN02283 dihydropteridine reductase 217 KLR28966 LEEM01000053 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLR30325 LEEL01000045 Enterobacter kobei dihydropteridine reductase 217 KLR40091 LEEN01000024 Enterobacter hormaechei subsp. steigerwaltii dihydropteridine reductase 217 KLW07972 LESZ01000004 Enterobacter cloacae oxygen - insensitive NAD(P)H nitroreductase 217 KLW13711 LETA01000002 Enterobacter cloacae oxygen - insensitive NAD(P)H nitroreductase 217 KLW18492 LETB01000003 Enterobacter sp. BWH52 oxygen - insensitive NAD(P)H nitroreductase 217 65 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KLW26047 LETD01000004 Enterobacter sp. BWH64 oxygen - insensitive NAD(P)H nitroreductase 217 KLW32531 LETE01000008 Enterobacter sp. MGH85 oxygen - insensitive NAD(P)H nitroreductase 217 KLW40786 LETG01000003 Enterobacter sp. MGH119 oxygen - insensitive NAD(P)H nitroreductase 217 KLW41347 LETH01000009 Enterobacter sp. MGH120 oxygen - insensitive NAD(P)H nitroreductase 217 KLW50123 LETF01000005 Enterobacter sp. MGH86 oxygen - insensitive NAD(P)H nitroreductase 217 KLW54414 LETI01000007 Enterobacter sp. MGH127 oxygen - insensitive NAD(P)H nitroreductase 217 KLW57527 LETJ01000002 Enterobacter sp. MGH128 oxygen - insensitive NAD(P)H nitroreductase 217 KLW59202 LETL01000008 Enterobacter sp. BIDMC93 oxygen - insensitive NAD(P)H nitroreductase 217 KLW73177 LETN01000002 Enterobacter sp. BIDMC99 oxygen - insensitive NAD(P)H nitroreductase 217 KLW81937 LETO01000020 Enterobacter sp. BIDMC100 oxygen - insensitive NAD(P)H nitroreductase 217 KLW90890 LETR01000001 Enterobacter sp. BIDMC92 oxygen - insensitive NAD(P)H nitroreductase 217 X A0A0J1VWE2 (UniProt) KML25527 LDWM01000001 Leclercia adecarboxylata dihydropteridine reductase 217 KMN67227 LDUO01000002 Leclercia sp. LK8 dihydropteridine reductase 217 KOQ80652 LDVU01000068 Enterobacter cloacae subsp. cloacae dihydropteridine reductase 217 KOQ87409 LDVV01000054 Enterobacter cloacae subsp. cloacae dihydropteridine reductase 217 KOQ93714 LDVX01000013 Enterobacter asburiae dihydropteridine reductase 217 KPR21094 LJDV01000008 Enterobacter cloacae subsp. cloacae dihydropteridine reductase 217 KPU04692 LJEZ01000041 Enterobacter cloacae subsp. cloacae dihydropteridine reductase 217 KRS24540 LLXN01000111 Enterobacter cloacae dihydropteridine reductase 217 KSX59031 LNHM01000049 Enterobacter sp. 50588862 NAD(P)H - dependent oxidoreductase 217 X A0A0V9DV66 (UniProt) KSZ07484 LNIO01000058 Enterobacter sp. 50858885 NAD(P)H - dependent oxidoreductase 217 66 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KTG80664 LPOA01000048 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTG84608 LPOC01000044 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTG86594 LPOB01000070 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTG91605 LPOD01000058 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTG99646 LPOE01000034 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTH08142 LPOF01000034 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTH09403 LPOG01000025 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTH14315 LPOH01000027 Enterobacter hormaechei NAD(P)H - dependent oxidoreductase 217 KTH18700 LPOK01000079 Enterobacter cloacae subsp. cloacae NAD(P)H - dependent oxidoreductase 217 KTH20759 LPOI01000057 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTH27214 LPOJ01000059 Enterobacter cloacae subsp. cloacae NAD(P)H - dependent oxidoreductase 217 KTH43167 LPON01000023 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTH48191 LPOP01000040 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTH52258 LPOO01000023 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTH57455 LPOQ01000078 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTH74797 LPOT01000013 Enterobacter cloacae subsp. cloacae NAD(P)H - dependent oxidoreductase 217 KTH87422 LPOV01000007 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 67 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KTH90299 LPOW01000033 Enterobacter cloacae subsp. cloacae NAD(P)H - dependent oxidoreductase 217 KTH92960 LPOY01000068 Enterobacter cloacae subsp. cloacae NAD(P)H - dependent oxidoreductase 217 KTI10466 LPPA01000028 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTI39611 LPPI01000062 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTI42379 LPPH01000030 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTI47583 LPPJ01000034 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTI52549 LPPK01000082 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTI60960 LPPL01000071 Enterobacter kobei NAD(P)H - dependent oxidoreductase 217 KTI63472 LPPN01000079 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTI63977 LPPM01000046 Enterobacter cloacae subsp. cloacae NAD(P)H - dependent oxidoreductase 217 KTI95150 LPPS01000046 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTI96293 LPPR01000005 Enterobacter hormaechei NAD(P)H - dependent oxidoreductase 217 KTJ14364 LPPX01000022 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTJ20738 LPPY01000061 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTJ24913 LPQA01000034 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTJ42059 LPQC01000035 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTJ70678 LPQK01000055 Enterobacter hormaechei NAD(P)H - dependent oxidoreductase 217 68 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KTJ82381 LPQI01000024 Enterobacter cloacae subsp. cloacae NAD(P)H - dependent oxidoreductase 217 KTJ85353 LPQL01000045 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTJ86452 LPQM01000034 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTJ86847 LPQN01000085 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTJ96976 LPQO01000045 Enterobacter hormaechei NAD(P)H - dependent oxidoreductase 217 KTK06233 LPQQ01000045 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTK10694 LPQS01000045 Enterobacter hormaechei NAD(P)H - dependent oxidoreductase 217 KTK11953 LPQR01000034 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KTK24322 LPQU01000045 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 X A0A0W1YUK5 (UniProt) KTK29608 LPQW01000086 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KTK35666 LPQV01000028 Enterobacter hormaechei NAD(P)H - dependent oxidoreductase 217 KTQ45547 LDQG01000023 Enterobacter cancerogenus dihydropteridine reductase 217 KTQ52338 LDQD01000040 Enterobacter asburiae dihydropteridine reductase 217 KTQ53424 LDQH01000007 Enterobacter cancerogenus dihydropteridine reductase 217 KTQ63824 LDQM01000003 Enterobacter xiangfangensis dihydropteridine reductase 217 KTQ66510 LDQE01000004 Enterobacter asburiae dihydropteridine reductase 217 KTQ71393 LDQK01000005 Enterobacter xiangfangensis dihydropteridine reductase 217 KTQ74078 LDQI01000007 Enterobacter cancerogenus dihydropteridine reductase 217 KTQ79589 LDQF01000011 Enterobacter asburiae dihydropteridine reductase 217 KTQ83662 LDQJ01000006 Enterobacter cancerogenus dihydropteridine reductase 217 KTQ93775 LDQO01000001 Enterobacter xiangfangensis dihydropteridine reductase 217 KTQ94732 LDQQ01000001 Enterobacter xiangfangensis dihydropteridine reductase 217 KTR00900 LDQS01000004 Enterobacter xiangfangensis dihydropteridine reductase 217 KTR13789 LDQP01000003 Enterobacter xiangfangensis dihydropteridine reductase 217 69 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KTR16152 LDQT01000012 Enterobacter xiangfangensis dihydropteridine reductase 217 KTR23491 LDQR01000005 Enterobacter xiangfangensis dihydropteridine reductase 217 KTR29804 LDQL01000036 Enterobacter xiangfangensis dihydropteridine reductase 217 KTR33302 LDQN01000023 Enterobacter xiangfangensis dihydropteridine reductase 217 KTR45762 LDQU01000004 Enterobacter xiangfangensis dihydropteridine reductase 217 KUH52752 LKUI01000012 Enterobacter cloacae subsp. cloacae dihydropteridine reductase 217 KUQ00846 LRBY01000036 Enterobacter kobei NAD(P)H - dependent oxidoreductase 217 KUQ05118 LRBZ01000098 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KUQ21814 LRCD01000196 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KUQ25915 LRCE01000130 Enterobacter asburiae NAD(P)H - dependent oxidoreductase 217 KUQ34672 LRCF01000046 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KUQ35170 LRCH01000146 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KUQ49934 LRCJ01000039 Enterobacter asburiae NAD(P)H - dependent oxidoreductase 217 KUQ63953 LRCM01000021 Enterobacter kobei NAD(P)H - dependent oxidoreductase 217 KUQ66043 LRCK01000024 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KUQ77117 LRCO01000001 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KUQ77282 LRCP01000012 Enterobacter kobei NAD(P)H - dependent oxidoreductase 217 KUQ82043 LRCN01000031 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KUQ87332 LRCQ01000042 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KUQ91727 LRCT01000115 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 70 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KUQ96880 LRCS01000043 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KUR08289 LRCU01000001 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KUR30674 LRCZ01000028 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVI51697 LRIO01000127 Enterobacter cloacae subsp. cloacae NAD(P)H - dependent oxidoreductase 217 KVI62307 LRIR01000050 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVI67583 LRIS01000034 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVI94875 LRIY01000034 Enterobacter hormaechei NAD(P)H - dependent oxidoreductase 217 KVJ04049 LRIZ01000023 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVJ05337 LRJA01000101 Enterobacter asburiae NAD(P)H - dependent oxidoreductase 217 KVJ12397 LRJB01000012 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVJ18342 LRJC01000012 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVJ24582 LRJD01000012 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVJ30752 LRJE01000012 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVJ36558 LRJH01000034 Enterobacter cloacae subsp. cloacae NAD(P)H - dependent oxidoreductase 217 KVJ37673 LRJF01000001 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVJ52360 LRJI01000001 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVJ68336 LRJM01000001 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 71 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KVJ71757 LRJL01000001 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVJ72350 LRJP01000086 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVJ75509 LRJN01000001 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVJ78395 LRJO01000006 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVJ82232 LRJQ01000052 Enterobacter asburiae NAD(P)H - dependent oxidoreductase 217 KVJ94409 LRJT01000045 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVK03536 LRJS01000001 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVK05669 LRJU01000028 Enterobacter hormaechei subsp. oharae NAD(P)H - dependent oxidoreductase 217 KVK07627 LRJV01000034 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVK13877 LRJY01000075 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVK18908 LRJX01000048 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVK25305 LRIN01000103 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 KVK30750 LREQ01000063 Enterobacter hormaechei NAD(P)H - dependent oxidoreductase 217 KYH19405 LUGN01000001 Enterobacter cloacae NAD(P)H - dependent oxidoreductase 217 KYJ78922 LOBN01000007 Enterobacter cloacae NAD(P)H - dependent oxidoreductase 217 KYO08671 LEDX02000020 Enterobacter ludwigii NAD(P)H - dependent oxidoreductase 217 KYO15497 LEDV02000008 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H - dependent oxidoreductase 217 72 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KYQ76748 LOMM01000013 Enterobacter sp. SENG - 6 NAD(P)H - dependent oxidoreductase 217 KZP49864 LVTX01000044 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZP50052 LVTR01000036 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZP59420 LVTS01000042 Enterobacter cloacae complex sp. GN04787 NAD(P)H nitroreductase 217 KZP70673 LVTZ01000030 Enterobacter cloacae subsp. dissolvens NAD(P)H nitroreductase 217 KZP71339 LVTV01000012 Enterobacter hormaechei subsp. oharae NAD(P)H nitroreductase 217 KZP74228 LVTP01000097 Enterobacter cloacae complex 'Hoffmann cluster IV' NAD(P)H nitroreductase 217 KZP84796 LVUH01000015 Enterobacter hormaechei subsp. oharae NAD(P)H nitroreductase 217 KZP87247 LVUA01000010 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZP87438 LVTT01000222 Enterobacter cloacae complex sp. GN04826 NAD(P)H nitroreductase 217 X A0A0X6Z623 (UniProt) KZP91282 LVUG01000049 Enterobacter asburiae NAD(P)H nitroreductase 217 KZQ01202 LVTU01000021 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZQ10184 LVUM01000022 Enterobacter kobei NAD(P)H nitroreductase 217 KZQ11844 LVTQ01000236 Enterobacter cloacae complex 'Hoffmann cluster IV' NAD(P)H nitroreductase 217 KZQ19715 LVUI01000022 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZQ24451 LVTY01000016 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZQ26350 LVUF01000120 Enterobacter cloacae complex sp. GN05526 NAD(P)H nitroreductase 217 KZQ41025 LVUS01000006 Enterobacter cloacae subsp. dissolvens NAD(P)H nitroreductase 217 KZQ42475 LVUD01000011 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZQ56649 LVUJ01000020 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZQ65574 LVUX01000034 Enterobacter kobei NAD(P)H nitroreductase 217 KZQ74737 LVUK01000184 Enterobacter cloacae complex 'Hoffmann cluster IV' NAD(P)H nitroreductase 217 73 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KZQ88537 LVUZ01000001 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZQ90092 LVUQ01000023 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZQ99425 LVVC01000001 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZQ99976 LVUU01000057 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZR24251 LVVD01000021 Enterobacter hormaechei subsp. steigerwaltii NAD(P)H nitroreductase 217 KZR30349 LVVA01000021 Enterobacter cloacae complex sp. GN06232 NAD(P)H nitroreductase 217 X A0A162E6U0 (UniProt) KZR40420 LVVI01000034 Enterobacter cloacae complex 'Hoffmann cluster IV' NAD(P)H nitroreductase 217 KZR42159 LVVE01000067 Enterobacter asburiae NAD(P)H nitroreductase 217 OAE36549 LXPT01000036 Enterobacter cloacae NAD(P)H nitroreductase 217 OAE65687 LXPZ01000039 Enterobacter cloacae NAD(P)H nitroreductase 217 OAH34057 LSTS01000023 Enterobacter xiangfangensis NAD(P)H - dependent oxidoreductase 217 OAR71532 LSUR01000039 Enterobacter cloacae NAD(P)H - dependent oxidoreductase 217 OAR85305 LSUS01000023 Enterobacter cloacae NAD(P)H - dependent oxidoreductase 217 OAT39275 LXES01000032 Enterobacter soli ATCC BAA - 2102 oxygen - insensitive NAD(P)H nitroreductase/dihydropteridine reductase 217 OAY17414 LZCS01000019 Enterobacter asburiae NAD(P)H nitroreductase 217 OAZ41327 LZEN01000029 Enterobacter cloacae NAD(P)H nitroreductase 217 OAZ94693 LYYC01000010 Enterobacter asburiae NAD(P)H nitroreductase 217 OBS89924 LSQV01000012 Enterobacter asburiae NAD(P)H nitroreductase 217 OEG80433 LJDP02000122 Enterobacter cloacae complex 'Hoffmann cluster III' NAD(P)H nitroreductase 217 OEG91103 LJDJ02000001 Enterobacter asburiae NAD(P)H nitroreductase 217 OEG95508 LJED02000157 Enterobacter kobei NAD(P)H nitroreductase 217 74 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) OEH05625 LJEO02000112 Enterobacter cloacae complex 'Hoffmann cluster IV' NAD(P)H nitroreductase 217 OEH07809 LJER02000001 Enterobacter asburiae NAD(P)H nitroreductase 217 X A0A1D3B951 (UniProt) OEH12797 LJEW02000099 Enterobacter kobei NAD(P)H nitroreductase 217 OEH18070 LJEY02000079 Enterobacter asburiae NAD(P)H nitroreductase 217 OEH22099 LJDO02000071 Enterobacter sp. ST121:950178628 NAD(P)H nitroreductase 217 OEI71308 MDVW01000019 Enterobacter sp. ku - bf2 NAD(P)H nitroreductase 217 OFU74459 LTZU01000008 Enterobacter sp. HMSC16D10 NAD(P)H - dependent oxidoreductase 217 OHY48340 MBTX01000065 Enterobacter cloacae complex 'Hoffmann cluster IV' NAD(P)H nitroreductase 217 OHY64544 MBMT01000089 Enterobacter cloacae complex 'Hoffmann cluster IV' NAD(P)H nitroreductase 217 OIR48669 MKER01000005 Lelliottia nimipressuralis NAD(P)H nitroreductase 217 OIR53486 MKEQ01000001 Enterobacter hormaechei ATCC 49162 NAD(P)H nitroreductase 217 OJH28178 JSVH01000104 Enterobacter kobei dihydropteridine reductase 217 OOC92560 MTFV01000010 Enterobacter cloacae NAD(P)H nitroreductase 217 OOK78025 MPZO01000003 Pedobacter himalayensis NAD(P)H nitroreductase 217 OOV73515 MWLI01000072 Enterobacter cloacae NAD(P)H nitroreductase 217 OQD51350 MTKD01000001 Enterobacter cancerogenus NAD(P)H nitroreductase 217 ORC20908 MWMD01000002 Enterobacter cloacae subsp. cloacae NAD(P)H nitroreductase 217 ORC31004 MWME01000004 Enterobacter cloacae subsp. cloacae NAD(P)H nitroreductase 217 ORD23492 NCRW01000002 Enterobacter cloacae NAD(P)H nitroreductase 217 SAA02736 FKDK01000001 Enterobacter cloacae nitroreductase 217 SAA04403 FKDF01000001 Enterobacter cloacae nitroreductase 217 SAA10055 FKBM01000001 Enterobacter cloacae nitroreductase 217 SAA11604 FKBN01000003 Enterobacter cloacae nitroreductase 217 SAA12898 FKBB01000015 Enterobacter cloacae nitroreductase 217 SAA13613 FKCA01000002 Enterobacter cloacae nitroreductase 217 SAA21926 FKAO01000008 Enterobacter cloacae nitroreductase 217 SAA27147 FKBJ01000006 Enterobacter cloacae nitroreductase 217 SAA27968 FKBO01000005 Enterobacter cloacae nitroreductase 217 SAA42770 FKCR01000005 Enterobacter cloacae nitroreductase 217 SAA52862 FKBG01000006 Enterobacter cloacae nitroreductase 217 75 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SAA55253 FKBI01000010 Enterobacter cloacae nitroreductase 217 SAA65037 FKCT01000006 Enterobacter cloacae nitroreductase 217 SAA91716 FKAY01000012 Enterobacter cloacae nitroreductase 217 SAB04137 FKCI01000001 [Enterobacter] aerogenes nitroreductase 217 SAB05543 FKBZ01000001 Enterobacter cloacae nitroreductase 217 SAB09583 FKCV01000001 Enterobacter cloacae nitroreductase 217 SAB12814 FKCO01000001 Enterobacter cloacae nitroreductase 217 SAB16149 FKBP01000031 Enterobacter cloacae nitroreductase 217 SAB23482 FKCD01000022 Enterobacter cloacae nitroreductase 217 SAB26478 FKBK01000023 Enterobacter cloacae nitroreductase 217 SAB29498 FKDC01000006 Enterobacter cloacae nitroreductase 217 SAB39454 FKCW01000003 Enterobacter cloacae nitroreductase 217 SAB49507 FKDI01000024 Enterobacter cloacae nitroreductase 217 SAB49971 FKCM01000004 [Enterobacter] aerogenes nitroreductase 217 SAB53712 FKCQ01000022 Enterobacter cloacae nitroreductase 217 SAB54213 FKCU01000005 Enterobacter cloacae nitroreductase 217 SAB57257 FKBY01000002 Enterobacter cloacae nitroreductase 217 SAB60405 FKCJ01000002 Enterobacter cloacae nitroreductase 217 SAB60523 FKCK01000001 Enterobacter cloacae nitroreductase 217 SAB61877 FKBW01000001 Enterobacter cloacae nitroreductase 217 SAB64872 FKCF01000003 Enterobacter cloacae nitroreductase 217 SAB66050 FKCE01000001 Enterobacter cloacae nitroreductase 217 SAB69185 FKBU01000001 Enterobacter cloacae nitroreductase 217 SAB71343 FKBX01000001 Enterobacter cloacae nitroreductase 217 SAB72692 FKDA01000001 Enterobacter cloacae nitroreductase 217 SAB73941 FKDB01000003 Enterobacter cloacae nitroreductase 217 SAB76967 FKCB01000005 Enterobacter cloacae nitroreductase 217 SAB78266 FKCN01000002 Enterobacter cloacae nitroreductase 217 SAB81491 FKDD01000004 Enterobacter cloacae nitroreductase 217 SAB83561 FKDJ01000001 Enterobacter cloacae nitroreductase 217 SAB91962 FKCL01000001 Enterobacter cloacae nitroreductase 217 SAB92460 FKDL01000005 Enterobacter cloacae nitroreductase 217 SAC00944 FKDH01000009 Enterobacter cloacae nitroreductase 217 SAC19627 FKCH01000002 Enterobacter cloacae nitroreductase 217 SAC22435 FKCC01000021 Enterobacter cloacae nitroreductase 217 76 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SAC23165 FKDE01000027 Enterobacter cloacae nitroreductase 217 SAC40547 FKDP01000002 Enterobacter cloacae nitroreductase 217 SAC52878 FKDQ01000001 Enterobacter cloacae nitroreductase 215 SAC58935 FKDS01000001 Enterobacter cloacae nitroreductase 215 SAC59184 FKDM01000001 Enterobacter cloacae nitroreductase 217 SAC61352 FKDR01000002 Enterobacter cloacae nitroreductase 217 SAC64471 FKDV01000001 Enterobacter cloacae nitroreductase 217 SAC64485 FKDN01000004 Enterobacter cloacae nitroreductase 217 SAC66563 FKDT01000001 Enterobacter cloacae nitroreductase 217 SAC72423 FKCY01000007 Enterobacter cloacae nitroreductase 217 SAC91605 FKCZ01000009 Enterobacter cloacae nitroreductase 217 SAC93538 FKDG01000029 Enterobacter cloacae nitroreductase 217 SAD00330 FKEA01000003 Enterobacter cloacae nitroreductase 217 SAD06557 FKDW01000002 Enterobacter cloacae nitroreductase 217 SAD20032 FKEM01000002 Enterobacter cloacae nitroreductase 217 X A0A155XML1 (UniProt) SAD20572 FKEG01000001 Enterobacter cloacae nitroreductase 217 X A0A0X4EH75 (UniProt) SAD25447 FKEJ01000001 Enterobacter cloacae nitroreductase 217 SAD28852 FKEF01000001 Enterobacter cloacae nitroreductase 217 SAD30055 FKDZ01000005 Enterobacter cloacae nitroreductase 217 SAD30063 FKEN01000001 Enterobacter cloacae nitroreductase 217 SAD38094 FKEB01000002 Enterobacter cloacae nitroreductase 217 SAD43294 FKER01000001 Enterobacter cloacae nitroreductase 217 SAD44220 FKEL01000005 Enterobacter cloacae nitroreductase 217 SAD47002 FKET01000001 Enterobacter cloacae nitroreductase 217 SAD52735 FKEC01000005 Enterobacter cloacae nitroreductase 217 SAD53317 FKEO01000008 Enterobacter cloacae nitroreductase 217 SAD55041 FKEV01000001 Enterobacter cloacae nitroreductase 217 SAD58068 FKEU01000002 Enterobacter cloacae nitroreductase 217 SAD61074 FKEW01000001 Enterobacter cloacae nitroreductase 217 SAD61815 FKEE01000002 Enterobacter cloacae nitroreductase 217 SAD65095 FKEI01000005 Enterobacter cloacae nitroreductase 217 SAD70767 FKES01000002 Enterobacter cloacae nitroreductase 217 SAD84247 FKDU01000008 Enterobacter cloacae nitroreductase 217 77 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SAD91670 FKEH01000007 Enterobacter cloacae nitroreductase 217 SAD95622 FKEZ01000001 Enterobacter cloacae nitroreductase 217 SAD95647 FKDO01000014 Enterobacter cloacae nitroreductase 217 SAE00086 FKEK01000010 Enterobacter cloacae nitroreductase 217 SAE13724 FKDY01000016 Enterobacter cloacae nitroreductase 217 SAE13834 FKEX01000002 Enterobacter cloacae nitroreductase 217 SAE24346 FKFJ01000001 Enterobacter cloacae nitroreductase 217 SAE29034 FKFG01000002 Enterobacter cloacae nitroreductase 217 SAE36397 FKFK01000001 Enterobacter cloacae nitroreductase 217 SAE39710 FKFF01000002 Enterobacter cloacae nitroreductase 217 SAE47717 FKFH01000002 Enterobacter cloacae nitroreductase 217 SAE49498 FKFI01000001 Enterobacter cloacae nitroreductase 217 SAE49828 FKFB01000003 Enterobacter cloacae nitroreductase 217 SAE51062 FKEP01000008 Enterobacter cloacae nitroreductase 217 SAE57309 FKFE01000009 Enterobacter cloacae nitroreductase 217 SAE67731 FKFC01000002 Enterobacter cloacae nitroreductase 217 SAE71105 FKFQ01000001 Enterobacter cloacae nitroreductase 217 SAE74949 FKEQ01000016 Enterobacter cloacae nitroreductase 217 SAE76723 FKFA01000011 Enterobacter cloacae nitroreductase 217 SAE80945 FKFM01000002 Enterobacter cloacae nitroreductase 217 SAE93277 FKFO01000001 Enterobacter cloacae nitroreductase 217 SAE94808 FKFR01000001 Enterobacter cloacae nitroreductase 217 SAE96551 FKFP01000002 Enterobacter cloacae nitroreductase 217 SAF01451 FKFD01000010 Enterobacter cloacae nitroreductase 217 SAF11915 FKFS01000007 Enterobacter cloacae nitroreductase 217 SAF35596 FKGF01000001 Enterobacter cloacae nitroreductase 217 SAF37355 FKFU01000002 Enterobacter cloacae nitroreductase 217 SAF37740 FKGG01000001 Enterobacter cloacae nitroreductase 217 SAF42163 FKGA01000001 Enterobacter cloacae nitroreductase 217 SAF45631 FKFW01000003 Enterobacter cloacae nitroreductase 217 SAF46279 FKFN01000008 Enterobacter cloacae nitroreductase 217 SAF50080 FKGK01000001 Enterobacter cloacae nitroreductase 217 SAF56133 FKFT01000005 Enterobacter cloacae nitroreductase 217 SAF67525 FKGL01000001 Enterobacter cloacae nitroreductase 217 SAF71051 FKGC01000008 Enterobacter cloacae nitroreductase 217 78 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SAF74441 FKGH01000002 Enterobacter cloacae nitroreductase 217 SAF75369 FKGT01000001 Enterobacter cloacae nitroreductase 217 SAF83751 FKFZ01000006 Enterobacter cloacae nitroreductase 217 SAF87875 FKGI01000002 Enterobacter cloacae nitroreductase 217 SAF90882 FKFV01000007 Enterobacter cloacae nitroreductase 217 SAF91144 FKGB01000003 Enterobacter cloacae nitroreductase 217 SAF96241 FKGP01000002 Enterobacter cloacae nitroreductase 217 SAF96936 FKFY01000005 Enterobacter cloacae nitroreductase 217 SAG02935 FKGO01000004 Enterobacter cloacae nitroreductase 217 SAG10926 FKGM01000002 Enterobacter cloacae nitroreductase 217 SAG12298 FKHA01000001 Enterobacter cloacae nitroreductase 217 SAG12543 FKGX01000001 Enterobacter cloacae nitroreductase 217 SAG29285 FKGS01000003 Enterobacter cloacae nitroreductase 217 SAG29371 FKHD01000001 Enterobacter cloacae nitroreductase 217 SAG36680 FKFX01000025 Enterobacter cloacae nitroreductase 217 SAG37487 FKGU01000005 Enterobacter cloacae nitroreductase 217 SAG41943 FKGZ01000002 Enterobacter cloacae nitroreductase 217 SAG44047 FKHC01000002 Enterobacter cloacae nitroreductase 217 SAG44883 FKGR01000002 Enterobacter cloacae nitroreductase 217 SAG54405 FKHF01000001 Enterobacter cloacae nitroreductase 215 SAG91102 FKHO01000001 Enterobacter cloacae nitroreductase 217 SAG94334 FKHN01000001 Enterobacter cloacae nitroreductase 217 SAH11013 FKHB01000008 Enterobacter cloacae nitroreductase 217 SAH11674 FKHI01000003 Enterobacter cloacae nitroreductase 217 SAH16545 FKGV01000011 Enterobacter cloacae nitroreductase 217 SAH16889 FKHP01000001 Enterobacter cloacae nitroreductase 217 SAH23588 FKGJ01000023 Enterobacter cloacae nitroreductase 217 SAH24378 FKHT01000001 Enterobacter cloacae nitroreductase 217 SAH36146 FKGE01000027 Enterobacter cloacae nitroreductase 217 SAH47684 FKHX01000001 Enterobacter cloacae nitroreductase 217 SAH59601 FKHE01000009 Enterobacter cloacae nitroreductase 217 SAH60362 FKHU01000003 Enterobacter cloacae nitroreductase 217 SAH64810 FKHY01000005 Enterobacter cloacae nitroreductase 217 SAH65951 FKHQ01000007 Enterobacter cloacae nitroreductase 217 SAH66437 FKID01000001 Enterobacter cloacae nitroreductase 217 79 Table A. 4 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SAH79547 FKHL01000008 Enterobacter cloacae nitroreductase 217 SAH81040 FKHK01000005 Enterobacter cloacae nitroreductase 217 SAH93782 FKIA01000005 Enterobacter cloacae nitroreductase 217 SAH93923 FKHV01000002 Enterobacter cloacae nitroreductase 217 SAI02736 FKIC01000004 Enterobacter cloacae nitroreductase 217 SAI09028 FKHR01000003 Enterobacter cloacae nitroreductase 217 SAI14426 FKHW01000005 Enterobacter cloacae nitroreductase 217 SAI14706 FKIB01000002 Enterobacter cloacae nitroreductase 217 X A0A145CU16 (UniProt) SAI49348 FKHS01000030 Enterobacter cloacae nitroreductase 217 SAI56111 FKHZ01000005 Enterobacter cloacae nitroreductase 217 SAJ03134 FKIW01000007 Enterobacter cloacae nitroreductase 217 SAJ04635 FKIS01000007 Enterobacter cloacae nitroreductase 217 SAJ07412 FKIX01000006 Enterobacter cloacae nitroreductase 215 SAJ14952 FKIU01000007 Enterobacter cloacae nitroreductase 217 SAJ19176 FKIQ01000008 Enterobacter cloacae nitroreductase 217 SAJ28726 FKIR01000014 Enterobacter cloacae nitroreductase 217 SAZ11274 FKLS01000001 Enterobacter kobei nitroreductase 217 SBL27928 FKZC01000001 Klebsiella oxytoca Oxygen - insensitive NAD(P)H nitroreductase 217 SCZ31110 FMVY01000002 Enterobacter sp. NFIX45 nitroreductase / dihydropteridine reductase 217 SEO76491 FODW01000001 Enterobacter sp. NFIX58 nitroreductase / dihydropteridine reductase 217 SFI03138 FOQN01000001 Enterobacter sp. NFIX59 nitroreductase / dihydropteridine reductase 217 SHG59995 FQWJ01000002 Pantoea sesami nitroreductase / dihydropteridine reductase 217 SMF89767 LT840187 Enterobacter cloacae nitroreductase / dihydropteridine reductase 217 80 Table A. 5 . FunGene and primer design sequences for pnrB Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AAM95987 AF532912 Pseudomonas putida oxygen - insensitive NADPH nitroreductase 217 X AAM95987.1 (NCBI) AAN68044 AE015451 Pseudomonas putida KT2440 6,7 - dihydropteridine reductase 217 ABQ79389 CP000712 Pseudomonas putida F1 dihydropteridine reductase 217 ABY97978 CP000926 Pseudomonas putida GB - 1 6,7 - dihydropteridine reductase 217 ADR60849 CP002290 Pseudomonas putida BIRD - 1 PnrB 217 X ADR60849.1 (NCBI) AFK67306 CP003588 Pseudomonas putida ND6 dihydropteridine reductase 217 AFO51068 CP003734 Pseudomonas putida DOT - T1E dihydropteridine reductase 217 AGA74160 CP003738 Pseudomonas putida HB3267 dihydropteridine reductase 217 AGN78716 CP005976 Pseudomonas putida H8234 dihydropteridine reductase 217 AJA15421 CP009974 Pseudomonas putida S12 dihydropteridine reductase 217 AJQ47913 CP010979 Pseudomonas putida S13.1.2 dihydropteridine reductase 217 ANC03605 CP011789 Pseudomonas putida dihydropteridine reductase 216 ANC82375 CP015202 Pseudomonas putida B6 - 2 NAD(P)H nitroreductase 217 ANI04291 CP015876 Pseudomonas putida SJTE - 1 NAD(P)H nitroreductase 217 ANI33970 CP011525 Pseudomonas sp. JY - Q dihydropteridine reductase 217 AOX09888 CP016212 Pseudomonas putida JB NAD(P)H nitroreductase 217 APE98345 CP017073 Pseudomonas putida NAD(P)H nitroreductase 217 APO83023 CP018743 Pseudomonas sp. DRA525 NAD(P)H nitroreductase 217 AUZ60049 CP026332 Pseudomonas sp. XWY - 1 Oxygen - insensitive NAD(P)H nitroreductase/ Dihydropteridine reductase 217 BAN55223 AP013070 Pseudomonas putida NBRC 14164 nitroreductase 217 CAK15120 CT573326 Pseudomonas entomophila L48 oxygen - insensitive NADPH nitroreductase 217 EJT82690 ALXA01000164 Pseudomonas putida S11 dihydropteridine reductase 217 EMR47500 ALPV02000011 Pseudomonas putida LS46 dihydropteridine reductase 217 ENY79724 APBQ01000004 Pseudomonas putida TRO1 dihydropteridine reductase 217 ERL01707 AOUR02000030 Pseudomonas putida LF54 dihydropteridine reductase 217 ERT16443 AXDX02000175 Pseudomonas putida SJ3 dihydropteridine reductase 217 ESP86245 AYKV01000005 Pseudomonas putida S12 dihydropteridine reductase 217 KIC80666 JWLN01000026 Pseudomonas sp. C5pp dihydropteridine reductase 217 KKO15840 AYRY01000007 Pseudomonas putida KG - 4 dihydropteridine reductase 217 81 Table A. 5 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KMU95024 LFYQ01000023 Pseudomonas putida dihydropteridine reductase 217 KMY34721 LDJF01000018 Pseudomonas putida dihydropteridine reductase 217 KPM64697 LKKT01000041 Pseudomonas putida dihydropteridine reductase 217 KPM68139 LKKS01000025 Pseudomonas putida dihydropteridine reductase 217 KWW19341 LNNI01000002 Stenotrophomonas rhizophila NAD(P)H - dependent oxidoreductase 217 KZO52039 LWDW01000078 Pseudomonas putida NAD(P)H nitroreductase 217 OAH42514 LSTU01000078 Pseudomonas monteilii NAD(P)H - dependent oxidoreductase 217 OAS06505 LSUZ01000180 Pseudomonas putida NAD(P)H - dependent oxidoreductase 217 OCT29702 MCBG01000022 Pseudomonas putida NAD(P)H nitroreductase 216 OCT31398 MCBH01000011 Pseudomonas putida NAD(P)H nitroreductase 216 OCT33640 MCBI01000011 Pseudomonas putida NAD(P)H nitroreductase 216 OCT40086 MCBJ01000011 Pseudomonas putida NAD(P)H nitroreductase 216 ORL51207 NBWB01000012 Pseudomonas putida oxygen - insensitive NAD(P)H - dependent nitroreductase NfsB 217 ORL69362 NBWA01000016 Pseudomonas putida oxygen - insensitive NAD(P)H - dependent nitroreductase NfsB 217 SIS12998 LT707061 Pseudomonas putida dihydropteridine reductase 217 SKB88244 LT799039 Pseudomonas putida dihydropteridine reductase 217 SMC56309 FWXQ01000002 Pseudomonas sp. URIL14HWK12:I5 dihydropteridine reductase 217 WP_043200951.1 1 NZ_JENB01000025.1: c56599 - 55946 Pseudomonas putida oxygen - insensitive NAD(P)H - dependent nitroreductase NfsB 217 X WP_043200951.1 (NCBI) WP_060497226.1 1 NZ_BCBJ01000003.1: c3014 - 2361 Pseudomonas sp. NBRC 111133 NAD(P)H nitroreductase 217 X WP_060497226.1 (NCBI) WP_060515567.1 1 NZ_BCBI01000057.1: c38546 - 37893 Pseudomonas sp. NBRC 111132 NAD(P)H nitroreductase 217 X WP_060515567.1 (NCBI) WP_063915060.1 1 NZ_LWBV01000011.1: c46838 - 46185 Pseudomonas sp. p21 NAD(P)H nitroreductase 217 X WP_063915060.1 (NCBI) WP_085599282.1 1 NZ_NEJU01000002.1: c242079 - 241426 Pseudomonas sp. B10(2017) NAD(P)H nitroreductase 217 X WP_085599282.1 (NCBI) WP_085615138.1 1 NZ_NEJL01000004.1: 218166 - 218819 Pseudomonas MULTISPECIES: NAD(P)H nitroreductase 217 X WP_085615138.1 (NCBI) 82 Table A. 5 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) WP_085624491.1 1 NZ_NEIN01000005.1: 167504 - 168157 Pseudomonas MULTISPECIES: NAD(P)H nitroreductase 217 X WP_085624491.1 (NCBI) WP_085665187.1 1 NZ_NEIR01000013.1: c164785 - 164132 Pseudomonas sp. B5(2017) NAD(P)H nitroreductase 217 X WP_085665187.1 (NCBI) WP_085706301.1 1 NZ_NEIO01000023.1: c44082 - 43429 Pseudomonas sp. B8(2017) NAD(P)H nitroreductase 217 X WP_085706301.1 (NCBI) WP_085720572.1 1 NZ_NEJN01000002.1: 424849 - 425502 Pseudomonas sp. B22(2017) NAD(P)H nitroreductase 217 X WP_085720572.1 (NCBI) 1 These sequences were not on FunGene so were added back in to the primer design sequence set manually . 83 Table A. 6 . FunGene and primer design sequences for xenA Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AAF02538 AF154061 Pseudomonas putida xenobiotic reductase A 363 X 5CPL (PDB) AAN66878 AE015451 Pseudomonas putida KT2440 XenA 363 AAO55915 AE016853 Pseudomonas syringae pv. tomato str. DC3000 xenobiotic reductase A 363 AAY37233 CP000075 Pseudomonas syringae pv. syringae B728a NADH:flavin oxidoreductase/NADH oxidase 363 AAY40303 AY957609 Pseudomonas putida DOT - T1E xenobiotic reductase 363 ABQ77442 CP000712 Pseudomonas putida F1 NADH:flavin oxidoreductase/NADH oxidase 363 ABZ00028 CP000926 Pseudomonas putida GB - 1 NADH:flavin oxidoreductase/NADH oxidase 363 ACA74393 CP000949 Pseudomonas putida W619 NADH:flavin oxidoreductase/NADH oxidase 363 ADR58970 CP002290 Pseudomonas putida BIRD - 1 XenA 363 AEJ14509 CP002870 Pseudomonas putida S16 xenobiotic reductase A 360 AFJ57172 CP003041 Pseudomonas fluorescens A506 xenobiotic reductase A 363 AFK70926 CP003588 Pseudomonas putida ND6 NADH:flavin oxidoreductase/NADH oxidase 363 AFO48989 CP003734 Pseudomonas putida DOT - T1E NADH:flavin oxidoreductase/NADH oxidase 363 AGA74837 CP003738 Pseudomonas putida HB3267 XenA 363 AGE26148 CP004045 Pseudomonas poae RE*1 - 1 - 14 xenobiotic reductase A 363 AGN80144 CP005976 Pseudomonas putida H8234 NADH:flavin oxidoreductase 363 AGZ36485 CP003961 Pseudomonas sp. VLB120 NADH:flavin oxidoreductase/NADH oxidase 363 AHC84020 CP006978 Pseudomonas monteilii SB3078 NADH:flavin oxidoreductase 363 AHC89391 CP006979 Pseudomonas monteilii SB3101 NADH:flavin oxidoreductase 363 AHD15905 CP007012 Pseudomonas sp. FGI182 NADH:flavin oxidoreductase 363 AHG40605 CP007014 Pseudomonas syringae CC1557 NADH:flavin oxidoreductase 363 AHH54488 KF827430 Pseudomonas putida xenobiotic reductase 363 AHZ78908 CP007620 Pseudomonas putida xenobiotic reductase A 363 84 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AIB36087 CP007637 Pseudomonas simiae NADH:flavin oxidoreductase 363 AIB41642 CP007638 Pseudomonas sp. WCS374 NADH:flavin oxidoreductase 363 AJA12330 CP009974 Pseudomonas putida S12 NADH:flavin oxidoreductase 363 AJG12026 CP010359 Pseudomonas plecoglossicida NADH:flavin oxidoreductase 363 AJP51761 CP010896 Pseudomonas fluorescens xenobiotic reductase A 363 AJQ50999 CP010979 Pseudomonas putida S13.1.2 NADH:flavin oxidoreductase 363 AJZ96515 CP005975 Pseudomonas fluorescens PICF7 NADH:flavin oxidoreductase 363 AKA86083 CP011117 Pseudomonas fluorescens xenobiotic reductase A 363 AKF45677 CP005969 Pseudomonas syringae pv. syringae B301D NADH:flavin oxidoreductase, Old Yellow Enzyme family 363 AKF51813 CP006256 Pseudomonas syringae pv. syringae HS191 NADH:flavin oxidoreductase, Old Yellow Enzymefamily 349 AKT31150 CP011972 Pseudomonas syringae pv. actinidiae ICMP 18884 NADH:flavin oxidoreductase 363 ALD96243 CP005970 Pseudomonas syringae UMAF0158 oxidoreductase 363 ALU60317 CP013183 Pseudomonas syringae pv. lapsa NADH:flavin oxidoreductase 363 AMA45169 CP013997 Pseudomonas monteilii NADH:flavin oxidoreductase 363 AMS21491 CP014868 Pseudomonas fluorescens NADH:flavin oxidoreductase 363 ANC81490 CP015202 Pseudomonas putida B6 - 2 NADH:flavin oxidoreductase 363 ANI02283 CP015876 Pseudomonas putida SJTE - 1 NADH:flavin oxidoreductase 363 ANI35820 CP011525 Pseudomonas sp. JY - Q NADH:flavin oxidoreductase 363 AOE57546 CP012179 Pseudomonas syringae pv. actinidiae ICMP 18708 NADH:flavin oxidoreductase 363 AOS73499 CP017296 Pseudomonas fluorescens NADH:flavin oxidoreductase 363 BAN53045 AP013070 Pseudomonas putida NBRC 14164 xenobiotic reductase A 369 X T2H290 (UniProt) BAP43811 AP014637 Pseudomonas sp. StFLB209 xenobiotic reductase A 363 CAK16750 CT573326 Pseudomonas entomophila L48 xenobiotic reductase A 363 CRM05256 CVTN01000034 Pseudomonas sp. 31 R 17 NADPH dehydrogenase 363 85 Table A. 6 . Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) CRM18142 CVTU01000039 Pseudomonas sp. 44 R 15 NADPH dehydrogenase 363 CRM29370 CVTJ01000044 Pseudomonas sp. 25 E 4 NADPH dehydrogenase 363 CRM64863 CVTK01000058 Pseudomonas sp. 28 E 9 NADPH dehydrogenase 363 CRM71811 CVTV01000057 Pseudomonas sp. 37 R 15 NADPH dehydrogenase 363 CRM85450 CVTO01000162 Pseudomonas sp. 24 E 13 NADPH dehydrogenase 363 CRN00641 CVTX01000123 Pseudomonas sp. 34 E 7 NADPH dehydrogenase 363 CRN04937 LN865164 Pseudomonas sp. URMO17WK12:I11 NADPH dehydrogenase 363 EEB57589 ABSM01000035 Pseudomonas syringae pv. tomato T1 xenobiotic reductase A 363 EFQ64130 AEAZ01000013 Pseudomonas fluorescens WH6 xenobiotic reductase A 363 EGC00475 AEWE02000142 Pseudomonas sp. TJI - 51 NADH:flavin oxidoreductase 363 EGH10960 AEAE01000300 Pseudomonas syringae pv. morsprunorum str. M302280 xenobiotic reductase A 363 EGH21037 AEAG01000233 Pseudomonas syringae pv. mori str. 301020 xenobiotic reductase A 363 EGH29481 AEAH01000519 Pseudomonas syringae pv. japonica str. M301072 NADH:flavin oxidoreductase/NADH oxidase 363 EGH52142 AEAJ01000375 Pseudomonas syringae Cit 7 NADH:flavin oxidoreductase/NADH oxidase 363 EGH59050 AEAK01000183 Pseudomonas syringae pv. maculicola str. ES4326 xenobiotic reductase A 363 EGH63875 AEAL01000063 Pseudomonas syringae pv. actinidiae str. M302091 xenobiotic reductase A 363 EGH74239 AEAO01000647 Pseudomonas syringae pv. aceris str. M302273 NADH:flavin oxidoreductase/NADH oxidase 363 EGH79809 AEAN01001032 Pseudomonas syringae pv. aptata str. DSM 50252 NADH:flavin oxidoreductase/NADH oxidase 363 EGH87466 AEAF01000431 Pseudomonas syringae pv. lachrymans str. M301315 xenobiotic reductase A 363 EGH90517 AEAP01000239 Pseudomonas syringae pv. tabaci str. ATCC 11528 xenobiotic reductase A 363 86 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) EGH99022 AEAM01000441 Pseudomonas syringae pv. lachrymans str. M302278 xenobiotic reductase A 363 EGI03899 ABZR01000560 Pseudomonas syringae pv. oryzae str. 1_6 xenobiotic reductase A 363 EIK59498 AHPN01000001 Pseudomonas fluorescens SS101 xenobiotic reductase A 363 EIK69323 AHPP01000002 Pseudomonas synxantha BG33R xenobiotic reductase A 363 EIK94421 AJWX01000024 Pseudomonas sp. M47T1 xenobiotic reductase A 363 EKG41581 AKCJ01000141 Pseudomonas syringae pv. avellanae str. ISPaVe013 xenobiotic reductase A 363 EKN47358 AMQP01000035 Pseudomonas viridiflava UASWS0038 XenA 363 EKX82219 AMWJ01000173 Pseudomonas putida CSV86 NADH:flavin oxidoreductase/NADH oxidase 363 ELP99815 AMXK01000040 Pseudomonas syringae BRIP34876 NADH:flavin oxidoreductase 363 ELQ06431 AMXL01000008 Pseudomonas syringae BRIP34881 NADH:flavin oxidoreductase 363 ELQ11581 AMZX01000026 Pseudomonas syringae BRIP39023 NADH:flavin oxidoreductase 363 EMR45280 ALPV02000017 Pseudomonas putida LS46 XenA 363 ENY77786 APBQ01000060 Pseudomonas putida TRO1 NADH:flavin oxidoreductase/NADH oxidase 363 EPJ87167 ATLM01000017 Pseudomonas sp. CFT9 putative xenobiotic reductase 363 EPJ95771 ATLR01000010 Pseudomonas sp. CF149 xenobiotic reductase A 363 EPL10706 ATLQ01000053 Pseudomonas sp. CF150 putative xenobiotic reductase 363 EPM44393 AOKE01000198 Pseudomonas syringae pv. actinidiae ICMP 19098 xenobiotic reductase A 363 EPM69780 AOJT01001410 Pseudomonas syringae pv. actinidiae ICMP 18886 xenobiotic reductase A 363 EPM72486 AOJV01000035 Pseudomonas syringae pv. theae ICMP 3923 xenobiotic reductase A 363 EPM89582 AOJX01000059 Pseudomonas syringae pv. actinidiae ICMP 19068 xenobiotic reductase A 363 EPM98481 AOJZ01000055 Pseudomonas syringae pv. actinidiae ICMP 19104 xenobiotic reductase A 363 87 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) EPN12185 AOKB01000050 Pseudomonas syringae pv. actinidiae ICMP 9855 xenobiotic reductase A 363 EPN15537 AOKC01000356 Pseudomonas syringae pv. actinidiae ICMP 19100 xenobiotic reductase A 363 EPN23908 AOKD01000233 Pseudomonas syringae pv. actinidiae ICMP 19099 xenobiotic reductase A 363 EPN31631 AOKH01000282 Pseudomonas syringae pv. actinidiae ICMP 18883 xenobiotic reductase A 363 EPN40178 AOKI01000532 Pseudomonas syringae pv. actinidiae ICMP 19095 xenobiotic reductase A 363 EPN53783 AOKJ01000053 Pseudomonas syringae pv. actinidiae ICMP 19094 xenobiotic reductase A 363 EPN67578 AOKM01001490 Pseudomonas syringae pv. actinidiae ICMP 19101 xenobiotic reductase A 363 EPN69472 AOKL01000421 Pseudomonas syringae pv. actinidiae ICMP 19079 xenobiotic reductase A 363 EPN76784 AOKQ01000469 Pseudomonas syringae pv. actinidiae ICMP 18801 xenobiotic reductase A 363 EPN80270 AOKN01000101 Pseudomonas syringae pv. actinidiae ICMP 19097 xenobiotic reductase A 363 ERH47346 AVQG01000058 Pseudomonas fluorescens EGD - AQ6 NADH:flavin oxidoreductase 363 ERL01089 AOUR02000063 Pseudomonas putida LF54 NADH:flavin oxidoreductase 363 EST13798 AYJQ01000069 Pseudomonas putida S610 flavin oxidoreductase / NADH oxidase family protein 363 ETK39784 AOJA01000060 Pseudomonas fluorescens FH5 NADH:flavin oxidoreductase 363 EXL33003 JALJ01000005 Pseudomonas syringae pv. syringae str. B301D - R NADH:flavin oxidoreductase, Old Yellow Enzyme family 363 EZI24854 AHIP01000041 Pseudomonas extremaustralis 14 - 3 substr. 14 - 3b NADH:flavin oxidoreductase 363 EZP67781 JFYX01000007 Pseudomonas sp. RIT357 putative xenobiotic reductase 363 GAO92257 BBWG01000029 Pseudomonas syringae pv. actinidiae xenobiotic reductase A 363 KAK39242 AOKO01000022 Pseudomonas syringae pv. actinidiae ICMP 18884 xenobiotic reductase A 363 88 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KCU97759 AOKP01000131 Pseudomonas syringae pv. actinidiae ICMP 9617 xenobiotic reductase A 363 KEY89873 JMIT01000001 Pseudomonas sp. WCS358 NADH:flavin oxidoreductase 363 KEZ27011 AJXI01000065 Pseudomonas syringae pv. tabaci str. 6605 NADH:flavin oxidoreductase 363 KEZ65469 ACHU02000949 Pseudomonas syringae pv. tabaci str. ATCC 11528 NADH:flavin oxidoreductase 363 KFE45277 JPQV01000033 Pseudomonas syringae NADH:flavin oxidoreductase 363 KFF42841 JPRX01000001 Pseudomonas sp. BRG - 100 NADH:flavin oxidoreductase 363 KFF83486 JPNN01000077 Pseudomonas syringae pv. syringae NADH:flavin oxidoreductase 363 KGF65078 JRMB01000001 Pseudomonas lutea NADH:flavin oxidoreductase 363 KGI92449 JRPO01000027 Pseudomonas sp. H2 NADH:flavin oxidoreductase 363 KGK26984 JOJY01000001 Pseudomonas plecoglossicida NADH:flavin oxidoreductase 363 KGK92869 JRRA01000032 Pseudomonas syringae pv. tomato NADH:flavin oxidoreductase 363 KHL75754 JTCJ01000042 Pseudomonas putida NADH:flavin oxidoreductase 363 KIC81698 JWLN01000016 Pseudomonas sp. C5pp NADH:flavin oxidoreductase 363 KIQ06542 JXQW01000001 Pseudomonas fulva NADH:flavin oxidoreductase 363 KIQ11179 JXQT01000019 Pseudomonas simiae NADH:flavin oxidoreductase 363 KIQ36105 JXQO01000012 Pseudomonas viridiflava NADH:flavin oxidoreductase 363 KIR19740 JXCQ01000069 Pseudomonas fluorescens NADPH dehydrogenase 363 KIU52287 JXOG01000163 Pseudomonas putida NADH:flavin oxidoreductase 363 KIY17823 JWJF01000103 Pseudomonas amygdali pv. tabaci NADH:flavin oxidoreductase 363 KKI23303 LAZV01000188 Pseudomonas syringae pv. persicae NADH:flavin oxidoreductase 363 KKO13881 AYRY01000025 Pseudomonas putida KG - 4 NADH:flavin oxidoreductase 363 KKY54047 LCWS01000002 Pseudomonas amygdali pv. tabaci str. ATCC 11528 NADH:flavin oxidoreductase 363 KKY58242 LCWT01000004 Pseudomonas amygdali pv. lachrymans NADH:flavin oxidoreductase 363 KMM79283 JYKX01000004 Pseudomonas deceptionensis NADH:flavin oxidoreductase 363 89 Table A. 6 . Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KMM85239 JYLA01000003 Pseudomonas taetrolens NADH:flavin oxidoreductase 363 KMM98537 JYKZ01000007 Pseudomonas psychrophila NADH:flavin oxidoreductase 363 KMU95130 LFYQ01000021 Pseudomonas putida NADH:flavin oxidoreductase 363 KMY03695 AYTM02000002 Pseudomonas syringae KCTC 12500 NADH:flavin oxidoreductase 363 KMY36699 LDJF01000007 Pseudomonas putida NADH:flavin oxidoreductase 363 KOG06597 LGAR01000010 Pseudomonas syringae pv. aceris NADHflavin oxidoreductase/NADH oxidase 363 KOP54490 JTHM01000050 Pseudomonas coronafaciens pv. porri NADH:flavin oxidoreductase 363 KOP61167 JUEU01000015 Pseudomonas coronafaciens pv. porri NADH:flavin oxidoreductase 363 KOX64919 LBHT01000065 Pseudomonas psychrophila NADH:flavin oxidoreductase 363 KPA91268 JSYZ01000007 Pseudomonas fuscovaginae NADH:flavin oxidoreductase 363 KPA99436 JTBY01000019 Pseudomonas fuscovaginae NADH:flavin oxidoreductase 363 KPB14520 LGKR01000102 Pseudomonas savastanoi xenobiotic reductase A 363 KPB17767 LGKT01000094 Pseudomonas syringae pv. syringae NADHflavin oxidoreductase 363 KPB26448 LGKU01000017 Pseudomonas syringae pv. syringae NADHflavin oxidoreductase 363 KPB51484 LGKZ01000009 Pseudomonas coronafaciens pv. oryzae Xenobiotic reductase A 363 KPB65778 LGLA01000016 Pseudomonas amygdali pv. myricae Xenobiotic reductase A 363 KPB72305 LGLD01000041 Pseudomonas syringae pv. maculicola Xenobiotic reductase A 363 KPB76072 LGLE01000038 Pseudomonas syringae pv. maculicola Xenobiotic reductase A 363 KPB90331 LGLH01000075 Pseudomonas syringae pv. maculicola Xenobiotic reductase A 363 KPB97366 LGLI01000078 Pseudomonas amygdali pv. lachrymans Xenobiotic reductase A 363 KPC09767 LGLJ01000060 Pseudomonas amygdali pv. lachrymans Xenobiotic reductase A 363 KPC20153 LGLK01000033 Pseudomonas amygdali pv. lachrymans Xenobiotic reductase A 363 90 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KPC27726 LGLN01000067 Pseudomonas syringae pv. cilantro Xenobiotic reductase A 363 KPC57944 LGLQ01000039 Pseudomonas amygdali pv. morsprunorum Xenobiotic reductase A 363 KPG76757 LHOY01000006 Pseudomonas libanensis NADH:flavin oxidoreductase 363 KPL65379 JRXH01000017 Pseudomonas viridiflava NADH:flavin oxidoreductase 363 KPM58581 LKKS01000137 Pseudomonas putida NADH:flavin oxidoreductase 363 KPM60167 LKKT01000088 Pseudomonas putida NADH:flavin oxidoreductase 363 KPW12300 LJPO01000074 Pseudomonas syringae pv. atrofaciens NADH:flavin oxidoreductase/NADH oxidase 363 KPW32857 LJPS01000116 Pseudomonas coronafaciens pv. atropurpurea Xenobiotic reductase A 370 X A0A0P9I8N7 (UniProt) KPW36430 LJPR01000084 Pseudomonas syringae pv. apii Xenobiotic reductase A 363 KPW36510 LJPQ01000094 Pseudomonas amygdali Xenobiotic reductase A 363 KPW43710 LJPT01000173 Pseudomonas syringae pv. antirrhini Xenobiotic reductase A 370 X A0A0N8QM38 (UniProt) KPW59084 LJPU01000046 Pseudomonas syringae pv. berberidis Xenobiotic reductase A 363 KPW59535 LJPW01000078 Pseudomonas caricapapayae Xenobiotic reductase A 363 KPW75204 LJPY01000106 Pseudomonas amygdali pv. ciccaronei Xenobiotic reductase A 363 KPW80628 LJPZ01000081 Pseudomonas syringae pv. coriandricola Xenobiotic reductase A 363 KPW86204 LJQB01000027 Pseudomonas congelans NADH:flavin oxidoreductase/NADH oxidase 374 X A0A0P9MDZ2 (UniProt) KPX08211 LJQE01000123 Pseudomonas syringae pv. cunninghamiae Xenobiotic reductase A 374 X A0A0P9R9Q3 (UniProt) KPX11492 LJQC01000036 Pseudomonas syringae pv. coryli Xenobiotic reductase A 363 KPX22183 LJQG01000076 Pseudomonas amygdali pv. dendropanacis Xenobiotic reductase A 363 KPX27260 LJQJ01000579 Pseudomonas ficuserectae xenobiotic reductase A 363 KPX34269 LJQK01000021 Pseudomonas coronafaciens pv. garcae Xenobiotic reductase A 372 X A0A0P9QQ78 (UniProt) 91 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KPX49079 LJQM01000029 Pseudomonas syringae pv. helianthi Xenobiotic reductase A 370 X A0A0N8RPQ9 (UniProt) KPX65160 LJQQ01000034 Pseudomonas syringae pv. lapsa NADH:flavin oxidoreductase/NADH oxidase 363 KPX69394 LJQP01000214 Pseudomonas amygdali pv. lachrymans Xenobiotic reductase A 374 KPX70385 LJQR01000162 Pseudomonas syringae pv. maculicola Xenobiotic reductase A 363 KPX70929 LJQO01000285 Pseudomonas amygdali pv. photiniae Xenobiotic reductase A 363 KPX86489 LJQS01000035 Pseudomonas amygdali pv. mellea Xenobiotic reductase A 363 KPX89519 LJQU01000429 Pseudomonas amygdali pv. mori Xenobiotic reductase A 363 KPX92876 LJQV01000249 Pseudomonas amygdali pv. myricae Xenobiotic reductase A 363 KPX96612 LJQW01000339 Pseudomonas savastanoi pv. nerii Xenobiotic reductase A 363 KPY06286 LJQX01000038 Pseudomonas coronafaciens pv. oryzae Xenobiotic reductase A 363 KPY11776 LJQY01000398 Pseudomonas syringae pv. philadelphi Xenobiotic reductase A 370 KPY23643 LJRA01000112 Pseudomonas coronafaciens pv. porri Xenobiotic reductase A 363 KPY25300 LJRB01000250 Pseudomonas syringae pv. papulans NADH:flavin oxidoreductase 374 X A0A0Q0CDH2 (UniProt) KPY31821 LJRC01000246 Pseudomonas syringae pv. primulae XenA 370 X A0A0P9YD88 (UniProt) KPY42460 LJRF01000212 Pseudomonas syringae pv. ribicola XenA 383 X A0A0P9YU58 (UniProt) KPY51316 LJRE01000090 Pseudomonas syringae pv. rhaphiolepidis Xenobiotic reductase A 363 KPY61375 LJRH01000089 Pseudomonas syringae pv. solidagae Xenobiotic reductase A 374 X 1 A0A0P9ZH38 (UniProt) KPY70038 LJRJ01000190 Pseudomonas savastanoi pv. savastanoi Xenobiotic reductase A 363 KPY74635 LJRK01000019 Pseudomonas syringae pv. syringae NADH:flavin oxidoreductase/NADH oxidase 363 92 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KPY84223 LJRL01000017 Pseudomonas amygdali pv. tabaci Xenobiotic reductase A 374 X A0A0Q0AXV3 (UniProt) KPY86895 LJRM01000070 Pseudomonas syringae pv. tagetis Xenobiotic reductase A 363 KPY92773 LJRO01000438 Pseudomonas tremae Xenobiotic reductase A 363 KPY97352 LJRN01000003 Pseudomonas syringae pv. tomato Xenobiotic reductase A 363 KPZ03551 LJRP01000027 Pseudomonas syringae pv. aptata NADH:flavin oxidoreductase/NADH oxidase 363 KPZ13212 LJRR01000300 Pseudomonas syringae pv. viburni Xenobiotic reductase A 363 KPZ23504 LJRT01000106 Pseudomonas coronafaciens pv. zizaniae Xenobiotic reductase A 363 KPZ26452 LJRS01000052 Pseudomonas viridiflava XenA 363 KPZ30393 LJRU01000298 Pseudomonas syringae pv. theae hypothetical protein 363 KQN59517 LMLL01000014 Pseudomonas sp. Leaf58 NADH:flavin oxidoreductase 363 KQQ66475 LMOA01000002 Pseudomonas sp. Leaf127 NADH:flavin oxidoreductase 363 KRA21433 LMGQ01000006 Pseudomonas sp. Root569 NADH:flavin oxidoreductase 363 KRD00894 LMIY01000006 Pseudomonas sp. Root9 NADH:flavin oxidoreductase 363 KRP45927 JYLH01000006 Pseudomonas libanensis NADH:flavin oxidoreductase 363 KRP46498 JYLJ01000023 Pseudomonas synxantha NADH:flavin oxidoreductase 363 KRP49716 JYLI01000011 Pseudomonas poae NADH:flavin oxidoreductase 363 KRP61178 JYLK01000004 Pseudomonas trivialis NADH:flavin oxidoreductase 363 KRP65217 JYLL01000044 Pseudomonas veronii NADH:flavin oxidoreductase 363 KRP65781 JYLM01000003 Pseudomonas orientalis NADH:flavin oxidoreductase 363 KRP66441 JYLM01000002 Pseudomonas orientalis NADH:flavin oxidoreductase 363 KRP69737 JYLN01000010 Pseudomonas sp. DSM 29164 NADH:flavin oxidoreductase 363 KRP82190 JYLO01000004 Pseudomonas sp. DSM 29167 NADH:flavin oxidoreductase 363 KRP90091 JYLP01000014 Pseudomonas sp. DSM 28142 NADH:flavin oxidoreductase 363 KTB59618 LKEJ01000147 Pseudomonas viridiflava ICMP 13104 NADH:flavin oxidoreductase 363 KTB74906 LKEK01000013 Pseudomonas sp. ICMP 3272 NADH:flavin oxidoreductase 363 93 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KTB76290 LKEO01000137 Pseudomonas syringae ICMP 13102 NADH:flavin oxidoreductase 363 KTB82988 LKEL01000109 Pseudomonas syringae pv. syringae PD2774 NADH:flavin oxidoreductase 363 KTB87381 LKEM01000009 Pseudomonas syringae pv. syringae PD2766 NADH:flavin oxidoreductase 363 KTB93776 LKEP01000009 Pseudomonas syringae ICMP 11293 NADH:flavin oxidoreductase 363 KTB99733 LKGU01000023 Pseudomonas syringae ICMP 11292 NADH:flavin oxidoreductase 363 KTC05025 LKGW01000110 Pseudomonas sp. ICMP 10191 NADH:flavin oxidoreductase 363 KTC07361 LKGV01000012 Pseudomonas syringae ICMP 11168 NADH:flavin oxidoreductase 363 KTC10692 LKGX01000091 Pseudomonas marginalis ICMP 11289 NADH:flavin oxidoreductase 363 KTC28179 LKBK01000086 Pseudomonas sp. ICMP 19500 NADH:flavin oxidoreductase 363 KTC43005 LKBL01000055 Pseudomonas sp. ABAC21 NADH:flavin oxidoreductase 363 KTC46527 LKBQ01000178 Pseudomonas syringae pv. actinidiae ICMP 19497 NADH:flavin oxidoreductase 363 KTC55621 LKCH01000071 Pseudomonas syringae ICMP 19498 NADH:flavin oxidoreductase 363 KTC61416 LKCI01000009 Pseudomonas syringae ICMP 19499 NADH:flavin oxidoreductase 363 KTS93911 LDSM01000036 Pseudomonas parafulva NADH:flavin oxidoreductase 363 KUR49064 LNKZ01000080 Pseudomonas syringae pv. tomato NADPH dehydrogenase 363 KUR49358 LNKY01000017 Pseudomonas syringae pv. tomato NADPH dehydrogenase 363 KWS08243 LIHR01000120 Pseudomonas syringae pv. syringae NADH:flavin oxidoreductase 363 KWS14735 LIHS01000025 Pseudomonas syringae pv. syringae NADH:flavin oxidoreductase 363 KWS21119 LIHT01000075 Pseudomonas syringae pv. syringae NADH:flavin oxidoreductase 363 KWS28871 LIHU01000017 Pseudomonas syringae pv. syringae NADH:flavin oxidoreductase 363 KWS32435 LIHW01000155 Pseudomonas syringae pv. papulans NADH:flavin oxidoreductase 363 KWS37873 LIHV01000184 Pseudomonas syringae pv. rhaphiolepidis NADH:flavin oxidoreductase 363 KWS44549 LIHY01000180 Pseudomonas amygdali pv. myricae NADH:flavin oxidoreductase 363 KWS47726 LIHX01000084 Pseudomonas savastanoi pv. nerii NADH:flavin oxidoreductase 363 94 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KWS54718 LIHZ01000086 Pseudomonas amygdali pv. morsprunorum NADH:flavin oxidoreductase 363 KWS65852 LIIA01000109 Pseudomonas amygdali pv. morsprunorum NADH:flavin oxidoreductase 363 KWS68338 LIIB01000018 Pseudomonas amygdali pv. morsprunorum NADH:flavin oxidoreductase 363 KWS80777 LIID01000003 Pseudomonas amygdali pv. eriobotryae NADH:flavin oxidoreductase 363 KWS85568 LIIE01000028 Pseudomonas amygdali pv. dendropanacis NADH:flavin oxidoreductase 363 KWT12181 LIIJ01000048 Pseudomonas syringae pv. avii NADH:flavin oxidoreductase 363 KWV68804 LCYB01000047 Pseudomonas fluorescens NADPH dehydrogenase 363 KWV71504 LCYC01000059 Pseudomonas fluorescens NADPH dehydrogenase 363 KWV78740 LCYD01000008 Pseudomonas fluorescens NADPH dehydrogenase 371 X A0A109L0F8 (UniProt) KWV89736 LCYA01000018 Pseudomonas fluorescens NADPH dehydrogenase 363 KWW14945 LNNI01000023 Stenotrophomonas rhizophila NADH:flavin oxidoreductase 363 KXK68425 JFBC01000827 Pseudomonas monteilii NADH:flavin oxidoreductase 358 KYC14061 LQXW01000089 Pseudomonas sp. ABFPK NADH:flavin oxidoreductase 363 KZL39595 LAGV01000018 Pseudomonas syringae pv. syringae NADH:flavin oxidoreductase 363 KZO51922 LWDW01000089 Pseudomonas putida NADH:flavin oxidoreductase 363 OAE17352 LVWZ01000003 Pseudomonas brenneri NADH:flavin oxidoreductase 363 OAG90515 LKEH01000025 Pseudomonas viridiflava NADH:flavin oxidoreductase 363 OAH45484 LSTU01000060 Pseudomonas monteilii NADH:flavin oxidoreductase 363 OAI88493 LUCV01000029 Pseudomonas putida NADH:flavin oxidoreductase 363 OAK57773 LVHH01000060 Pseudomonas putida NADH:flavin oxidoreductase 363 OAS18859 LSUZ01000066 Pseudomonas putida NADH:flavin oxidoreductase 363 OBS40910 LYUP01000003 Pseudomonas syringae pv. syringae NADH:flavin oxidoreductase 363 OCW24493 MAUE01000026 Pseudomonas sp. S1E40 NADH:flavin oxidoreductase 363 OCX20361 MDEN01000062 Pseudomonas graminis NADH:flavin oxidoreductase 363 95 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) ODJ90924 MBPF01000034 Pseudomonas viridiflava NADH:flavin oxidoreductase 363 OEC35283 LYSO01000199 Pseudomonas sp. AP42 NADH:flavin oxidoreductase 363 OHC34750 MHZO01000006 Pseudomonadales bacterium RIFCSPLOWO2_12_60_38 NADH:flavin oxidoreductase 363 OHC39719 MHZP01000088 Pseudomonadales bacterium RIFCSPLOWO2_12_FULL_59_450 NADH:flavin oxidoreductase 363 OHW41413 MIFU01000003 Pseudomonas sp. 06C 126 NADH:flavin oxidoreductase 363 OJT21760 MPOR01000060 Pseudomonas sp. FSL W5 - 0203 NADH:flavin oxidoreductase 363 OKO48625 MPBM01000021 Pseudomonas sp. BTN1 NADH:flavin oxidoreductase 363 OKP71313 MRXZ01000008 Pseudomonas fluorescens NADH:flavin oxidoreductase 363 OKS49835 MOMJ01000096 Pseudomonas syringae pv. actinidiae NADH:flavin oxidoreductase 363 OKS50752 MOMK01000092 Pseudomonas syringae pv. actinidiae NADH:flavin oxidoreductase 363 OKS53916 MOML01000088 Pseudomonas syringae pv. actinidiae NADH:flavin oxidoreductase 363 OKS69485 MOMM01000098 Pseudomonas syringae pv. actinidiae NADH:flavin oxidoreductase 363 OKS71121 MOMN01000097 Pseudomonas syringae pv. actinidiae NADH:flavin oxidoreductase 363 OLS64627 MKZO01000004 Pseudomonas sp. GM4FR NADPH dehydrogenase 363 OLY75578 LPNO01000009 Pseudomonas sp. ATCC PTA - 122608 NADH:flavin oxidoreductase 363 ONH40299 MNPU01000025 Pseudomonas gessardii NADH:flavin oxidoreductase 363 ONH50659 MNPW01000014 Pseudomonas cedrina subsp. cedrina NADH:flavin oxidoreductase 363 SCX36276 FMUC01000016 Pseudomonas sp. NFACC25 2,4 - dienoyl - CoA reductase 363 SDE90858 FNBM01000001 Pseudomonas seleniipraecipitans 2,4 - dienoyl - CoA reductase 363 SDG60929 FNCO01000002 Pseudomonas abietaniphila 2,4 - dienoyl - CoA reductase 363 SDH68626 FNCX01000005 Pseudomonas sp. BS3767 2,4 - dienoyl - CoA reductase 363 SDN38921 FNHT01000005 Pseudomonas sp. BS3759 2,4 - dienoyl - CoA reductase 363 SDN46203 FNHM01000008 Pseudomonas syringae 2,4 - dienoyl - CoA reductase 363 SDO43711 FNJH01000001 Pseudomonas congelans 2,4 - dienoyl - CoA reductase 363 SDQ45065 FNKR01000003 Pseudomonas gessardii 2,4 - dienoyl - CoA reductase 363 96 Table A. 6 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SDR29758 FNKM01000002 Pseudomonas grimontii 2,4 - dienoyl - CoA reductase 363 SDW49665 FNNY01000004 Pseudomonas syringae 2,4 - dienoyl - CoA reductase 363 SDZ12916 FNPP01000010 Pseudomonas syringae 2,4 - dienoyl - CoA reductase 363 SEC23539 FNSP01000004 Pseudomonas panacis 2,4 - dienoyl - CoA reductase 363 SEC41669 FNRS01000001 Pseudomonas taetrolens 2,4 - dienoyl - CoA reductase 363 SEE40898 FNTR01000004 Pseudomonas proteolytica 2,4 - dienoyl - CoA reductase 363 SEF02887 FNUD01000002 Pseudomonas deceptionensis 2,4 - dienoyl - CoA reductase 363 SEI50856 FNYJ01000001 Pseudomonas sp. NFR16 2,4 - dienoyl - CoA reductase 363 SEQ15057 FOEO01000004 Pseudomonas sp. NFACC02 2,4 - dienoyl - CoA reductase 363 SES71110 FOHW01000001 Pseudomonas graminis hypothetical protein 363 SFB33384 FOKB01000004 Pseudomonas simiae 2,4 - dienoyl - CoA reductase 363 SFH24896 FOPR01000003 Pseudomonas syringae 2,4 - dienoyl - CoA reductase 363 SFI81213 FOQB01000011 Pseudomonas syringae 2,4 - dienoyl - CoA reductase 363 SFL75637 FOTU01000004 Pseudomonas syringae 2,4 - dienoyl - CoA reductase 363 SFN84275 FOVV01000004 Pseudomonas syringae 2,4 - dienoyl - CoA reductase 363 SFO25780 FOWH01000003 Pseudomonas syringae 2,4 - dienoyl - CoA reductase 363 SFW23264 FPJB01000001 Pseudomonas sp. NFACC10 - 1 2,4 - dienoyl - CoA reductase 363 SFY24249 FPJI01000017 Pseudomonas sp. NFPP02 2,4 - dienoyl - CoA reductase 363 SFY40182 FPJJ01000010 Pseudomonas sp. NFR02 2,4 - dienoyl - CoA reductase 363 1 This sequence was not included in the final primer design sequence set because it did not meet the stricter criteria used in the f inal review. 97 Table A. 7 . FunGene and primer design sequences for xenB Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AAF02539 AF154062 Pseudomonas fluorescens xenobiotic reductase B 349 X Q9RPM1 (UniProt) AAN66545 AE015451 Pseudomonas putida KT2440 Xenobiotic reductase B 349 AAY90620 CP000076 Pseudomonas protegens Pf - 5 xenobiotic reductase B 349 ABA73025 CP000094 Pseudomonas fluorescens Pf0 - 1 putative reductase 349 X Q3KGT1 (UniProt) ABP83887 CP000680 Pseudomonas mendocina ymp NADH:flavin oxidoreductase/NADH oxidase 349 ABQ77120 CP000712 Pseudomonas putida F1 NADH:flavin oxidoreductase/NADH oxidase 349 ABZ00357 CP000926 Pseudomonas putida GB - 1 NADH:flavin oxidoreductase/NADH oxidase 349 ACA71491 CP000949 Pseudomonas putida W619 NADH:flavin oxidoreductase/NADH oxidase 349 ADR58656 CP002290 Pseudomonas putida BIRD - 1 XenB 349 AEA67461 CP002585 Pseudomonas brassicacearum subsp. brassicacearum NFM421 putative reductase 349 AEB57203 CP002620 Pseudomonas mendocina NK - 01 NADH:flavin oxidoreductase/NADH oxidase 349 AEJ14844 CP002870 Pseudomonas putida S16 NADH:flavin oxidoreductase/NADH oxidase 349 AEV61380 CP003150 Pseudomonas fluorescens F113 NADH:flavin oxidoreductase, Old Yellow Enzyme family 349 AFJ54804 CP003041 Pseudomonas fluorescens A506 xenobiotic reductase B 351 AFK70508 CP003588 Pseudomonas putida ND6 NADH:flavin oxidoreductase/NADH oxidase 349 98 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AGA75223 CP003738 Pseudomonas putida HB3267 NADH:flavin oxidoreductase 349 AGD93208 JX406851 uncultured bacterium xenobiotic reductase B 349 X L7Y4X7 (UniProt) AGE24813 CP004045 Pseudomonas poae RE*1 - 1 - 14 xenobiotic reductase B 351 AGL83161 CP003190 Pseudomonas protegens CHA0 N - ethylmaleimide reductase NemA 349 AGN80394 CP005976 Pseudomonas putida H8234 NADH:flavin oxidoreductase 349 AGS77941 KF055345 Pseudomonas putida xenobiotic reductase B 349 AGZ33755 CP003961 Pseudomonas sp. VLB120 NADH:flavin oxidoreductase 349 AHC34158 CP006852 Pseudomonas sp. TKP NADH:flavin oxidoreductase 351 AHC84358 CP006978 Pseudomonas monteilii SB3078 NADH:flavin oxidoreductase 349 AHC89729 CP006979 Pseudomonas monteilii SB3101 NADH:flavin oxidoreductase 349 AHD16245 CP007012 Pseudomonas sp. FGI182 NADH:flavin oxidoreductase 349 AHL32686 CP007410 Pseudomonas brassicacearum NADH:flavin oxidoreductase 349 AHZ69210 CP005960 Pseudomonas mandelii JR - 1 xenobiotic reductase B 349 X A0A024E9G9 (UniProt) AHZ79197 CP007620 Pseudomonas putida NADH:flavin oxidoreductase 349 AIB35284 CP007637 Pseudomonas simiae NADH:flavin oxidoreductase 351 AIB40980 CP007638 Pseudomonas sp. WCS374 NADH:flavin oxidoreductase 351 AIC18479 CP008696 Pseudomonas chlororaphis NADH:flavin oxidoreductase 349 AIG05209 CP008896 Pseudomonas fluorescens NADH:flavin oxidoreductase 351 99 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AIN56779 CP009365 Pseudomonas mosselii SJ10 NADH:flavin oxidoreductase 349 AIS14518 CP009290 Pseudomonas chlororaphis subsp. aurantiaca NADH:flavin oxidoreductase 349 AJA12635 CP009974 Pseudomonas putida S12 NADH:flavin oxidoreductase 349 AJG11724 CP010359 Pseudomonas plecoglossicida NADH:flavin oxidoreductase 349 AJO77016 CP010892 Pseudomonas sp. MRSN12121 NADH:flavin oxidoreductase 349 AJP51036 CP010896 Pseudomonas fluorescens xenobiotic reductase B 351 AJQ50713 CP010979 Pseudomonas putida S13.1.2 NADH:flavin oxidoreductase 349 AJZ92320 CP005975 Pseudomonas fluorescens PICF7 NADH:flavin oxidoreductase 351 AKA26193 CP011110 Pseudomonas chlororaphis NADH:flavin oxidoreductase 349 AKA85083 CP011117 Pseudomonas fluorescens NADH:flavin oxidoreductase, Old Yellow Enzyme family 351 AKK00454 CP011020 Pseudomonas chlororaphis NADH:flavin oxidoreductase 349 AKS05896 CP011507 Pseudomonas trivialis NADH:flavin oxidoreductase 351 AKV07122 CP010945 Pseudomonas fluorescens NCIMB 11764 NADH:flavin oxidoreductase 349 X A0A0K1QN81 (UniProt) ALE89407 CP012676 Pseudomonas sp. L10.10 NADH:flavin oxidoreductase 349 ALI02756 CP012830 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 X A0A0N7H0C5 (UniProt) ALI06144 CP012831 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 ALN20415 CP013124 Pseudomonas mendocina S5.2 alkene reductase 349 ALQ02030 CP012680 Pseudomonas brassicacearum NADH:flavin oxidoreductase 349 100 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AMB80784 CP013861 Pseudomonas fragi alkene reductase 349 AMB85847 CP014135 Pseudomonas agarici alkene reductase 349 AMK29616 CP014343 Pseudomonas putida NADH:flavin oxidoreductase 349 AMN79393 CP014546 Pseudomonas azotoformans alkene reductase 351 AMO78612 CP014158 Pseudomonas citronellolis N - ethylmaleimide reductase 349 AMQ86030 CP014205 Pseudomonas sp. MS586 alkene reductase 349 AMS13897 CP014867 Pseudomonas chlororaphis alkene reductase 349 AMS20787 CP014868 Pseudomonas fluorescens alkene reductase 351 AMT87710 CP014947 Pseudomonas koreensis alkene reductase 349 AMW85394 CP012400 Pseudomonas fluorescens NADH:flavin oxidoreductase, Old Yellow Enzyme family 351 X A0A143GMM1 (UniProt) AMZ72190 CP015225 Pseudomonas fluorescens alkene reductase 349 ANC84610 CP015202 Pseudomonas putida B6 - 2 alkene reductase 349 ANF84946 CP015600 Pseudomonas antarctica NADH:flavin oxidoreductase 351 ANH97038 CP015852 Pseudomonas koreensis alkene reductase 349 X A0A1A9JD21 (UniProt) ANI01920 CP015876 Pseudomonas putida SJTE - 1 alkene reductase 349 ANI17272 CP015878 Pseudomonas citronellolis alkene reductase 349 ANI36191 CP011525 Pseudomonas sp. JY - Q NADH:flavin oxidoreductase 349 ANI53064 CP011566 Pseudomonas sp. DR 5 - 09 NADH:flavin oxidoreductase 349 ANI62042 CP011567 Pseudomonas sp. GR 6 - 02 NADH:flavin oxidoreductase 349 X A0A173JP35 (UniProt) ANJ57850 CP014870 Pseudomonas sp. A3(2016) alkene reductase 349 ANY88072 CP016634 Pseudomonas putida N - ethylmaleimide reductase 349 101 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AOE64385 CP014262 Pseudomonas corrugata alkene reductase 349 X A0A1B3CH67 (UniProt) AOE66706 CP015637 Pseudomonas fluorescens alkene reductase 351 AOE72527 CP015638 Pseudomonas fluorescens alkene reductase 351 AOE78173 CP015639 Pseudomonas fluorescens alkene reductase 351 AOS39011 CP014693 Pseudomonas brassicacearum alkene reductase 349 AOS72848 CP017296 Pseudomonas fluorescens alkene reductase 351 APC15562 1 CP017886 (1521004 - 1522053) Pseudomonas frederiksbergensis Alkene reductase 349 X A0A1J0EHH6 (UniProt) BAN52746 AP013070 Pseudomonas putida NBRC 14164 xenobiotic reductase B 349 BAO60736 AP014522 Pseudomonas protegens Cab57 xenobiotic reductase B 349 BAQ73051 AP014627 Pseudomonas sp. Os17 xenobiotic reductase B 349 BAQ79279 AP014628 Pseudomonas sp. St29 xenobiotic reductase B 349 BAV29269 AP017423 Pseudomonas sp. LAB - 08 NADH:flavin oxidoreductase 349 BAV73391 AP014623 Pseudomonas chlororaphis subsp. aurantiaca xenobiotic reductase B 349 CAY47641 AM181176 Pseudomonas fluorescens SBW25 putative reductase 351 CCH36864 CAIG01000006 Pseudomonas pseudoalcaligenes CECT 5344 NADH:flavin oxidoreductase/NADH oxidase 349 CDF93320 CBLV010000095 Pseudomonas sp. SHC52 NADH:flavin oxidoreductases, Old Yellow Enzyme family 349 X A0A0A1HSW6 (UniProt) CDM41878 HG916826 Pseudomonas pseudoalcaligenes CECT 5344 NADH:flavin oxidoreductase/NADH oxidase 349 CDR92503 LK391695 Pseudomonas pseudoalcaligenes NADH:flavin oxidoreductase/NADH oxidase 349 CEL27966 CDMF01000001 Pseudomonas fluorescens N - ethylmaleimide reductase 349 X A0A0B7D4B5 (UniProt) 102 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) CRI58840 LN847264 Pseudomonas sp. CCOS 191 N - ethylmaleimide reductase 349 CRL48432 LN854573 Pseudomonas sp. URMO17WK12:I11 N - ethylmaleimide reductase 349 X A0A0S4HRC9 (UniProt) CRM01181 CVTI01000009 Pseudomonas sp. 24 R 17 N - ethylmaleimide reductase 351 CRM02824 CVTK01000026 Pseudomonas sp. 28 E 9 N - ethylmaleimide reductase 351 CRM03580 CVTJ01000021 Pseudomonas sp. 25 E 4 N - ethylmaleimide reductase 351 CRM17195 CVTM01000031 Pseudomonas sp. 31 E 5 N - ethylmaleimide reductase 349 CRM27102 CVTL01000022 Pseudomonas sp. 31 E 6 N - ethylmaleimide reductase 349 CRM28733 CVTT01000038 Pseudomonas sp. 58 R 3 N - ethylmaleimide reductase 351 X A0A1B5D928 (UniProt) CRM30878 CVTG01000018 Pseudomonas sp. 24 E 1 N - ethylmaleimide reductase 351 CRM45217 CVTU01000090 Pseudomonas sp. 44 R 15 N - ethylmaleimide reductase 351 CRM47242 CVTP01000021 Pseudomonas sp. 58 R 12 N - ethylmaleimide reductase 351 CRM51727 CVTN01000065 Pseudomonas sp. 31 R 17 N - ethylmaleimide reductase 351 CRM55034 CVTS01000040 Pseudomonas sp. 25 R 14 N - ethylmaleimide reductase 351 CRM56446 CVTO01000117 Pseudomonas sp. 24 E 13 N - ethylmaleimide reductase 351 CRM60541 CVTV01000050 Pseudomonas sp. 37 R 15 N - ethylmaleimide reductase 351 CRM85006 CVTH01000092 Pseudomonas sp. 8 R 14 N - ethylmaleimide reductase 351 CRM94062 CVTW01001110 Pseudomonas sp. 22 E 5 N - ethylmaleimide reductase 351 103 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) CRM95738 CVTW01001144 Pseudomonas sp. 22 E 5 N - ethylmaleimide reductase 349 X A0A1B5FAM7 (UniProt) CRN01384 CVTX01000141 Pseudomonas sp. 34 E 7 N - ethylmaleimide reductase 351 EFQ64781 AEAZ01000007 Pseudomonas fluorescens WH6 NADH:flavin oxidoreductase/NADH oxidase 349 X E2XN25 (UniProt) EGB95234 AEWE02000091 Pseudomonas sp. TJI - 51 NADH:flavin oxidoreductase 349 EIK63596 AHPN01000001 Pseudomonas fluorescens SS101 xenobiotic reductase B 351 EIK68118 AHPP01000002 Pseudomonas synxantha BG33R xenobiotic reductase B 351 EIK70206 AHPO01000001 Pseudomonas fluorescens Q8r1 - 96 xenobiotic reductase B 349 EIK98083 AJWX01000002 Pseudomonas sp. M47T1 xenobiotic reductase B 349 EIM14524 AHOT01000027 Pseudomonas chlororaphis O6 xenobiotic reductase B 349 EJF73535 AKVH01000058 Pseudomonas sp. Ag1 xenobiotic reductase B 351 X J1IR65 (UniProt) EJL01909 AGBM01000001 Pseudomonas fluorescens Q2 - 87 xenobiotic reductase B 349 X J2Y524 (UniProt) EJL08155 AHHJ01000004 Pseudomonas chlororaphis subsp. aureofaciens 30 - 84 xenobiotic reductase B 349 EJL97622 AKJB01000085 Pseudomonas sp. GM102 NADH:flavin oxidoreductase 349 X J2V2F2 (UniProt) EJL98803 AKJV01000061 Pseudomonas sp. GM16 NADH:flavin oxidoreductase 349 X J2MLJ3 (UniProt) EJM14726 AKJS01000132 Pseudomonas sp. GM21 NADH:flavin oxidoreductase 349 EJM19669 AKJT01000028 Pseudomonas sp. GM18 NADH:flavin oxidoreductase 349 X J2P8U7 (UniProt) EJM24862 AKJQ01000056 Pseudomonas sp. GM25 NADH:flavin oxidoreductase 349 EJM45429 AKJR01000019 Pseudomonas sp. GM24 NADH:flavin oxidoreductase 349 EJM64761 AKJK01000070 Pseudomonas sp. GM50 NADH:flavin oxidoreductase 349 X J2SCW5 (UniProt) 104 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) EJM86044 AKJI01000027 Pseudomonas sp. GM60 NADH:flavin oxidoreductase 349 EJM89053 AKJH01000036 Pseudomonas sp. GM67 NADH:flavin oxidoreductase 349 EJN19686 AKJE01000087 Pseudomonas sp. GM79 NADH:flavin oxidoreductase 349 X J3I9U5 (UniProt) EJN30950 AKJF01000061 Pseudomonas sp. GM78 NADH:flavin oxidoreductase 349 EJN32926 AKJD01000056 Pseudomonas sp. GM80 NADH:flavin oxidoreductase 349 X J3IPY2 (UniProt) EJN39968 AKJC01000028 Pseudomonas sp. GM84 NADH:flavin oxidoreductase 349 EJO92734 ALKM01000018 Pseudomonas mendocina DLHK NADH:flavin oxidoreductase 349 EJZ56958 1 CM001561 (1441846 - 1440797) Pseudomonas fluorescens R124 NADH:flavin oxidoreductase 349 X K0WBQ2 (UniProt) EKN47447 AMQP01000035 Pseudomonas viridiflava UASWS0038 NADH:flavin oxidoreductase 349 ELQ17266 AMZW01000070 Pseudomonas fluorescens BRIP34879 xenobiotic reductase B 351 EMI07731 AOGS01000008 Pseudomonas sp. Lz4W NADH:flavin oxidoreductase/NADH oxidase 349 EMR45607 ALPV02000017 Pseudomonas putida LS46 NADH:flavin oxidoreductase 349 ENY79318 APBQ01000013 Pseudomonas putida TRO1 NADH:flavin oxidoreductase/NADH oxidase 349 EPA95962 APIO01000051 Pseudomonas sp. G5(2012) NADH:flavin oxidoreductase 349 EPB93281 ASJX01000087 Pseudomonas plecoglossicida NB2011 NADH:flavin oxidoreductase 349 EPJ83344 ATLM01000040 Pseudomonas sp. CFT9 putative reductase 351 EPJ87362 ATLN01000082 Pseudomonas sp. CFII68 xenobiotic reductase B 349 105 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) EPJ96296 ATLR01000008 Pseudomonas sp. CF149 xenobiotic reductase B 349 EPL13953 ATLQ01000011 Pseudomonas sp. CF150 putative reductase 351 ERH53411 AVQF01000001 Pseudomonas mendocina EGD - AQ5 NADH:flavin oxidoreductase 349 ERH56204 AVQG01000023 Pseudomonas fluorescens EGD - AQ6 NADH:flavin oxidoreductase 351 ERI51154 AVOF01000414 Pseudomonas sp. EGD - AK9 NADH:flavin oxidoreductase 349 ERL02865 AOUR02000011 Pseudomonas putida LF54 NADH:flavin oxidoreductase 349 ERT19414 AXDX02000028 Pseudomonas putida SJ3 NADH:flavin oxidoreductase 349 ESP81685 AYKV01000339 Pseudomonas putida S12 NADH:flavin oxidoreductase 349 ESU56671 AXUO01000023 Pseudomonas mosselii SJ10 NADH:flavin oxidoreductase 349 ESW55952 AKXH02000078 Pseudomonas fluorescens BBc6R8 NADH:flavin oxidoreductase, Old Yellow Enzyme family 351 X V7E6V6 (UniProt) ETD36775 AYUD01000017 Pseudomonas chlororaphis subsp. aurantiaca PB - St2 NADH:flavin oxidoreductase 349 ETF08564 AYMZ01000003 Pseudomonas moraviensis R28 - S NADH:flavin oxidoreductase 349 ETK14997 AOHM01000129 Pseudomonas sp. FH1 putative reductase 351 ETK20007 AOHN01000007 Pseudomonas sp. FH4 xenobiotic reductase B 351 ETK40031 AOJA01000053 Pseudomonas fluorescens FH5 NADH:flavin oxidoreductase 351 ETM67429 ANIR02000002 Pseudomonas sp. M1 NADH:flavin oxidoreductase 349 EUB72080 AKJN02000008 Pseudomonas sp. GM41(2012) 12 - oxophytodienoate reductase 349 EUB88069 AKJP02000001 Pseudomonas sp. GM30 12 - oxophytodienoate reductase 349 X W6VUU6 (UniProt) 106 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) EXF91143 AFOY02000028 Pseudomonas fluorescens HK44 NADH:flavin oxidoreductase 349 X A0A010SIM9 (UniProt) EZI29412 AHIP01000004 Pseudomonas extremaustralis 14 - 3 substr. 14 - 3b NADH:flavin oxidoreductase 351 EZP27080 JFYN01000041 Pseudomonas sp. RIT288 putative reductase 349 X A0A031FTW4 (UniProt) EZP65263 JFYX01000027 Pseudomonas sp. RIT357 putative reductase 351 X A0A031IVN5 (UniProt) KDD66191 AZQQ01000100 Pseudomonas mandelii PD30 NADH:flavin oxidoreductase 349 X A0A059KW36 (UniProt) KER98673 JOVT01000001 Pseudomonas mendocina NADH:flavin oxidoreductase 349 KES20626 JNCW01000027 Pseudomonas sp. AAC NADH:flavin oxidoreductase 349 KEX92696 JOJW01000600 Pseudomonas putida NADH:flavin oxidoreductase 349 KEY85454 JMIT01000004 Pseudomonas sp. WCS358 NADH:flavin oxidoreductase 349 KFB13754 AMCD02000001 Pseudomonas mendocina S5.2 NADH:flavin oxidoreductase 349 KFF43528 JPRX01000001 Pseudomonas sp. BRG - 100 NADH:flavin oxidoreductase 351 KFX68328 AWSQ01000006 Pseudomonas taeanensis MS - 3 NADH:flavin oxidoreductase 349 KGE65450 ASGY01000188 Pseudomonas fluorescens LMG 5329 NADH:flavin oxidoreductase 351 KGF63359 JRMB01000002 Pseudomonas lutea NADH:flavin oxidoreductase 349 KGI93390 JRPO01000017 Pseudomonas sp. H2 NADH:flavin oxidoreductase 349 KGK27307 JOJY01000001 Pseudomonas plecoglossicida NADH:flavin oxidoreductase 349 KGU84577 AUPB01000037 Pseudomonas mediterranea CFBP 5447 NADH:flavin oxidoreductase 349 107 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KHA75274 JSFK01000001 Pseudomonas chlororaphis NADH:flavin oxidoreductase 349 X A0A0A6DL87 (UniProt) KHK62616 JQGJ02000014 Pseudomonas frederiksbergensis NADH:flavin oxidoreductase 349 X A0A0B1Z038 (UniProt) KHL72640 JTCJ01000582 Pseudomonas putida NADH:flavin oxidoreductase 349 KIC82144 JWLN01000013 Pseudomonas sp. C5pp NADH:flavin oxidoreductase 349 KIF57554 JTGH01000018 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 KIF57970 JTGG01000017 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 KIH84784 JXDG01000014 Pseudomonas batumici NADH:flavin oxidoreductase, old yellow enzyme family 349 X A0A0C2EFI2 (UniProt) KII35693 JSAL01000011 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 KII36337 JSAK01000003 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 KIK86261 JTKF01000026 Pseudomonas sp. W15Feb9B NADH:flavin oxidoreductase 349 X A0A0C2R9U2 (UniProt) KIP94325 JXQY01000013 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 KIQ06552 JXQW01000001 Pseudomonas fulva NADH:flavin oxidoreductase 349 X A0A0D0L692 (UniProt) KIQ15503 JXQT01000001 Pseudomonas simiae NADH:flavin oxidoreductase 351 KIQ34136 JXQO01000022 Pseudomonas viridiflava NADH:flavin oxidoreductase 349 KIQ58286 JXNZ01000156 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 KIR14312 JXOE01000052 Pseudomonas fluorescens N - ethylmaleimide reductase 349 KIR20920 JXCQ01000036 Pseudomonas fluorescens N - ethylmaleimide reductase 351 108 Table A. 7 . Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KIY40023 JYKS01000023 Pseudomonas sp. 10 - 1B NADH:flavin oxidoreductase 349 KJH75266 JZRI01000119 Pseudomonas sp. ES3 - 33 NADH:flavin oxidoreductase 349 KJH87387 JYHW01000021 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 KJU76655 JTFL01000133 Pseudomonas pseudoalcaligenes NADH:flavin oxidoreductase 349 KJZ38146 LACE01000008 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 KJZ45649 LACD01000008 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 KJZ51510 LACF01000033 Pseudomonas fluorescens NADH:flavin oxidoreductase 351 KJZ60693 LACG01000012 Pseudomonas fluorescens NADH:flavin oxidoreductase 351 KJZ63606 LACH01000053 Pseudomonas fluorescens NADH:flavin oxidoreductase 349 X A0A0F4V499 (UniProt) KKA05202 JZXC01000030 Pseudomonas kilonensis NADH:flavin oxidoreductase 349 KKO13364 AYRY01000036 Pseudomonas putida KG - 4 NADH:flavin oxidoreductase 349 KKX64351 JTEN01000036 Pseudomonas putida NADH:flavin oxidoreductase 349 KMM80195 JYKX01000002 Pseudomonas deceptionensis NADH:flavin oxidoreductase 349 KMM90164 JYKY01000010 Pseudomonas lundensis NADH:flavin oxidoreductase 349 KMM94028 JYLB01000002 Pseudomonas lini NADH:flavin oxidoreductase 349 KMN01515 JYKZ01000002 Pseudomonas psychrophila NADH:flavin oxidoreductase 349 KMN14846 JYLF01000002 Pseudomonas sp. DSM 29166 NADH:flavin oxidoreductase 349 109 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KMN19599 JYLG01000002 Pseudomonas sp. DSM 28140 NADH:flavin oxidoreductase 349 KMT57316 LFMW01000001 Pseudomonas sp. KG01 NADH:flavin oxidoreductase 351 KMU97538 LFYQ01000002 Pseudomonas putida NADH:flavin oxidoreductase 349 KMY37166 LDJF01000005 Pseudomonas putida NADH:flavin oxidoreductase 349 KNH21892 LFQK01000047 Pseudomonas syringae NADH:flavin oxidoreductase 349 X A0A0L1M0A7 (UniProt) KNH46712 LFQO01000022 Pseudomonas lini NADH:flavin oxidoreductase 349 KNX79790 JHEE01000030 Pseudomonas sp. 250J NADH:flavin oxidoreductase 349 KOX63247 LBHT01000123 Pseudomonas psychrophila NADH:flavin oxidoreductase 349 KOY02903 LIUV01000004 Pseudomonas sp. 655 NADH:flavin oxidoreductase 349 X A0A0M9B180 (UniProt) KPG71943 LHOY01000037 Pseudomonas libanensis NADH:flavin oxidoreductase 351 KPG81095 LHPA01000026 Pseudomonas sp. RIT - PI - o NADH:flavin oxidoreductase 349 KPG89508 LHPC01000077 Pseudomonas sp. RIT - PI - q NADH:flavin oxidoreductase 349 KPG90359 LIGE01000047 Pseudomonas sp. RIT - PI - r NADH:flavin oxidoreductase 349 X A0A0P6RJL3 (UniProt) KPL63051 JRXH01000090 Pseudomonas viridiflava NADH:flavin oxidoreductase 349 KPM61121 LKKS01000116 Pseudomonas putida NADH:flavin oxidoreductase 353 KPM63840 LKKT01000050 Pseudomonas putida NADH:flavin oxidoreductase 349 KPN92360 LIRD01000002 Pseudomonas sp. In5 NADH:flavin oxidoreductase 349 110 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KPW58318 LJPW01000107 Pseudomonas caricapapayae NADH:flavin oxidoreductase 349 KPX45515 LJQM01000119 Pseudomonas syringae pv. helianthi NADH:flavin oxidoreductase 349 KPY47232 LJRF01000107 Pseudomonas syringae pv. ribicola NADH:flavin oxidoreductase 349 KPY82151 LJRM01000168 Pseudomonas syringae pv. tagetis NADH:flavin oxidoreductase 349 KPZ26549 LJRS01000052 Pseudomonas viridiflava NADH:flavin oxidoreductase 349 KQB53953 LLWH01000124 Pseudomonas endophytica NADH:flavin oxidoreductase 349 KQM55866 LMKI01000001 Pseudomonas sp. Leaf15 alkene reductase 351 KQN52963 LMLH01000007 Pseudomonas sp. Leaf48 alkene reductase 349 X A0A0S8ZHZ0 (UniProt) KQO43820 LMMB01000001 Pseudomonas sp. Leaf83 alkene reductase 349 KQT66828 LMQZ01000001 Pseudomonas sp. Leaf434 alkene reductase 349 KQV22463 LMCV01000002 Pseudomonas sp. Root329 alkene reductase 349 KQW33059 LMDO01000014 Pseudomonas sp. Root401 alkene reductase 349 KQZ80538 LMGK01000024 Pseudomonas sp. Root562 alkene reductase 349 KRA08390 LMGQ01000025 Pseudomonas sp. Root569 alkene reductase 351 KRC91733 LMIY01000033 Pseudomonas sp. Root9 alkene reductase 351 KRP46219 JYLH01000005 Pseudomonas libanensis NADH:flavin oxidoreductase 351 KRP52035 JYLI01000005 Pseudomonas poae NADH:flavin oxidoreductase 351 KRP56740 JYLJ01000002 Pseudomonas synxantha NADH:flavin oxidoreductase 351 KRP60927 JYLK01000005 Pseudomonas trivialis NADH:flavin oxidoreductase 351 KRP66147 JYLM01000003 Pseudomonas orientalis NADH:flavin oxidoreductase 351 111 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KRP72691 JYLO01000021 Pseudomonas sp. DSM 29167 NADH:flavin oxidoreductase 351 KRP73729 JYLN01000002 Pseudomonas sp. DSM 29164 NADH:flavin oxidoreductase 351 X A0A0R3AUE0 (UniProt) KRP78024 JYLL01000009 Pseudomonas veronii NADH:flavin oxidoreductase 351 KRP95344 JYLP01000006 Pseudomonas sp. DSM 28142 NADH:flavin oxidoreductase 351 KRV76043 LKJO01000074 Pseudomonas citronellolis NADH:flavin oxidoreductase 349 KRW79973 LKKN01000043 Pseudomonas citronellolis NADH:flavin oxidoreductase 349 KSW24251 LKAX01000007 Pseudomonas sp. ADP NADH:flavin oxidoreductase 349 KTB60191 LKEF01000041 Pseudomonas fluorescens ICMP 11288 NADH:flavin oxidoreductase 351 X A0A0W0HHA2 (UniProt) KTB63178 LKEI01000025 Pseudomonas fluorescens NADH:flavin oxidoreductase 351 KTC10619 LKGX01000091 Pseudomonas marginalis ICMP 11289 NADH:flavin oxidoreductase 349 KTC17938 LKGY01000077 Pseudomonas marginalis ICMP 9505 NADH:flavin oxidoreductase 351 KTC30003 LKBK01000063 Pseudomonas sp. ICMP 19500 NADH:flavin oxidoreductase 351 KTC30506 LKBM01003010 Pseudomonas sp. ABAC61 NADH:flavin oxidoreductase 349 KTC64218 LKBO01000144 Pseudomonas fluorescens ABAC62 NADH:flavin oxidoreductase 351 KUJ91836 LGEP01000163 Pseudomonas sp. 63_8 NADH:flavin oxidoreductase/NADH oxidase 349 KVV02262 LLWI01000080 Pseudomonas sp. TAD18 N - ethylmaleimide reductase 349 KVV03976 LLWJ01000070 Pseudomonas sp. TAA207 N - ethylmaleimide reductase 349 112 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) KWR82998 JWMC01000019 Pseudomonas sp. PI1 alkene reductase 349 KWU51469 LRMR01000007 Pseudomonas fluorescens alkene reductase 351 X A0A0X7K752 (UniProt) KWV70004 LCYB01000031 Pseudomonas fluorescens N - ethylmaleimide reductase 351 KWV73294 LCYC01000048 Pseudomonas fluorescens N - ethylmaleimide reductase 351 KWV82444 LCYD01000004 Pseudomonas fluorescens N - ethylmaleimide reductase 351 KWW17213 LNNI01000011 Stenotrophomonas rhizophila alkene reductase 349 KXK70423 JFBC01000463 Pseudomonas monteilii NADH:flavin oxidoreductase 349 KYC15044 LQXW01000067 Pseudomonas sp. ABFPK alkene reductase 349 KZN18510 LUKJ01000003 Pseudomonas fluorescens alkene reductase 349 X A0A166P6Y4 (UniProt) KZO57322 LWDW01000001 Pseudomonas putida alkene reductase 349 OAB51206 LRSO01000018 Pseudomonas thivervalensis alkene reductase 349 OAE13969 LUXZ01000001 Pseudomonas simiae alkene reductase 351 OAG90436 LKEH01000025 Pseudomonas viridiflava NADH:flavin oxidoreductase 349 OAH44117 LSTU01000067 Pseudomonas monteilii alkene reductase 349 OAJ46344 LKEG01000053 Pseudomonas marginalis NADH:flavin oxidoreductase 351 OAK58391 LVHH01000054 Pseudomonas putida alkene reductase 349 OAS06649 LSUZ01000179 Pseudomonas putida alkene reductase 349 OBP13010 LZYC01000004 Pseudomonas sp. EGD - AKN5 alkene reductase 349 OBZ25625 MAUK01000008 Pseudomonas protegens alkene reductase 349 OBZ31261 MAUL01000003 Pseudomonas protegens alkene reductase 349 OCT21678 MCBI01000076 Pseudomonas putida alkene reductase 349 OCT22860 MCBH01000057 Pseudomonas putida alkene reductase 349 OCT23449 MCBG01000061 Pseudomonas putida alkene reductase 349 113 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) OCT36411 MCBJ01000053 Pseudomonas putida alkene reductase 349 OCW26635 MBDT01000005 Pseudomonas sp. S3E12 alkene reductase 351 OCW30538 MAUE01000001 Pseudomonas sp. S1E40 alkene reductase 351 OCX11190 MDEN01000069 Pseudomonas graminis alkene reductase 349 ODJ90848 MBPF01000034 Pseudomonas viridiflava alkene reductase 349 OEC50081 LYSO01000044 Pseudomonas sp. AP42 alkene reductase 351 OEC69559 LXJP01000033 Pseudomonas sp. AP19 alkene reductase 351 OFJ41211 MKQJ01000035 Pseudomonas koreensis alkene reductase 349 OFS70239 LWMZ01000089 Pseudomonas sp. HMSC08G10 alkene reductase 349 OHC26220 MHZK01000211 Pseudomonadales bacterium RIFCSPHIGHO2_02_FULL_60_43 alkene reductase 349 OHC32763 MHZO01000025 Pseudomonadales bacterium RIFCSPLOWO2_12_60_38 alkene reductase 351 OHC41724 MHZP01000028 Pseudomonadales bacterium RIFCSPLOWO2_12_FULL_59_450 alkene reductase 351 OHR79983 LTNB01000814 Pseudomonas sp. HMSC75E02 alkene reductase 349 OHW38538 MIFU01000097 Pseudomonas sp. 06C 126 alkene reductase 351 OIN11636 MDGK01000015 Pseudomonas extremorientalis alkene reductase 351 OIN44232 MDDQ01000035 Pseudomonas azotoformans alkene reductase 351 OIN52527 MDDR01000026 Pseudomonas costantinii alkene reductase 351 OJT28512 MPOR01000026 Pseudomonas sp. FSL W5 - 0203 alkene reductase 351 OJT48555 MQUK01000011 Pseudomonas moraviensis alkene reductase 349 OKA19855 MPJC01000009 Pseudomonas sp. A4R1.5 alkene reductase 349 OKA23865 MPJD01000018 Pseudomonas sp. A4R1.12 alkene reductase 349 OKK42947 MRUA01000010 Pseudomonas protegens alkene reductase 349 OKK46238 MRTZ01000005 Pseudomonas protegens alkene reductase 349 OKK61234 MRUB01000004 Pseudomonas protegens alkene reductase 349 OKK65769 MRUC01000003 Pseudomonas protegens alkene reductase 349 114 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) OKO46042 MPBM01000048 Pseudomonas sp. BTN1 alkene reductase 351 OKP74931 MRXZ01000001 Pseudomonas fluorescens alkene reductase 351 OLU02012 MSTQ01000009 Pseudomonas reinekei alkene reductase 349 OLY74077 LPNO01000020 Pseudomonas sp. ATCC PTA - 122608 alkene reductase 351 OMQ37512 MNAH01000003 Pseudomonas putida alkene reductase 349 ONH37534 MNPU01000059 Pseudomonas gessardii alkene reductase 351 ONH42989 MNPV01000006 Pseudomonas azotoformans alkene reductase 351 ONH46592 MNPV01000002 Pseudomonas azotoformans alkene reductase 351 ONH56488 MNPW01000002 Pseudomonas cedrina subsp. cedrina alkene reductase 351 SAM32360 FKLB01000037 Pseudomonas sp. 1 R 17 N - ethylmaleimide reductase 351 SBW79344 LT599583 Pseudomonas veronii 1YdBTEX2 N - ethylmaleimide reductase 351 SCW41458 FMTL01000001 Pseudomonas peli 2,4 - dienoyl - CoA reductase 349 SCW83011 FMTR01000011 Pseudomonas sp. NFACC05 - 1 2,4 - dienoyl - CoA reductase 349 SCW93541 FMTK01000013 Pseudomonas sp. NFACC56 - 3 2,4 - dienoyl - CoA reductase 349 SCX20837 FMUC01000003 Pseudomonas sp. NFACC25 2,4 - dienoyl - CoA reductase 351 SCX70584 FMUL01000016 Pseudomonas sp. NFACC32 - 1 2,4 - dienoyl - CoA reductase 349 SCY98277 FMUZ01000023 Pseudomonas sp. NFACC37 - 1 2,4 - dienoyl - CoA reductase 349 SCZ23436 FMVX01000002 Pseudomonas sp. NFIX46 2,4 - dienoyl - CoA reductase 349 SCZ41724 FMWA01000021 Pseudomonas sp. NFACC44 - 2 2,4 - dienoyl - CoA reductase 349 SCZ67909 FMWI01000003 Pseudomonas sp. NFPP17 2,4 - dienoyl - CoA reductase 349 SDA21722 FMWW01000003 Pseudomonas sp. NFPP12 2,4 - dienoyl - CoA reductase 349 SDA53725 FMXG01000001 Pseudomonas sp. NFPP33 2,4 - dienoyl - CoA reductase 349 SDA63826 FMXL01000003 Pseudomonas sp. NFPP15 2,4 - dienoyl - CoA reductase 349 SDA88071 FMXH01000017 Pseudomonas sp. NFACC51 2,4 - dienoyl - CoA reductase 349 SDA90200 FMXJ01000025 Pseudomonas sp. NFACC15 - 1 2,4 - dienoyl - CoA reductase 349 115 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SDB42891 FMXV01000011 Pseudomonas sp. NFACC13 - 1 2,4 - dienoyl - CoA reductase 349 SDB51512 FMYA01000031 Pseudomonas sp. NFACC17 - 2 2,4 - dienoyl - CoA reductase 349 SDB56938 FMXW01000008 Pseudomonas putida 2,4 - dienoyl - CoA reductase 349 SDC59335 FMZQ01000004 Pseudomonas chengduensis 2,4 - dienoyl - CoA reductase 349 SDD13725 FMYX01000006 Pseudomonas guariconensis 2,4 - dienoyl - CoA reductase 349 SDD61794 FNAE01000001 Pseudomonas alcaliphila 2,4 - dienoyl - CoA reductase 349 SDI29899 FNCO01000012 Pseudomonas abietaniphila 2,4 - dienoyl - CoA reductase 349 SDQ02331 FNJJ01000019 Pseudomonas guguanensis 2,4 - dienoyl - CoA reductase 349 SDQ15778 FNKJ01000002 Pseudomonas moorei 2,4 - dienoyl - CoA reductase 349 SDQ53006 FNLA01000002 Pseudomonas lundensis 2,4 - dienoyl - CoA reductase 349 SDQ86728 FNKR01000003 Pseudomonas gessardii 2,4 - dienoyl - CoA reductase 351 SDR39561 FNKM01000002 Pseudomonas grimontii 2,4 - dienoyl - CoA reductase 351 SDU06377 1 LT629796 (677132 - 678181) Pseudomonas mandelii dienoyl - CoA reductase 349 X A0A1H2FG62 (UniProt) SDU07035 1 LT629793 (1929328 - 1930383) Pseudomonas yamanorum dienoyl - CoA reductase 351 X A0A1H2FI93 (UniProt) SDV02422 1 LT629802 (3538029 - 3539084) Pseudomonas mucidolens dienoyl - CoA reductase 351 X A0A1H2NAM4 (UniProt) SDV13310 1 LT629803 (4140557 - 4141606) Pseudomonas vancouverensis N3_9PSED/1 - 349 2,4 - dienoyl - CoA reductase 349 X A0A1H2P6N3 (UniProt) SDY29456 FNNL01000015 Pseudomonas sp. NFACC08 - 1 2,4 - dienoyl - CoA reductase 349 SDY38662 FNOX01000003 Pseudomonas salomonii 2,4 - dienoyl - CoA reductase 351 X SEE22002.1 (NCBI) SDY97247 FNOA01000023 Pseudomonas sp. NFACC14 2,4 - dienoyl - CoA reductase 349 SDZ13240 FNPO01000009 Pseudomonas sp. PDC86 2,4 - dienoyl - CoA reductase 351 SEB38831 FNSU01000001 Pseudomonas marginalis 2,4 - dienoyl - CoA reductase 351 SEC04133 FNSP01000004 Pseudomonas panacis 2,4 - dienoyl - CoA reductase 351 SEC92950 FNTF01000002 Pseudomonas frederiksbergensis 2,4 - dienoyl - CoA reductase 349 SED08754 FNTJ01000002 Pseudomonas saponiphila 2,4 - dienoyl - CoA reductase 349 116 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SED22682 FNRV01000001 Pseudomonas mohnii 2,4 - dienoyl - CoA reductase 349 SED88337 FNTR01000004 Pseudomonas proteolytica 2,4 - dienoyl - CoA reductase 351 SEE19543 FNTY01000002 Pseudomonas migulae 2,4 - dienoyl - CoA reductase 349 SEE22002 FNUA01000002 Pseudomonas palleroniana 2,4 - dienoyl - CoA reductase 351 X A0A1K2CJ75 (UniProt) SEE30391 FNTS01000002 Pseudomonas costantinii 2,4 - dienoyl - CoA reductase 351 SEE31900 FNTT01000002 Pseudomonas kilonensis 2,4 - dienoyl - CoA reductase 349 SEF08692 FNUD01000002 Pseudomonas deceptionensis 2,4 - dienoyl - CoA reductase 349 SEJ13562 FNYJ01000002 Pseudomonas sp. NFR16 2,4 - dienoyl - CoA reductase 349 SEJ70746 FNZL01000028 Pseudomonas sp. NFACC23 - 1 2,4 - dienoyl - CoA reductase 349 SEJ85890 FNYG01000018 Pseudomonas sp. NFACC07 - 1 2,4 - dienoyl - CoA reductase 349 SEJ85899 FNZC01000053 Pseudomonas oleovorans 2,4 - dienoyl - CoA reductase 349 SEK47782 FOAR01000003 Pseudomonas agarici 2,4 - dienoyl - CoA reductase 349 SEL12555 FNZP01000003 Pseudomonas sp. NFPP18 2,4 - dienoyl - CoA reductase 349 SEL40389 FOAQ01000003 Pseudomonas sp. NFPP10 2,4 - dienoyl - CoA reductase 349 SEL93493 FOAE01000004 Pseudomonas sp. NFACC41 - 3 2,4 - dienoyl - CoA reductase 349 SEM36820 FOCB01000001 Pseudomonas sp. ok272 2,4 - dienoyl - CoA reductase 349 SEP06262 FOCU01000013 Pseudomonas sp. ok266 2,4 - dienoyl - CoA reductase 349 SEP82239 FOEQ01000001 Pseudomonas soli 2,4 - dienoyl - CoA reductase 349 SEQ26195 FOEY01000002 Pseudomonas sp. NFPP19 2,4 - dienoyl - CoA reductase 349 SER46881 FOEO01000032 Pseudomonas sp. NFACC02 2,4 - dienoyl - CoA reductase 349 SES68392 FOIH01000001 Pseudomonas sp. NFR09 2,4 - dienoyl - CoA reductase 351 SES82024 FOHW01000002 Pseudomonas graminis 2,4 - dienoyl - CoA reductase 349 SFA61815 FOJR01000003 Pseudomonas sp. NFPP13 2,4 - dienoyl - CoA reductase 349 SFA84395 FOKD01000002 Pseudomonas sp. NFPP24 2,4 - dienoyl - CoA reductase 351 SFB07440 FOKB01000002 Pseudomonas simiae 2,4 - dienoyl - CoA reductase 351 SFB52511 FOKH01000032 Pseudomonas sp. NFIX10 2,4 - dienoyl - CoA reductase 349 117 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SFD28559 FOLS01000021 Pseudomonas citronellolis 2,4 - dienoyl - CoA reductase 349 SFF35749 FONJ01000028 Pseudomonas sp. NFACC06 - 1 2,4 - dienoyl - CoA reductase 349 SFG82382 FOPO01000003 Pseudomonas sp. NFACC45 2,4 - dienoyl - CoA reductase 349 SFH82384 FOPT01000010 Pseudomonas sp. NFACC54 2,4 - dienoyl - CoA reductase 349 SFJ05126 FOQR01000003 Pseudomonas sp. NFPP08 2,4 - dienoyl - CoA reductase 349 SFK51295 FOSO01000009 Pseudomonas sp. NFACC52 2,4 - dienoyl - CoA reductase 349 SFL26404 FOTH01000004 Pseudomonas sp. NFACC46 - 3 2,4 - dienoyl - CoA reductase 349 SFM37229 FOUL01000002 Pseudomonas sp. ok602 2,4 - dienoyl - CoA reductase 349 SFM67152 FOUH01000003 Pseudomonas sp. NFPP05 2,4 - dienoyl - CoA reductase 349 SFO79162 FOWT01000001 Pseudomonas sp. NFPP28 2,4 - dienoyl - CoA reductase 351 SFO92034 FOVS01000046 Pseudomonas sp. NFACC24 - 1 2,4 - dienoyl - CoA reductase 349 SFP15761 FOXK01000001 Pseudomonas toyotomiensis 2,4 - dienoyl - CoA reductase 349 SFP52819 FOXG01000002 Pseudomonas sp. NFPP07 2,4 - dienoyl - CoA reductase 349 SFQ74454 FOYE01000002 Pseudomonas sp. NFIX49 2,4 - dienoyl - CoA reductase 349 X A0A1K2EIT8 (UniProt) SFS25730 FOZO01000003 Pseudomonas sp. NFACC42 - 2 2,4 - dienoyl - CoA reductase 351 SFS51188 FPAL01000002 Pseudomonas sp. NFACC48 - 1 2,4 - dienoyl - CoA reductase 349 SFT80513 FPBG01000003 Pseudomonas sp. NFPP25 2,4 - dienoyl - CoA reductase 349 SFU96342 FPCE01000007 Pseudomonas sp. OV546 2,4 - dienoyl - CoA reductase 351 SFW17210 FPJD01000001 Pseudomonas sp. NFACC19 - 2 2,4 - dienoyl - CoA reductase 349 SFW82632 FPJC01000034 Pseudomonas sp. NFACC04 - 2 2,4 - dienoyl - CoA reductase 349 SFW85607 FPIX01000031 Pseudomonas sp. NFACC16 - 2 2,4 - dienoyl - CoA reductase 349 SFW90883 FPIV01000029 Pseudomonas sp. NFACC09 - 4 2,4 - dienoyl - CoA reductase 349 SFX48187 FPJV01000003 Pseudomonas sp. NFPP09 2,4 - dienoyl - CoA reductase 349 SFX55506 FPKJ01000003 Pseudomonas sp. NFPP16 2,4 - dienoyl - CoA reductase 349 SFX60828 FPJI01000005 Pseudomonas sp. NFPP02 2,4 - dienoyl - CoA reductase 351 SFX62077 FPJJ01000002 Pseudomonas sp. NFR02 2,4 - dienoyl - CoA reductase 351 118 Table A. 7 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) SFX83486 FPKG01000003 Pseudomonas sp. NFPP14 2,4 - dienoyl - CoA reductase 349 SFY08697 FPKC01000017 Pseudomonas sp. NFACC47 - 1 2,4 - dienoyl - CoA reductase 349 SFY10961 FPKL01000019 Pseudomonas sp. NFACC49 - 2 2,4 - dienoyl - CoA reductase 349 X WP_017137250.1 (NCBI) SFY21049 FPKB01000018 Pseudomonas sp. NFACC36 2,4 - dienoyl - CoA reductase 349 SFY34800 FPKK01000014 Pseudomonas sp. NFACC43 2,4 - dienoyl - CoA reductase 349 X WP_034118542.1 (NCBI) SHN11077 FRDA01000008 Pseudomonas asturiensis hypothetical protein 349 WP_017137250.1 1 NZ_JH730961 (275353 - 274298) Pseudomonas fluorescens alkene reductase 351 X WP_069788488.1 (NCBI) WP_034118542.1 1 NZ_JRYA01000006 (787835 - 786780) Pseudomonas fluorescens alkene reductase 351 X WP_074691045.1 (NCBI) WP_078741200.1 1 NZ_MSDF01000022 (24368 - 23313) Pseudomonas fluorescens alkene reductase 351 X WP_078741200.1 (NCBI) 1 These sequences were not on FunGene so were added back in to the primer design sequence set manually . 119 Table A. 8 . FunGene and primer design sequences for xplA Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AAN27917 AF449421 Rhodococcus rhodochrous cytochrome P450 - like protein XplA 552 X AAN27917.1 (NCBI) AAQ03207 AF406983 Rhodococcus sp. YH1 inducible cytochrome P - 450 317 ABC17843 DQ277702 Rhodococcus sp. HS1 cytochrome P450 307 ABC17844 DQ277703 Rhodococcus erythropolis cytochrome P450 308 ABC17845 DQ277704 Rhodococcus erythropolis cytochrome P450 240 ABC17846 DQ277705 Rhodococcus erythropolis cytochrome P450 220 ABC17847 DQ277706 Rhodococcus erythropolis cytochrome P450 306 ABC17848 DQ277707 Rhodococcus sp. HS10 cytochrome P450 276 ABC17850 DQ277709 Rhodococcus sp. DN22 cytochrome P450 236 ABF83916 DQ487126 Rhodococcus sp. HS3 cytochrome P450 302 ABF83917 DQ487127 Rhodococcus sp. HS6 cytochrome P450 307 ABF83919 DQ487129 Rhodococcus sp. HS11 cytochrome P450 302 ABF83920 DQ487130 Rhodococcus sp. HS13 cytochrome P450 294 ABF83921 DQ487131 Rhodococcus sp. HS14 cytochrome P450 294 ABF83922 DQ487132 Rhodococcus sp. HS15 cytochrome P450 307 ABF83923 DQ487133 Rhodococcus sp. HS16 cytochrome P450 279 ABF83925 DQ487135 Rhodococcus sp. HS18 cytochrome P450 306 ABF83926 DQ487136 Rhodococcus sp. HS19 cytochrome P450 310 ABF83927 DQ487137 Rhodococcus sp. AM1 cytochrome P450 289 ACH54163 EU919740 uncultured bacterium XplA 177 ACH54165 EU919742 uncultured bacterium XplA 177 ACH54167 EU919744 uncultured bacterium XplA 179 ACO88874 FJ577793 Microbacterium sp. MA1 XplA 552 X ACO88874.1 (NCBI) ADK69005.2 1 CP002112.1 Gordonia sp. KTR9 flavodoxin - cytochrome P450 XplA (plasmid) 556 X ADK69005.2 (NCBI) AGU90203 KF571922 Williamsia sp. EG1 RDX degrading cytochrome P450 532 X AGU90203.1 (NCBI) AGU90209 KF571925 Rhodococcus sp. EG2 RDX degrading cytochrome P450 121 AGU90210 KF571926 Rhodococcus sp. EG2 RDX degradation cytochrome P450 377 AIA70903 CP007744 Pectobacterium atrosepticum hypothetical protein 395 AIK14324 CP009125 Pectobacterium atrosepticum putative cytochrome P450 395 ALU73572 CP007257 Rhodococcus erythropolis R138 nitric oxide synthase 552 X WP_059016415.1 (NCBI) 120 Table A. 8 Protein Accession No. Nucleotide Accession No. Organism Definition Size (aa) FunGene Sequence Set FunGene Sequence ID (Database) AMU10882 CP015035 Burkholderia cenocepacia hypothetical protein 361 ANY22184 CP016594 Gordonia terrae hypothetical protein 553 CAG74973 BX950851 Pectobacterium atrosepticum SCRI1043 putative cytochrome P450 405 EWD70452 JCML01000015 Klebsiella pneumoniae UCI 18 hypothetical protein 395 EZP26651 JFYN01000042 Pseudomonas sp. RIT288 cytochrome P450n 392 GAB46351 BAFD01000115 Gordonia terrae NBRC 100016 putative cytochrome P450 553 X WP_004023245.1 (NCBI) KFX12950 JQHK01000010 Pectobacterium atrosepticum hypothetical protein 395 KFX25849 JQHO01000001 Pectobacterium atrosepticum hypothetical protein 395 KJF70888 JWIT01000025 Agrobacterium sp. KFB 330 hypothetical protein 396 KKA09922 JZXC01000001 Pseudomonas kilonensis hypothetical protein 337 KMK87888 ALIV01000006 Pectobacterium atrosepticum ICMP 1526 putative cytochrome P450 395 KQP18769 LMMT01000008 Pseudorhodoferax sp. Leaf265 hypothetical protein 399 KQS65700 LMQC01000021 Modestobacter sp. Leaf380 hypothetical protein 396 KRC65821 LMIS01000001 Aeromicrobium sp. Root236 hypothetical protein 396 KVS24837 LPCW01000072 Burkholderia cepacia hypothetical protein 398 KWU25137 LNCR01000033 Burkholderia cenocepacia hypothetical protein 401 OCB11957 MBDY01000043 Mycobacterium vulneris hypothetical protein 551 X WP_065514957.1 (NCBI) SAQ11870 FLAC01000032 Klebsiella oxytoca Biotin biosynthesis cytochrome P450 395 SFO04715 FOUV01000049 Streptomyces sp. cf124 Cytochrome P450 403 1 Th is sequence w as not on FunGene so was added back into the primer design sequence set manually . 121 APPENDIX B Assay Validation 122 All newly designed and currently published primer sets were tested against serial plasmid dilutions on a C1000 Touch thermal cycler with CFX96 real - time platform (Bio - Rad, Hercules , C A) before proceeding with running them on the SmartChip. Reactions were r un in 0.2 mL low profile, white 8 - tube PCR strips ( TLS0851 , Bio - Rad) with optical, ultraclear flat PCR tube 8 - cap strips (TCS 0803 , Bio - Rad). This test was to confirm the primer sets would amplify efficiently . All primers were manufactured by Integrated DNA Technologies (IDT, Coralville, IA) and tested against plasmids containing a partial gene sequence (GenScript, Piscataway, NJ). Gene copy number for the plasmids was calculated as described by Ritalahti et al. (34 ) . Primer and plasmid sequence information is provided in Appendix C. Individual reactions consisted of a total volume of 20 µL with 1X LightCycler 480 SYBR Green I Master (Roche Applied Sciences, Indianapolis, IN), 0.5 µM each of the forward and reverse primers, plasmid DNA, and balance PCR grade water. In the case of 2 for ward and/or reverse primers, 0.25 µM was used of each individual primer for a total of 0.5 µM. Cycling was performed following the SmartChip protocol described by Wang et al. (35) : initial enzyme activation at 95°C for 10 min followed by 40 cycles of denat uration at 95°C for 30 s and annealing at 60°C for 3 0 s. A melting curve analysis was also performed from 55°C to 95°C with reads every 0.5°C for 5 s following amplification. Reactions were run in triplicate for 7 10 - fold serial plasmid dilutions (5·10 1 to 5·10 7 copies/reaction) to use later in generating a standard curve. A reaction blank was included for 123 each trial and most runs also incorporated triplicate negative controls using a plasmid with either an aceA or etnC gene insert ( 5·10 5 copies/reaction ) . The standard curve characteristics for each assay are reported in Table B.1. Eight designed assays were removed from their respective suites based on these results. From the xplA suite, PS1 was eliminated due to low efficiency and PS2 for scattered repl icates. PS13 from the xenA suite and PS12, PS13 and PS18 from the xenB suite were eliminated due to the linear dynamic range not extending to 10 3 copies per reaction. Also, PS3 and PS15 from the xenB suite were removed due to all of the reactions amplifyin g with or after the no template and negative controls. No assays were removed from diaA , nfsI , and pnrB . Three published assays also performed poorly in terms of either having low amplification efficiencies or most of the reactions amplifying with or after the no template and negative controls. These were Pub2 from xenA , Pub2 from xenB , and Pub5 from xplA , and they were therefore not used for the SmartChip runs . 124 Table B. 1 . Characteristics for the Standard Curves of perform well enough to generate a standard curve. Gene Primer Set Log (Linear Range) Slope Y - Intercept Efficiency (%) R 2 Use? diaA 1 7 to 1 - 3.28 38.7 102.0 0.997 Y 3 7 to 1 - 3.15 37.9 107.7 0.996 Y 4 7 to 1 - 3.28 39.2 101.8 0.996 Y nfsI 1 7 to 1 - 3.08 38.8 111.2 0.992 Y 2 7 to 1 - 3.07 38.5 111.5 0.991 Y 3 7 to 1 - 3.06 38.7 112.3 0.989 Y 4 7 to 1 - 3.05 39.2 112.6 0.983 Y 5 6 to 3 - 3.04 40.5 113.5 0.984 Y 6 7 to 1 - 3.09 39.5 110.5 0.982 Y 7 7 to 2 - 4.31 45.0 70.7 0.979 Y 8 7 to 1 - 4.05 43.1 76.5 0.994 Y Pub1 7 to 1 - 3.17 38.1 106.9 0.996 Y Pub2 7 to 1 - 3.50 39.5 93.0 0.992 Y pnrB 1 7 to 2 - 3.09 35.2 110.9 0.997 Y 2 7 to 2 - 3.45 40.7 95.1 0.991 Y 3 7 to 1 - 3.22 38.2 104.5 0.992 Y 4 7 to 1 - 3.21 35.5 104.7 0.998 Y 5 7 to 1 - 3.25 39.3 103.0 0.995 Y Pub1 7 to 1 - 3.25 38.6 103.0 0.998 Y Pub2 7 to 1 - 3.17 38.7 106.9 0.996 Y xenA 1 7 to 3 - 3.62 42.0 89.0 0.998 Y 2 7 to 2 - 3.83 47.3 82.4 0.957 Y 3 7 to 2 - 3.27 43.3 102.1 0.987 Y 4 7 to 1 - 3.37 40.7 98.0 0.993 Y 5 7 to 2 - 3.67 43.1 87.3 0.996 Y 6 7 to 1 - 3.86 43.3 81.5 0.993 Y 7 7 to 2 - 3.53 43.4 91.9 0.987 Y 8 7 to 1 - 3.52 41.2 92.4 0.996 Y 9 7 to 2 - 3.44 40.7 95.2 0.992 Y 10 7 to 1 - 3.43 40.2 95.5 0.996 Y 11 7 to 1 - 3.46 40.0 94.5 0.996 Y 12 7 to 1 - 3.38 39.9 97.5 0.962 Y 13 N/A N/A N/A N/A N/A N 14 7 to 1 - 3.67 43.8 87.1 0.993 Y 15 7 to 1 - 3.58 41.6 90.2 0.997 Y 16 7 to 1 - 3.51 41.2 92.6 0.998 Y 17 7 to 1 - 3.22 38.8 104.4 0.993 Y Pub1 7 to 1 - 4.20 47.3 73.0 0.970 Y Pub2 N/A N/A N/A N/A N/A N xenB 1 7 to 1 - 3.77 44.5 84.2 0.994 Y 2 7 to 1 - 3.06 40.3 112.4 0.982 Y 3 N/A N/A N/A N/A N/A N 4 7 to 1 - 3.59 43.0 89.8 0.994 Y 5 7 to 1 - 3.32 39.2 100.2 0.993 Y 6 7 to 2 - 3.35 46.4 98.9 0.978 Y 7 7 to 1 - 3.42 39.8 96.2 0.993 Y 8 7 to 1 - 3.58 42.2 90.4 0.993 Y 9 7 to 1 - 3.36 42.2 98.6 0.992 Y 10 7 to 2 - 3.61 47.8 89.3 0.964 Y 11 7 to 1 - 3.20 40.5 105.2 0.988 Y 12 N/A N/A N/A N/A N/A N 125 Table B. 1 . Gene Primer Set Log (Linear Range) Slope Y - Intercept Efficiency (%) R 2 Use? xenB 13 N/A N/A N/A N/A N/A N 14 7 to 1 - 3.20 42.3 105.2 0.981 Y 15 N/A N/A N/A N/A N/A N 16 7 to 2 - 2.99 44.9 116.1 0.991 Y 17 7 to 1 - 3.74 45.7 85.2 0.977 Y 18 N/A N/A N/A N/A N/A N 19 7 to 1 - 3.13 41.3 108.7 0.988 Y Pub1 7 to 1 - 3.83 43.9 82.4 0.957 Y Pub2 7 to 5 - 4.91 54.4 59.9 0.997 N xplA 1 7 to 2 - 5.07 49.7 57.5 0.975 N 2 7 to 2 - 3.85 43.7 81.9 0.971 N 3 7 to 3 - 4.13 45.1 74.7 0.989 Y 4 7 to 2 - 4.24 48.7 72.0 0.995 Y 5 7 to 1 - 3.24 41.0 103.7 0.973 Y Pub1 7 to 1 - 3.37 40.6 98.0 0.997 Y Pub2 7 to 1 - 3.55 40.1 91.3 0.989 Y Pub3 7 to 2 - 3.56 45.4 90.9 0.997 Y Pub4 7 to 2 - 3.17 37.9 106.6 0.997 Y Pub5 7 to 1 - 4.38 48.4 69.1 0.997 N Pub6 7 to 1 - 4.18 46.4 73.4 0.995 Y 16S rRNA 16S_rRNA 7 to 3 - 2.97 37.8 117.0 0.996 Y 126 APPENDIX C Primer and Plasmid Sequence Information 127 This appendix provides the sequence information for all primers used in the qPCR experiments in Tables C .1 - C .7. For the designed assays, forward and reverse end positions are specified, and these positions reference the sequence used for the plasmid that t he primer targets. Also, the total number of sequences expected to be amplified by each primer set according to the EcoFunPrimer tool (design) and the SeqFilters and ProbeMatch tools (theoretical) is listed. Table C .8 provides the insert sequence accession and positions for the plasmids used in the qPCR experiments. Table C. 1 . Designed assays for diaA Primer Set Forward End Reverse End Target Plasmid Sequences Hit Design Theo - retical 1 CCACAGACCTCACCAACTCATG 23,689 CACCAGAATCAGGATCTCCAACTG 23,764 CP012395 5 10 CACCTAAGGTTGAAGCAAACATCC CTGAGCCAGGATCTCCAACAG 2 GGAATCTCACCTAAGGTTGAAGCA 23,682 CTGAGTCAGGATCTCCAACAGC 23,763 CP012395 3 7 3 GGTAAATCTCCACAGACCTCACC 2,798,247 TCCCAGCTCTTAGGCAAATCTTC 2,798,374 AP009049 2 3 128 Table C. 2 . Designed assays for nfsI Primer Set Forward End Reverse End Target Plasmid Sequences Hit Design Theo - retical 1 TTCTGTCGCCCTGAAACGC 3,220,727 CCACATGAGAGGCGTCCAG 3,220,922 CP001918 401 722 ACCACGTGAGATGCATCCAG 2 ACTCAACCAAGGCGTTCGAC 3,220,748 TTCGCGCAGAACACCACTAC 3,220,937 CP001918 132 721 TTCGCGCAGAACACTACCAC 3 ACTCGACCAAGGCGTTCG 3,220,746 CACCACCACGTGAGAAGCG 3,220,927 CP001918 59 649 CACCACCACATGAGAGGCA 4 CCCTGAAACGCCACTCTACC 3,220,736 TCGCGCAGAACACTACTACG 3,220,936 CP001918 34 629 5 GCCCTGAAACGCCACTCA 3,220,733 CGCAGAACACCACCACATGA 3,220,933 CP001918 25 646 GCCCTGAAACGCCACTCC 6 AACTGACCGCGGAAGAAGC 3,220,777 CAGCCAGGCGTCATCCATC 3,220,963 CP001918 7 158 7 AACGCCACTCCACGAAGG 3,220,740 CCACATGAGAGGCGTCCAG 3,220,922 CP001918 7 490 8 AAACGCCACTCCACTAAGGC 3,220,741 CACCACCACGTGAGAAGCG 3,220,927 CP001918 4 643 CACCACCACATGAGAGGCA Table C. 3 . Designed assays for pnrB Primer Set Forward End Reverse End Target Plasmid Sequences Hit Design Theo - retical 1 CCACCAAAGCTTACGATGCC 2,716,115 ACGATCACATGCGACGCA 2,716,291 CP010979 27 50 CACAATCACGTGCGAGGCA 2 CGCCGCTACACCACCAAA 321,986 GCGAGGCGTTGAGAATCTTG 322,159 MCBI01000011 6 6 CGCCGCTACACCACCAAG GGGAGGCGTTGAGGATCTTC 3 TACCGTCACCCTGGCCAA 2,716,084 TCTTCGGCGAATTGTAGGCA 2,716,264 CP010979 5 22 CTTCGGCGAGTTGTAGGCA 4 ACCACCAAAGCCTACGATGC 2,716,114 GAGCAGTTTCGGCGTGTTG 2,716,270 CP010979 5 12 CGAGGAGCTTGGGTGTGTTG 5 AAGCGCCGTTACACTACCAA 2,716,102 CACGATCACATGCGAGGCA 2,716,291 CP010979 4 49 129 Table C. 4 . Designed assays for xenA Primer Set Forward End Reverse End Target Plasmid Sequences Hit Design Theo - retical 1 GATGCGGGCTTTGAGTGGA 2,411,045 CTGCCAGAGTCTGCTCGTC 2,411,242 CP004045 99 168 CGATGCGGGCTTTGAATGGA CGGTGAGAGTCTGCTCGTC 2 GCCGGCTTCGAGTGGATAG 1,370,729 CCAGCGTCTGCTCATCGC 1,370,921 AP013070 38 242 TGCCGGCTTTGAATGGATCG GGTGAGGGTCTGCTCATCAC 3 GCCGGCTTTGAATGGATCG 1,370,729 TCGATTCTGCCAGAGTCTGC 1,370,928 AP013070 16 52 TGCCGGCTTTGAATGGATCG CGACTCTGCCAGAGTCTGC 4 GCCGGTTTCGAATGGATCGA 2,411,049 CTCGGCAAGCGTCTGCTC 2,411,245 CP004045 25 115 CCGGTTTCGAGTGGATCGA ACTCGGTCAGGGTCTGCTC 5 GCCGGCTTTGAATGGATCG 1,370,729 TCGACTCTTCCAGGGTCTGC 1,370,928 AP013070 22 93 CCGGCTTCGAGTGGATCG TGGATTCGGTAAGCGTCTGC 6 CGTGATGCAGGCTTTGAGTG 2,411,043 GGTGAGGGTCTGCTCGTC 2,411,242 CP004045 11 137 TGACGCCGGCTTTGAGTG TCGGTGAGGGTTTGTTCGTC 7 GCGGGCTTTGAATGGATCGA 1,370,730 TGCAAGCGTCTGCTCGTC 1,370,923 AP013070 9 78 CCGGCTTCGAATGGATCGA TCTTCGAGGGTTTGCTCGTC 8 CCGTGATGCGGGCTTTGA 2,411,040 GAGGGTCTGCTCGTCACG 2,411,239 CP004045 6 113 GTGAGGGTCTGCTCATCACG 9 CCGGCTTCGAATGGATCGA 178,844 CTCCAGGGTCTGTTCGTCAC 179,035 FOEO01000004 5 91 GCTGGCTTTGAGTGGATCGA GGTCAGGGTCTGTTCGTCAC 10 GCAGGCTTTGAGTGGATCGA 2,411,049 ATCGACTCGGTGAGGGTCTG 2,411,248 CP004045 5 167 TCGACTCTGCAAGCGTCTG 11 GCGGGCTTTGAATGGATCGA 178,844 CCAGCGTTTGCTCATCACG 179,034 FOEO01000004 3 21 CGGGCTTTGAGTGGATCGA CCAGCGTCTGTTCGTCACG 12 GCCGGCTTTGAATGGATCG 2,411,048 GGTGAGGGTCTGCTCGTC 2,411,242 CP004045 3 212 TCTGCCAAAGTCTGCTCGTC 13 GGGCTTCGAGTGGATCGAA 178,845 TCGATTCTGCCAGAGTCTGC 179,042 FOEO01000004 2 109 CCGGCTTTGAGTGGATCGAG GACTCGGTCAGCGTCTGC 14 GCGAAGTCGGGTTTGAATGG 1,370,725 CACTGAGCGTCTGCTCGTC 1,370,923 AP013070 2 12 TGACGCCGGTTTCGAATGG CTTCGAGGGTCTGCTCGTC 15 CCGGCTTCGAGTGGATCG 2,411,048 GATTCGGCGAGGGTCTGC 2,411,247 CP004045 2 85 GACTCGGTCAGCGTCTGC 16 GCAGGCTTCGAGTGGATCG 2,411,048 ATGGATTCGCTGAGGGTCTG 2,411,248 CP004045 2 88 CGCTGGCTTTGAATGGATCG ATGGATTCGGTCAGCGTCTG 17 CCCGTGAAGCAGGCTTTGAA 178,836 CAGCGTCTGTTCGTCACGA 179,033 FOEO01000004 2 31 CGCGAAGCTGGCTTCGAA GAGGGTCTGCTCGTCACGA 130 Table C. 5 . Designed assays for xenB Primer Set Forward End Reverse End Target Plasmid Sequences Hit Design Theo - retical 1 GCTGGACCAGTTCCTGCA 5,114,440 GGTTCTCGTCGCCCATGTC 5,114,585 CP014205 179 330 AGGTTGTCGTCACCCATGTC 2 GCTGGACCAGTTCCTGCA 5,114,440 CAGTTCACGGGCCACGTAG 5,114,620 CP014205 55 245 AGTTCACGGGCGACGTAG 3 GACCAGTTCCTGCAGAGCAG 5,114,446 GGTTGGCGTCACCCATGTC 5,114,585 CP014205 31 298 GTCAGCGTCGCCCATGTC 4 GCTGGACCAGTTCCTGCAA 5,114,441 CGCCCATGTCGTGAGAGTC 5,114,576 CP014205 27 203 TGCTCGATCAGTTCCTGCAA 5 GCTGGACCAGTTCCTGCAA 5,114,441 CAGTTCACGGGCGACGTA 5,114,621 CP014205 18 259 TGCTCGATCAGTTCCTGCAA CAGTTCGCTGGCCACGTA 6 GCTACCTGCTCGACCAGTTC 5,114,435 CGCCCATGTCGTGAGAGTC 5,114,576 CP014205 17 295 GCCCATGTCGTGGGAGTC 7 GCTGGACCAGTTCCTGCA 5,114,440 CGAGCCACGTAGGTGAAGG 5,114,613 CP014205 12 315 CTGGCGACGTAGGTGAAGG 8 CGACCAGTTCCTGCAAAGCA 5,114,445 CCCATGTCGTGGGAATCGG 5,114,574 CP014205 8 279 9 GCTGGACCAGTTCCTGCA 5,114,440 TGAAGGTTTCGGCCGGATTG 5,114,599 CP014205 6 44 10 TGCTCGATCAGTTCCTGCAA 1,276,288 GCCCAGTTCACGGGCTAC 1,276,471 CP002620 5 132 11 GCTGGACCAGTTCCTGCAA 5,114,441 AGGTGAAGGTTTCGGACAGG 5,114,602 CP014205 4 139 TGCTCGATCAGTTCCTGCAA 12 CCTGCTCGACCAGTTTCTGC 1,276,286 GCGACGTAGCCGAAGGTC 1,276,458 CP002620 4 103 13 TGCTCGATCAGTTCCTGCAA 1,276,288 GCTCGCGGGCTACATAACC 1,276,465 CP002620 4 20 TTCACGGGCGACATAGCC 14 GCTGGACCAGTTCCTGCA 5,114,440 CCAGTTCACTGGCGACGTA 5,114,621 CP014205 3 163 15 GACCAGTTCCTGCAGAGCAG 5,114,446 CGACGTAGCCGAAGGTTTCG 5,114,608 CP014205 3 244 CGACGTAGGTGAAGGTCTCG 16 GCTGGACCAGTTCCTGCA 5,114,440 TCACGGGCGACGTAGGTAA 5,114,616 CP014205 2 82 17 CTACCTGGTCGACCAGTTCC 1,276,283 AGCTCACGTGCCACGTAG 1,276,467 CP002620 2 56 18 GCTCGACCAGTTTCTGCAGA 1,276,289 GCTCTCGTGCCACATAGCC 1,276,465 CP002620 2 11 TTCACGGGCGACATAGCC 19 CGACCAGTTCCTGCAAAGCA 5,114,445 CGAGCCACGTAGGTGAAGG 5,114,613 CP014205 2 308 CGACCAGTTCCTCCAAAGCA CTGGCGACGTAGGTGAAGG 131 Table C. 6 . Designed assays for xplA Primer Set Forward End Reverse End Target Plasmid Sequences Hit Design Theo - retical 1 CACGGTCGTGCTCGTCAC 1,932 TCGCTCCTGCGTCTGTCA 2,097 AF449421 23 23 2 AGGTCGCGGTGCTGATCA 98,438 CGAGTTCGACGAGCAGGTTC 98,596 BAFD01000115 3 3 GAGTTGGCGGTGCTCATCA CGAGGTCGACCAGTAGGTTC 3 GCTCAACTCGCAGTCGTGA 326,330 CGCCGAGTTCGATCAGCA 326,496 CP007257 1 1 4 1 CGGTCGTGCTCGTCACC 1,933 GCTCCTGCGTCTGTCACC 2095 AF449421 2 23 5 1 GAAGGTCGCGGTGCTGA 98,435 AGGTTCGCCATCGTCTTGAT 98,582 BAFD01000115 1 2 1 PS4 and PS5 were designed with the NCBI Primer - BLAST tool to replace PS1 and PS2, respectively Table C. 7 . qPCR Primer Sets from Literature. The primer set(s) with the highest theoretical coverage of the relevant primer design seque nce set is marked with an asterisk (*). Gene ID Target Plasmid Sequences Hit Reference nfsI Pub1* ACACGCCGGAAGCCAAA GGTGCATGTCGGCGAAGTA M63808 206 (66) Pub2 TTCAACACGCCAGAAGCCA AGCACTCGGTCACAATCGT M63808 198 (67) pnrB Pub1 CGAGATGACTGAAGAACACCTGAAC GTAGTGACGGCGGCTCTGG AF532912 22 (9) Pub2* CCCACGCTCACGCAAACCC GTACTGCCTTGCTGGGTGCG AF532912 33 (68) xenA Pub1* AGCACTCCAACAAGCGTAC ACCGACACCAGGTCCAACT AF154061 34 (23) Pub2 CACCATTCCCGAGACCAACA TTTAGATTCGGGGGCTGCTG AF154061 5 (30) xenB Pub1* TTGCTGGAAGTGACTGATG TGCCATAGAACAGCTCAGG AF154062 11 (23) Pub2 ACCTTCACCTATGTTGCTCGC CGTTTCTAGCGTTTCATGCGGT AF154062 2 (30) xplA Pub1* CCGAGTGGGCCAAACAGT TCCTCCTCGTCGAGTTCGAT AF449421 23 (69) Pub2* CGACGAGGAGGACATGAGATG GCAGTCGCCTATACCAGGGATA AF449421 23 (70) Pub3* CTACGGACAGGGTGAACTG TCCTGTTGCAGTCGCCTAT AF449421 23 (12) Pub4* GATGACCGCTGCGTCCATCGAT CCTGTTGCAGTCGCCTATACC AF449421 23 (71) Pub5 AGGCTATCGCCACGATTCTG ATCTGTCCCGCACAGGAATG AF449421 _ 1139 - 1520 7 (30) Pub6 CAACAACGCGATCGACATCC TCGAACATCGCCTCCATCAC AF449421 _ 300 - 959 5 (67) 16S rRNA 16S_ rRNA GGGTTGCGCTCGTTGC ATGGYTGTCGTCAGCTCGTG KX858536 N/A (36) 132 Table C. 8 . Plasmid sequence insert information Gene Sequence Accession Start Position End Position Complement? diaA AP009049 2,798,225 2,798,397 No CP012395 23,659 23,831 Yes nfsI CP001918 3,220,709 3,220,982 No M63808 609 939 No pnrB AF532912 364 646 No CP010979 2,716,067 2,716,310 Yes MCBI01000011 321,950 322,193 No xenA AF154061 674 1,232 No AP013070 1,370,703 1,370,949 No CP004045 2,411,022 2,411,268 Yes FOEO01000004 178,817 179,063 Yes xenB AF154062 820 1281 No CP002620 1,276,263 1,276,489 Yes CP014205 5,113,862 5,114,088 Yes xplA AF449421 1,915 2,334 No AF449421 _ 300 - 959 2,065 2,724 No AF449421 _ 1139 - 1520 2,904 3,285 No BAFD01000115 98,417 98,618 No CP007257 326,312 326,513 No 16S rRNA KX858536 992 1 , 051 No etnC DQ264722 362 609 No aceA CP000316 2,189,466 2,189,604 No 133 APPENDIX D SmartChip Data and Standard Curves 134 Table D. 1 . SmartChip Ct values for diaA assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cycle threshold of 28 and a zero value indicates no amplification. Sample PS1 PS2 PS3 Plasmid Standards 10 1 copies per rxn 0.00 0.00 26.97 27.82 26.87 0.00 25.65 26.21 0.00 10 2 copies per rxn 22.83 0.00 25.90 23.19 23.20 24.35 22.76 23.12 25.32 10 3 copies per rxn 19.99 20.06 23.27 20.15 20.06 23.60 19.91 19.68 23.90 10 4 copies per rxn 16.28 16.25 18.34 16.29 16.59 18.26 16.45 16.51 0.00 10 5 copies per rxn 13.05 12.95 15.08 13.04 13.07 0.00 13.01 12.82 15.13 10 6 copies per rxn 10.31 10.17 10.01 10.57 10.46 10.13 10.32 10.62 9.99 10 7 copies per rxn 6.97 6.88 6.05 6.75 6.92 6.31 6.83 6.72 6.04 Shallow Zone Aquifer MW32 (Pre) 0.00 34.90 0.00 0.00 0.00 32.19 0.00 0.00 0.00 MW32 (Post) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 MW62 (Pre) 0.00 0.00 0.00 0.00 0.00 32.46 0.00 34.45 0.00 MW62 (Post) 0.00 0.00 0.00 34.30 34.89 0.00 0.00 0.00 0.00 Sample PS1 PS2 PS3 Perched Zone Aquifer MW48 (Pre) 0.00 0.00 0.00 33.63 0.00 0.00 34.48 0.00 0.00 MW48 (Post) 0.00 0.00 0.00 0.00 0.00 0.00 MW60 R (Pre) 25.47 24.89 0.00 0.00 0.00 0.00 0.00 0.00 0.00 MW60 R (Post) 0.00 0.00 0.00 0.00 0.00 0.00 MW64 (Pre) 0.00 0.00 0.00 33.04 0.00 0.00 0.00 0.00 0.00 Sample PS1 PS2 PS3 MW64 (Post) 34.90 0.00 0.00 0.00 0.00 0.00 MW66 (Pre) 0.00 32.19 34.79 0.00 34.89 0.00 30.93 33.09 0.00 MW66 (Post) 0.00 0.00 0.00 0.00 0.00 33.94 MW67 (Pre) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 MW67 (Post) 0.00 0.00 0.00 0.00 0.00 0.00 Red Cedar River Red Cedar 0.00 0.00 0.00 0.00 0.00 0.00 Sediment MW71 0.00 33.54 0.00 0.00 0.00 34.41 0.00 0.00 31.40 Agricultural Soils MSU Soil E 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 MSU Soil F 0.00 33.70 0.00 33.69 0.00 0.00 0.00 0.00 0.00 KBS T1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 KBS T2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 KBS T3 28.54 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 KBS T4 0.00 0.00 27.72 0.00 0.00 0.00 0.00 0.00 0.00 Negative and No Template Controls aceA 0.00 0.00 0.00 NTC 0.00 0.00 0.00 135 Table D. 2 . SmartChip Ct values for nfsI assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cycle t hreshold of 28 and a zero value indicates no amplification Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 Pub1 Pub2 Plasmid Standards 10 1 copies per rxn 0.00 0.00 0.00 26.45 27.51 27.25 0.00 26.85 27.46 25.69 29.36 0.00 27.19 26.98 27.84 27.13 29.23 0.00 26.41 0.00 29.56 26.88 28.13 25.91 0.00 0.00 29.33 26.73 28.43 0.00 10 2 copies per rxn 0.00 24.43 0.00 26.37 25.21 26.39 27.93 25.06 23.65 24.75 26.56 24.49 24.28 24.57 25.98 26.03 27.97 27.13 24.35 24.99 26.30 0.00 24.33 23.89 24.16 24.22 0.00 23.36 23.98 23.84 10 3 copies per rxn 22.86 20.68 21.12 20.55 20.92 20.91 0.00 20.50 20.92 20.55 22.35 20.88 20.86 20.60 21.09 21.01 22.02 20.50 21.20 20.59 21.84 19.78 20.58 0.00 20.08 0.00 21.75 0.00 21.21 20.87 10 4 copies per rxn 17.29 15.64 15.89 0.00 0.00 16.14 17.60 15.34 23.21 0.00 17.77 16.05 16.30 16.03 16.50 16.53 17.53 16.12 0.00 22.50 17.54 16.07 16.20 16.13 16.50 16.15 17.56 15.95 23.05 22.24 10 5 copies per rxn 13.25 11.94 12.30 11.86 12.46 12.29 13.38 11.88 13.87 12.15 13.22 12.01 12.45 11.93 12.52 12.41 13.16 12.03 13.93 12.42 13.40 11.92 12.54 11.89 12.59 12.36 13.61 11.97 13.93 12.25 10 6 copies per rxn 10.59 9.10 9.39 8.94 9.29 9.47 10.47 9.01 10.54 8.51 10.56 9.22 9.51 9.12 9.41 9.63 10.62 9.23 10.64 8.67 10.35 8.94 9.13 8.89 9.08 9.37 10.25 8.89 10.70 8.84 10 7 copies per rxn 7.09 5.60 5.80 5.67 5.94 6.11 6.89 5.60 6.92 4.72 6.80 5.50 5.96 5.48 5.84 5.98 6.69 5.68 7.12 5.07 6.85 5.72 5.98 5.57 5.96 6.05 6.72 5.83 7.13 5.09 Shallow Zone Aquifer MW32 (Pre) 24.70 0.00 27.99 0.00 31.41 25.40 0.00 0.00 0.00 29.22 24.75 0.00 0.00 0.00 34.07 25.64 24.98 0.00 27.39 28.74 24.80 0.00 27.60 0.00 31.70 25.49 0.00 0.00 0.00 28.90 MW32 (Post) 0.00 30.70 29.83 0.00 27.40 30.60 28.58 33.99 31.57 32.44 0.00 0.00 0.00 0.00 29.88 29.44 0.00 34.13 0.00 34.57 0.00 32.12 29.45 0.00 28.00 28.79 30.18 0.00 0.00 0.00 MW62 (Pre) 22.55 0.00 27.97 0.00 0.00 23.07 0.00 31.74 26.68 30.73 22.41 0.00 29.57 0.00 0.00 23.59 0.00 0.00 26.91 31.99 22.77 28.03 27.96 0.00 0.00 0.00 0.00 0.00 27.12 0.00 MW62 (Post) 26.91 0.00 0.00 0.00 0.00 29.50 31.15 0.00 34.30 0.00 27.65 28.17 0.00 0.00 0.00 32.71 31.04 0.00 28.87 0.00 26.88 0.00 33.66 0.00 0.00 0.00 33.46 0.00 30.33 0.00 Perched Zone Aquifer MW48 (Pre) 34.19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 21.03 0.00 0.00 0.00 31.04 26.74 26.94 0.00 0.00 0.00 20.22 0.00 29.11 0.00 0.00 0.00 25.73 0.00 27.25 30.16 MW48 (Post) 27.29 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 28.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 32.81 0.00 MW60R (Pre) 24.89 0.00 0.00 23.60 24.26 24.07 24.79 22.74 0.00 33.70 33.93 0.00 0.00 0.00 0.00 34.30 0.00 0.00 0.00 0.00 0.00 0.00 30.64 0.00 0.00 0.00 34.21 0.00 0.00 0.00 MW60R (Post) 23.24 0.00 31.27 0.00 0.00 0.00 28.04 0.00 0.00 28.92 23.59 0.00 0.00 0.00 34.49 0.00 28.40 0.00 0.00 30.06 MW64 (Pre) 0.00 28.86 0.00 0.00 31.62 27.26 27.28 0.00 0.00 0.00 0.00 33.89 0.00 0.00 33.64 28.57 29.24 0.00 0.00 0.00 0.00 0.00 27.84 0.00 34.10 0.00 0.00 0.00 28.93 0.00 136 Table D. 2 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 Pub1 Pub2 MW64 (Post) 26.35 29.39 28.20 0.00 31.31 0.00 26.30 32.12 0.00 29.67 0.00 31.33 29.52 0.00 32.89 0.00 26.75 0.00 28.26 32.42 MW66 (Pre) 23.52 26.85 26.15 31.91 32.21 25.04 24.33 0.00 27.51 0.00 24.54 28.55 27.20 0.00 31.19 0.00 25.36 0.00 0.00 30.17 24.47 0.00 0.00 0.00 0.00 0.00 24.86 0.00 26.28 0.00 MW66 (Post) 0.00 31.25 0.00 0.00 0.00 0.00 27.55 0.00 0.00 32.38 0.00 29.28 0.00 0.00 0.00 26.87 26.15 30.86 0.00 0.00 MW67 (Pre) 24.52 29.94 28.07 0.00 0.00 0.00 26.64 0.00 29.07 30.82 25.01 28.55 27.79 0.00 0.00 27.59 26.86 0.00 0.00 30.36 23.99 28.28 0.00 0.00 0.00 0.00 27.11 29.16 0.00 30.27 MW67 (Post) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Red Cedar River Red Cedar 25.10 0.00 28.03 0.00 0.00 0.00 25.93 31.42 27.35 30.64 0.00 27.47 0.00 0.00 0.00 24.47 25.75 29.41 0.00 26.64 Sediment MW71 0.00 32.35 34.23 0.00 33.77 0.00 27.76 0.00 0.00 0.00 29.85 34.08 0.00 0.00 29.70 28.53 27.79 0.00 0.00 31.71 0.00 34.91 0.00 0.00 0.00 34.26 0.00 0.00 0.00 0.00 Agricultural Soils MSU Soil E 25.31 0.00 27.62 32.58 31.46 25.62 0.00 0.00 28.15 29.43 26.19 29.04 29.67 0.00 0.00 27.37 0.00 0.00 0.00 28.99 25.97 0.00 29.24 0.00 33.27 27.54 27.42 0.00 28.14 28.68 MSU Soil F 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 26.90 0.00 0.00 0.00 30.35 25.92 28.92 0.00 0.00 0.00 0.00 27.37 30.35 26.33 27.28 KBS T1 25.47 0.00 28.17 0.00 0.00 0.00 26.58 32.82 27.81 28.69 26.51 27.73 27.64 0.00 33.20 25.79 27.40 29.67 0.00 29.99 24.51 0.00 0.00 0.00 0.00 25.14 0.00 0.00 26.99 28.56 KBS T2 0.00 28.20 0.00 0.00 33.19 26.20 0.00 31.21 28.79 30.37 0.00 26.73 28.10 32.62 30.93 25.66 0.00 34.11 0.00 28.93 0.00 0.00 28.26 0.00 0.00 25.87 27.54 31.01 0.00 31.20 KBS T3 25.75 0.00 0.00 0.00 0.00 25.84 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 26.46 0.00 0.00 28.61 24.97 26.04 27.86 0.00 32.82 26.48 26.24 0.00 27.51 28.34 KBS T4 24.93 0.00 0.00 0.00 33.27 26.22 27.09 31.96 27.88 27.08 0.00 0.00 27.09 0.00 33.27 25.17 25.90 0.00 27.24 30.18 24.52 0.00 0.00 0.00 31.86 25.22 25.42 0.00 26.06 28.38 Negative and No Template Controls aceA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 26.61 0.00 NTC 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 137 Table D. 3 . SmartChip Ct values for pnrB assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cycle threshold of 28 and a zero value indicates no amplification Sample PS1 PS2 PS3 PS4 PS5 Pub1 Pub2 Plasmid Standards 10 1 copies per rxn 28.90 27.35 0.00 0.00 33.44 0.00 0.00 27.85 32.24 33.96 26.72 0.00 0.00 0.00 27.42 29.88 32.03 27.00 30.85 0.00 0.00 10 2 copies per rxn 23.90 27.47 0.00 23.63 26.80 24.60 24.30 23.76 33.56 28.90 22.78 26.62 23.33 23.75 24.24 0.00 29.11 23.85 0.00 23.58 24.02 10 3 copies per rxn 0.00 26.41 25.69 20.19 23.59 19.74 20.60 20.31 26.81 25.02 18.95 0.00 21.02 21.04 20.29 0.00 26.29 19.57 23.71 20.38 20.64 10 4 copies per rxn 17.46 19.51 22.47 17.08 19.69 20.81 19.25 17.44 19.30 22.37 0.00 17.55 19.31 18.98 0.00 19.00 22.67 16.89 20.44 18.72 19.98 10 5 copies per rxn 12.35 15.15 17.05 11.61 15.40 13.62 13.86 12.38 0.00 17.48 12.03 15.41 13.81 13.97 12.31 15.28 17.85 12.31 15.54 13.53 13.70 10 6 copies per rxn 9.19 10.58 14.34 9.05 12.49 10.07 10.00 9.37 10.82 14.50 9.02 12.56 10.05 10.13 8.81 10.74 14.33 0.00 12.36 9.94 10.22 10 7 copies per rxn 6.03 5.92 10.73 5.80 9.27 6.32 6.34 5.72 6.12 10.93 5.71 9.32 6.43 6.54 5.85 5.94 10.91 5.53 9.08 6.23 6.30 Shallow Zone Aquifer MW32 (Pre) 26.13 24.59 26.28 26.20 26.90 27.53 21.73 25.47 25.13 25.77 0.00 26.58 27.11 21.50 0.00 24.97 26.38 24.81 0.00 27.89 22.23 MW32 (Post) 29.14 34.87 25.76 29.17 0.00 0.00 0.00 0.00 28.06 26.01 28.01 0.00 0.00 23.87 29.05 28.10 25.85 27.60 33.67 0.00 23.61 MW62 (Pre) 25.78 23.52 22.00 0.00 0.00 25.96 0.00 25.99 23.50 21.68 0.00 0.00 25.72 0.00 26.14 23.64 22.01 24.00 0.00 26.50 0.00 MW62 (Post) 30.82 26.41 32.93 0.00 0.00 0.00 0.00 28.99 24.10 32.51 33.64 0.00 0.00 0.00 27.25 24.22 30.82 27.36 28.98 0.00 0.00 Perched Zone Aquifer MW48 (Pre) 32.63 32.72 0.00 33.70 0.00 0.00 29.15 0.00 24.12 0.00 25.73 0.00 28.66 22.16 0.00 22.57 22.62 25.37 29.08 27.67 21.95 MW48 (Post) 31.20 24.90 33.63 28.80 34.62 0.00 25.48 31.95 25.71 0.00 28.96 0.00 32.91 26.38 MW60R (Pre) 28.55 21.55 26.69 26.73 29.36 0.00 23.16 0.00 31.96 33.95 0.00 0.00 0.00 0.00 0.00 24.26 0.00 29.16 0.00 0.00 28.95 MW60R (Post) 0.00 24.19 0.00 0.00 0.00 0.00 24.12 25.90 27.19 0.00 0.00 28.24 27.70 24.54 MW64 (Pre) 0.00 0.00 0.00 0.00 0.00 0.00 24.25 0.00 0.00 0.00 29.79 0.00 0.00 27.27 27.38 26.49 26.06 28.48 0.00 0.00 25.18 138 Table D. 3 Sample PS1 PS2 PS3 PS4 PS5 Pub1 Pub2 MW64 (Post) 0.00 21.06 26.90 0.00 29.73 0.00 24.58 0.00 21.07 26.21 0.00 0.00 28.12 24.78 MW66 (Pre) 0.00 25.96 25.70 25.74 27.82 0.00 22.70 26.23 25.57 25.23 0.00 27.45 0.00 23.13 0.00 25.48 26.38 0.00 28.25 0.00 24.30 MW66 (Post) 28.66 23.98 0.00 0.00 31.14 0.00 0.00 27.06 22.28 0.00 26.89 0.00 27.51 23.98 MW67 (Pre) 0.00 27.82 0.00 0.00 28.33 29.30 23.88 0.00 25.40 26.84 0.00 0.00 28.13 23.25 0.00 0.00 0.00 0.00 27.89 28.64 23.38 MW67 (Post) 33.75 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 29.59 0.00 0.00 0.00 Red Cedar River Red Cedar 0.00 0.00 26.43 25.85 0.00 0.00 22.47 25.22 0.00 26.91 24.88 25.62 0.00 21.76 Sediment MW71 0.00 0.00 0.00 34.42 0.00 31.49 0.00 0.00 33.14 0.00 28.14 31.41 31.59 27.26 0.00 0.00 0.00 34.54 0.00 31.44 27.19 Agricultural Soils MSU Soil E 0.00 29.94 27.54 0.00 28.70 0.00 24.69 0.00 28.07 0.00 28.05 29.23 0.00 0.00 0.00 27.72 0.00 0.00 0.00 28.81 25.29 MSU Soil F 0.00 28.64 0.00 0.00 29.72 0.00 24.67 0.00 0.00 0.00 0.00 30.19 28.81 24.67 27.21 25.98 0.00 0.00 29.34 27.04 25.11 KBS T1 27.15 29.60 0.00 0.00 29.04 29.21 23.65 28.21 28.88 0.00 28.82 29.59 28.04 24.76 0.00 0.00 26.19 0.00 28.47 0.00 0.00 KBS T2 0.00 28.08 0.00 0.00 0.00 0.00 24.37 0.00 0.00 26.12 26.31 29.36 0.00 0.00 0.00 28.06 27.37 26.80 0.00 27.42 24.24 KBS T3 26.73 0.00 0.00 0.00 29.72 25.37 24.22 26.47 29.12 0.00 26.00 28.27 27.71 25.22 26.21 0.00 0.00 25.77 26.84 25.65 23.52 KBS T4 27.55 30.03 0.00 27.99 28.84 25.82 24.58 0.00 28.89 0.00 0.00 0.00 26.63 24.30 0.00 27.94 26.23 27.22 28.08 0.00 24.48 Negative and No Template Controls aceA 0.00 0.00 0.00 0.00 0.00 0.00 34.78 NTC 28.45 31.30 0.00 0.00 0.00 0.00 0.00 139 Table D. 4 . SmartChip Ct values for xenA assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standar d curve or is above the cycle threshold of 28 and a zero value indicates no amplification. Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 Plasmid Standards 10 1 copies per rxn 0.00 0.00 0.00 33.90 27.42 0.00 31.28 28.57 27.88 32.26 32.08 27.69 0.00 0.00 29.64 27.13 29.64 27.48 0.00 0.00 0.00 28.17 27.91 28.07 29.14 0.00 0.00 0.00 27.00 0.00 28.19 0.00 28.28 0.00 28.66 0.00 33.65 0.00 0.00 27.86 0.00 0.00 0.00 0.00 0.00 27.18 27.81 28.30 0.00 0.00 29.53 10 2 copies per rxn 27.08 33.43 31.66 33.32 24.31 25.05 27.14 28.43 28.03 26.03 23.73 24.98 27.59 25.59 25.73 24.34 26.37 27.93 31.75 30.58 28.11 24.35 24.96 0.00 27.63 26.86 24.66 23.51 0.00 0.00 26.35 25.38 24.90 26.48 26.65 30.22 0.00 30.74 0.00 0.00 27.25 25.67 25.46 24.93 24.48 24.84 27.31 25.34 25.95 23.89 0.00 10 3 copies per rxn 21.72 0.00 0.00 26.45 27.71 20.91 0.00 23.39 21.09 21.96 20.78 21.22 29.76 22.67 22.41 20.55 20.31 21.93 34.18 0.00 26.36 27.47 20.97 0.00 23.04 21.30 21.58 20.22 21.12 34.31 22.10 22.16 20.57 19.92 21.77 0.00 0.00 0.00 0.00 21.18 29.74 23.30 21.67 22.12 20.35 21.30 28.60 21.65 22.57 20.94 20.49 10 4 copies per rxn 17.59 22.34 22.99 22.08 16.93 16.88 18.86 18.76 18.10 17.43 17.64 16.96 18.33 17.52 18.03 18.06 15.07 17.42 22.39 22.86 21.83 16.68 16.84 18.61 18.78 18.17 17.38 17.44 16.69 18.18 17.52 17.80 17.66 14.87 17.55 22.37 23.10 21.82 16.93 16.80 18.73 18.80 0.00 17.59 17.81 16.85 18.35 17.56 18.08 17.98 14.86 10 5 copies per rxn 14.47 18.83 19.02 18.74 13.07 13.81 14.88 15.56 13.66 14.21 12.84 13.75 14.53 14.30 14.85 12.97 11.33 14.57 18.81 19.21 19.05 13.03 13.89 14.91 15.74 13.55 14.31 12.73 13.85 14.48 14.44 14.95 12.94 11.24 14.40 19.17 19.31 18.68 13.23 13.76 15.00 15.62 13.69 14.26 12.57 13.75 14.51 14.35 14.84 12.88 11.41 10 6 copies per rxn 10.96 15.90 16.23 15.83 10.29 10.24 11.90 12.30 10.46 10.82 9.62 10.33 11.54 10.95 11.56 9.79 7.73 10.82 15.89 16.24 15.40 10.27 10.26 11.77 12.19 10.45 10.74 9.55 10.29 11.64 10.91 11.40 9.75 7.49 11.09 15.99 16.16 15.32 10.41 10.63 11.94 12.18 10.61 10.86 9.73 10.37 11.55 10.94 11.48 9.93 7.86 10 7 copies per rxn 7.42 12.04 12.38 11.64 6.28 6.40 7.88 8.44 6.80 6.96 6.08 6.58 7.57 7.10 7.68 6.18 0.00 7.16 11.98 12.30 11.43 6.40 6.49 7.91 8.38 6.76 6.99 6.00 6.43 7.66 7.04 7.56 6.13 0.00 7.29 12.20 12.35 11.80 6.45 6.41 7.94 8.52 6.92 7.07 6.33 6.48 7.61 7.12 7.66 6.44 0.00 Shallow Zone Aquifer MW32 (Pre) 27.03 25.04 32.90 22.90 26.35 24.89 25.34 0.00 24.83 23.91 25.84 23.63 25.05 22.17 23.66 0.00 0.00 25.88 25.13 34.45 23.38 26.99 24.21 24.70 26.07 25.64 24.65 25.75 22.97 25.15 22.16 23.97 0.00 33.91 25.63 25.54 0.00 22.77 25.43 23.85 24.83 0.00 24.12 24.87 25.86 23.57 24.59 22.19 22.78 0.00 31.03 MW32 (Post) 0.00 0.00 0.00 24.17 28.87 27.40 28.78 30.33 29.32 31.65 0.00 0.00 30.47 0.00 28.62 0.00 0.00 0.00 0.00 32.27 23.91 30.09 28.22 30.19 0.00 0.00 0.00 28.41 33.27 29.52 26.04 27.30 32.99 0.00 32.56 0.00 34.91 24.29 0.00 28.72 29.06 30.26 31.18 0.00 0.00 0.00 31.64 26.27 0.00 32.99 0.00 MW62 (Pre) 26.37 29.13 0.00 23.54 0.00 27.01 27.49 24.59 28.26 0.00 27.11 0.00 0.00 21.67 0.00 0.00 34.78 25.33 0.00 30.57 23.21 0.00 25.97 26.88 24.95 0.00 25.95 0.00 27.93 28.27 21.80 0.00 0.00 28.64 25.82 27.43 31.20 23.40 26.71 26.25 27.59 25.02 0.00 0.00 27.76 0.00 27.42 22.39 0.00 28.47 0.00 MW62 (Post) 0.00 32.45 0.00 26.54 28.43 25.65 0.00 29.10 32.62 29.69 27.34 31.09 27.46 24.79 29.70 34.47 31.99 30.26 0.00 32.91 26.59 27.00 25.10 29.92 29.02 29.91 0.00 28.07 27.71 26.74 25.15 28.56 0.00 0.00 0.00 31.90 0.00 26.20 25.85 24.78 27.97 30.37 28.66 31.90 26.51 26.34 26.36 24.55 29.55 0.00 0.00 140 Table D. 4 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 Perched Zone Aquifer MW48 (Pre) 0.00 0.00 0.00 0.00 34.57 0.00 32.62 0.00 0.00 33.52 0.00 32.29 0.00 0.00 30.76 0.00 0.00 27.17 22.41 0.00 20.15 20.44 22.58 23.34 0.00 23.06 24.42 20.90 21.51 21.59 22.00 0.00 0.00 0.00 27.08 0.00 0.00 18.78 19.02 20.70 21.91 26.10 22.08 23.91 19.87 20.33 19.90 21.78 0.00 34.20 28.26 MW48 (Post) 0.00 0.00 0.00 0.00 27.54 31.52 0.00 0.00 33.19 33.14 26.90 0.00 31.83 0.00 0.00 0.00 0.00 34.10 0.00 0.00 29.27 29.20 33.87 0.00 30.48 30.95 29.29 27.88 31.26 0.00 26.27 28.62 33.27 30.96 MW60R (Pre) 30.51 31.31 0.00 25.68 27.07 26.51 30.32 26.03 27.50 27.42 26.36 31.34 26.57 23.45 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 34.16 0.00 34.53 0.00 0.00 0.00 0.00 29.32 30.68 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 MW60R (Post) 29.80 30.55 0.00 26.11 0.00 24.66 29.52 27.16 0.00 0.00 27.64 27.11 0.00 23.80 26.70 31.52 29.87 28.41 0.00 0.00 25.60 0.00 25.44 28.69 26.71 30.38 28.02 28.13 26.24 0.00 23.88 26.93 28.56 0.00 MW64 (Pre) 0.00 28.31 0.00 24.29 27.92 28.08 0.00 26.56 25.60 27.15 25.96 27.50 26.99 22.74 26.43 30.13 0.00 30.92 0.00 0.00 26.67 29.46 29.85 0.00 28.02 32.71 31.21 29.35 28.62 29.07 24.90 28.44 34.44 0.00 31.53 29.57 0.00 25.75 29.34 26.98 28.52 27.60 27.49 29.24 27.34 29.80 28.45 24.52 26.65 31.38 30.41 MW64 (Post) 28.72 24.94 0.00 24.78 24.91 22.13 26.56 0.00 27.76 0.00 26.24 26.83 23.91 23.69 25.88 0.00 0.00 0.00 24.30 34.96 25.03 25.51 22.47 28.03 0.00 0.00 30.40 26.08 26.28 24.10 23.87 26.65 0.00 0.00 MW66 (Pre) 26.85 24.68 0.00 22.56 25.46 24.89 27.12 0.00 24.96 26.33 23.84 0.00 24.35 21.18 0.00 0.00 0.00 0.00 24.16 0.00 22.45 24.61 24.93 26.13 24.69 25.52 26.12 23.99 24.01 24.07 21.55 24.86 0.00 28.20 0.00 26.66 0.00 24.03 26.63 25.93 28.55 24.90 0.00 0.00 24.38 25.89 27.13 21.99 25.93 29.88 30.48 MW66 (Post) 28.20 28.38 32.13 25.33 29.13 25.19 28.04 28.02 0.00 29.89 27.17 30.51 26.70 23.74 0.00 0.00 0.00 27.87 0.00 33.00 25.06 26.38 24.38 0.00 25.92 26.91 0.00 26.48 25.69 26.73 22.73 0.00 31.65 0.00 MW67 (Pre) 25.51 27.28 0.00 25.65 28.62 0.00 26.09 24.22 0.00 29.94 0.00 22.67 26.30 22.89 25.40 30.74 0.00 0.00 27.04 0.00 25.01 0.00 23.64 25.98 23.32 23.93 29.57 0.00 22.12 27.23 22.53 0.00 0.00 30.23 26.35 26.27 0.00 24.87 27.93 23.62 24.88 22.88 23.52 32.32 23.35 21.75 29.02 23.23 0.00 0.00 0.00 MW67 (Post) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 33.38 0.00 0.00 0.00 30.11 0.00 0.00 0.00 0.00 0.00 0.00 32.14 0.00 0.00 34.76 0.00 0.00 33.94 0.00 0.00 0.00 Red Cedar River Red Cedar 27.59 27.02 0.00 22.56 24.99 26.35 28.02 25.74 27.97 0.00 25.18 26.82 28.11 22.18 0.00 28.66 0.00 28.47 26.68 0.00 22.27 25.16 25.43 28.46 25.38 28.11 27.61 0.00 27.30 27.81 21.86 25.51 28.21 30.26 Sediment MW71 31.82 34.25 31.30 0.00 0.00 0.00 0.00 0.00 30.97 0.00 0.00 0.00 0.00 30.33 29.31 0.00 0.00 28.15 31.19 0.00 0.00 0.00 0.00 0.00 34.15 0.00 0.00 0.00 27.20 31.36 29.43 28.67 0.00 0.00 28.25 29.60 0.00 29.18 30.67 0.00 30.32 31.67 28.16 0.00 0.00 0.00 31.52 29.63 27.21 0.00 0.00 141 Table D. 4 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 Agricultural Soil MSU Soil E 27.39 26.70 32.62 24.18 26.94 0.00 27.31 25.50 28.37 0.00 0.00 26.55 0.00 21.83 0.00 29.65 0.00 0.00 28.28 0.00 25.24 0.00 27.22 28.44 26.51 0.00 30.02 0.00 0.00 0.00 23.20 0.00 0.00 0.00 0.00 0.00 34.50 24.84 27.78 26.90 0.00 27.21 29.28 29.30 0.00 26.13 27.28 22.91 27.51 0.00 34.35 MSU Soil F 0.00 0.00 33.61 0.00 0.00 26.99 28.22 25.57 0.00 28.01 27.12 0.00 26.67 22.05 26.14 29.80 0.00 0.00 27.58 0.00 25.49 29.45 27.88 0.00 26.74 29.77 30.28 28.53 0.00 0.00 0.00 27.74 0.00 0.00 29.58 28.20 0.00 0.00 29.10 28.69 0.00 27.03 29.15 29.76 0.00 0.00 0.00 23.91 27.51 0.00 29.33 KBS T1 27.18 0.00 31.64 24.25 26.66 25.87 27.19 25.43 0.00 27.49 25.54 27.52 26.37 21.55 0.00 28.28 0.00 28.07 28.95 30.90 25.23 0.00 26.75 28.47 0.00 0.00 28.61 0.00 0.00 0.00 22.89 0.00 29.57 0.00 28.26 27.16 0.00 23.99 0.00 0.00 0.00 25.62 27.70 27.58 26.86 23.22 0.00 22.22 0.00 0.00 28.83 KBS T2 0.00 26.79 0.00 25.11 28.31 27.00 28.68 26.73 28.47 28.86 0.00 0.00 26.18 22.43 0.00 0.00 0.00 28.58 0.00 0.00 24.34 26.53 26.45 28.28 25.08 28.07 28.85 0.00 27.46 25.91 0.00 25.15 28.89 0.00 28.65 27.18 0.00 24.54 27.22 26.40 0.00 0.00 28.63 27.46 0.00 27.56 26.04 22.45 0.00 29.06 31.84 KBS T3 28.68 27.59 0.00 0.00 0.00 26.70 27.15 26.48 28.44 28.69 0.00 28.66 0.00 22.44 0.00 28.94 0.00 28.96 0.00 30.85 25.21 27.84 0.00 29.26 0.00 0.00 28.01 0.00 28.80 0.00 22.53 26.67 30.83 0.00 0.00 0.00 33.04 23.13 26.57 0.00 0.00 24.89 29.45 0.00 0.00 26.04 0.00 22.06 0.00 28.19 0.00 KBS T4 28.05 0.00 32.43 0.00 27.28 26.34 0.00 25.58 0.00 27.48 26.49 0.00 27.39 21.96 26.30 29.92 0.00 27.10 0.00 0.00 24.10 26.68 26.36 0.00 0.00 27.50 26.91 25.70 27.25 0.00 21.66 25.45 29.18 0.00 27.29 25.57 0.00 23.47 0.00 25.60 27.47 25.15 26.82 26.96 26.29 25.89 26.44 21.75 25.75 27.55 35.00 Negative and No Template Controls aceA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 30.03 NTC 0.00 0.00 0.00 34.08 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 142 Table D. 5 . SmartChip Ct values for xenB assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standar d curve or is above the cycle threshold of 28 and a zero value indicates no amplification. Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 Plasmid Standards 10 1 copies per rxn 0.00 0.00 0.00 28.83 0.00 26.56 0.00 0.00 34.14 27.88 0.00 0.00 30.73 26.82 0.00 0.00 26.88 30.77 27.34 30.47 27.71 0.00 0.00 0.00 26.15 0.00 0.00 34.78 0.00 28.53 0.00 0.00 0.00 26.77 32.01 0.00 0.00 0.00 31.89 26.66 0.00 0.00 31.52 26.77 29.54 10 2 copies per rxn 27.53 25.29 27.47 24.63 0.00 25.37 27.64 29.22 0.00 24.98 29.00 0.00 29.27 25.59 26.64 0.00 26.19 27.04 25.35 28.05 23.89 27.73 28.39 30.34 25.07 29.35 28.36 30.80 24.13 0.00 27.59 25.94 29.49 26.75 27.94 26.41 27.86 27.16 29.91 0.00 28.40 30.54 27.64 25.17 0.00 10 3 copies per rxn 23.55 21.27 24.04 21.54 25.36 21.67 23.01 23.52 26.82 0.00 25.65 0.00 24.56 21.72 23.17 24.22 21.64 25.45 22.25 26.52 22.23 24.02 25.39 26.61 22.77 25.93 28.00 24.57 22.33 22.95 24.92 21.80 23.31 21.32 25.01 22.62 23.34 25.87 27.05 22.75 26.75 28.05 24.38 22.52 0.00 10 4 copies per rxn 19.29 17.69 20.60 18.09 20.21 17.46 0.00 20.89 21.65 17.49 21.59 0.00 19.74 17.38 18.50 19.49 17.76 20.62 18.04 20.61 17.99 19.84 21.21 21.94 17.79 21.85 23.40 19.81 17.66 18.40 19.67 0.00 0.00 0.00 21.05 18.43 19.80 20.63 21.64 17.82 21.59 23.56 19.53 18.13 18.44 10 5 copies per rxn 14.74 13.43 15.87 13.22 15.62 13.39 14.24 15.97 18.67 13.42 17.09 18.42 16.39 13.16 12.58 14.82 13.60 15.93 13.67 15.83 13.54 14.53 16.24 18.47 13.69 17.36 18.65 16.20 13.33 12.74 14.94 13.56 16.19 13.30 15.77 13.41 0.00 16.02 18.61 13.59 17.40 18.61 16.38 13.28 12.69 10 6 copies per rxn 11.64 10.52 12.92 10.53 12.32 10.70 12.12 13.46 15.22 10.75 14.52 15.77 12.92 10.51 9.41 11.93 10.93 13.32 0.00 12.61 10.90 11.65 13.76 15.11 11.01 14.61 16.11 12.76 10.72 9.61 11.68 10.53 12.41 10.51 11.92 10.53 11.23 12.63 15.10 10.46 0.00 15.14 12.73 10.30 9.60 10 7 copies per rxn 7.52 6.79 9.17 6.53 7.61 6.50 6.85 9.32 12.07 6.49 10.19 11.65 0.00 6.39 5.28 7.68 6.72 8.96 6.57 7.45 6.48 6.74 9.17 11.63 6.44 9.85 11.49 8.87 6.37 5.18 7.80 6.85 8.80 6.57 7.82 6.62 6.85 9.31 11.53 6.55 9.99 11.87 8.87 6.53 5.40 Shallow Zone Aquifer MW32 (Pre) 21.67 22.31 24.54 22.41 22.65 20.92 24.22 0.00 26.06 23.26 23.76 23.33 24.83 20.90 28.21 21.88 22.10 24.15 22.43 23.37 21.13 24.43 24.39 25.64 23.74 23.55 22.54 25.25 21.33 29.43 21.43 22.19 23.52 22.00 22.85 21.11 23.85 24.35 25.95 23.20 22.95 22.63 24.75 21.18 26.99 MW32 (Post) 27.46 0.00 31.31 27.17 33.56 29.88 30.22 0.00 31.36 26.52 30.99 0.00 31.13 26.14 29.89 27.97 31.99 0.00 27.39 0.00 0.00 0.00 26.62 0.00 25.68 28.56 0.00 32.62 0.00 28.84 28.62 0.00 31.03 27.61 0.00 26.08 28.14 26.86 0.00 25.44 27.69 33.79 0.00 27.43 27.74 MW62 (Pre) 22.95 25.12 28.80 25.09 22.44 23.50 27.19 22.41 25.49 0.00 25.49 25.58 26.47 26.31 32.52 22.79 25.40 28.06 0.00 21.79 23.80 27.29 22.47 26.10 26.18 24.83 25.88 26.88 26.07 0.00 23.04 24.92 26.73 24.77 22.66 23.82 27.29 22.68 26.14 25.59 25.16 26.44 26.64 26.26 28.05 MW62 (Post) 25.56 24.96 27.52 25.61 29.39 25.08 32.08 0.00 0.00 26.51 26.10 26.45 0.00 25.30 34.27 27.36 25.07 27.54 25.58 26.61 24.46 29.43 0.00 0.00 27.40 25.11 25.50 33.48 24.06 30.22 25.79 25.39 26.56 24.58 26.19 24.43 26.90 26.09 0.00 25.33 26.44 24.96 30.28 23.92 30.78 143 Table D. 5 . Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 Perched Zone Aquifer MW48 (Pre) 28.81 0.00 0.00 0.00 34.16 30.43 0.00 0.00 0.00 30.56 0.00 0.00 0.00 28.64 0.00 0.00 18.42 21.87 17.84 21.13 17.77 20.01 22.32 21.44 17.58 19.72 20.40 23.65 17.70 27.04 14.92 17.13 20.55 16.55 19.87 16.54 0.00 21.59 20.65 16.24 18.13 18.74 22.88 16.29 26.05 MW48 (Post) 24.91 26.44 0.00 26.80 29.24 27.48 31.47 0.00 0.00 0.00 32.24 0.00 29.52 26.95 27.87 25.55 28.76 0.00 27.88 30.40 28.91 32.19 0.00 30.48 26.09 31.74 0.00 0.00 0.00 0.00 MW60R (Pre) 23.64 23.87 25.54 24.38 23.32 22.89 25.48 24.83 0.00 25.78 24.72 26.52 26.94 22.70 25.49 26.65 27.01 0.00 26.58 30.23 26.58 28.88 0.00 0.00 26.20 0.00 32.57 34.90 25.24 0.00 26.08 24.51 28.66 25.46 30.35 25.62 29.99 27.92 0.00 25.61 29.64 33.80 0.00 25.49 30.13 MW60R (Post) 24.28 24.11 27.13 25.74 24.13 25.46 30.24 23.27 30.92 0.00 29.09 32.35 0.00 26.03 31.87 24.72 24.25 29.08 26.30 25.74 26.18 29.77 0.00 34.36 29.76 29.58 34.07 0.00 27.21 33.18 MW64 (Pre) 24.12 24.53 26.97 24.36 23.86 23.13 25.60 0.00 28.68 27.53 24.70 24.81 26.15 23.36 32.01 25.45 22.95 27.03 23.53 25.86 23.87 25.22 26.28 0.00 23.49 26.50 26.89 29.89 23.22 0.00 25.36 25.58 26.78 26.27 24.64 24.19 26.87 0.00 32.55 0.00 26.03 27.64 0.00 24.64 32.82 MW64 (Post) 25.04 0.00 30.46 23.75 25.27 23.39 29.08 0.00 29.63 24.19 26.18 26.93 28.99 23.93 31.57 25.10 24.44 28.68 23.91 25.39 23.10 29.13 0.00 28.78 24.06 26.91 28.33 0.00 24.08 0.00 MW66 (Pre) 22.00 22.34 24.19 22.30 23.09 21.47 23.82 0.00 27.67 23.77 22.38 22.52 26.20 21.22 27.37 22.14 22.12 24.14 22.31 22.61 21.12 23.67 0.00 30.41 0.00 22.49 22.26 25.09 21.53 27.66 23.43 24.70 26.60 24.68 24.28 23.68 27.35 23.82 31.63 26.31 24.99 24.91 26.23 24.62 0.00 MW66 (Post) 23.69 25.34 26.20 25.44 23.16 23.22 25.18 24.42 28.51 24.66 26.09 26.21 0.00 23.44 0.00 22.35 23.61 25.11 23.56 21.73 22.30 25.07 0.00 27.47 24.34 25.45 24.51 0.00 23.04 0.00 MW67 (Pre) 22.37 22.61 24.49 22.42 22.14 21.65 23.24 21.80 28.13 24.30 22.56 21.89 0.00 21.16 0.00 21.96 22.12 24.36 22.62 22.00 21.96 24.26 22.02 30.39 24.39 23.17 23.27 0.00 22.02 0.00 21.71 21.75 23.50 21.89 21.40 21.47 22.76 21.78 28.19 22.87 22.64 21.72 24.02 20.96 0.00 MW67 (Post) 0.00 0.00 0.00 0.00 0.00 29.63 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 33.04 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 29.08 Red Cedar River Red Cedar 23.20 25.58 27.97 25.66 27.12 25.34 29.06 25.54 30.03 0.00 28.24 29.35 27.72 27.91 27.20 22.80 0.00 29.94 24.68 25.80 24.12 33.28 24.45 28.83 0.00 27.51 27.19 28.40 26.50 30.34 Sediment MW71 0.00 26.91 29.86 0.00 0.00 34.39 0.00 0.00 33.27 0.00 33.31 0.00 0.00 27.55 31.84 28.03 27.20 27.53 31.67 28.98 27.62 28.47 31.41 29.08 27.85 32.44 33.92 0.00 27.81 25.70 28.03 26.61 0.00 27.85 32.16 27.22 0.00 31.23 0.00 33.77 29.55 29.49 0.00 27.28 31.24 144 Table D. 5 Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 Agricultural Soil MSU Soil E 26.02 24.64 33.78 24.99 27.89 25.15 33.79 25.62 32.79 26.56 28.06 0.00 27.74 26.52 0.00 28.39 26.66 29.81 26.55 28.76 26.10 0.00 27.06 0.00 0.00 29.67 0.00 27.90 30.43 0.00 25.92 25.59 0.00 25.60 26.96 25.75 30.92 26.18 31.91 28.26 28.83 32.91 26.87 26.43 0.00 MSU Soil F 24.86 25.43 27.94 24.80 26.10 24.91 28.14 0.00 34.10 26.11 0.00 32.43 27.89 27.73 34.29 26.11 26.44 32.25 26.03 26.97 25.39 0.00 27.26 28.74 28.11 0.00 30.18 26.17 27.42 0.00 24.48 26.38 29.13 25.59 25.80 25.31 0.00 26.18 0.00 28.88 30.54 32.04 27.02 27.83 0.00 KBS T1 24.36 24.83 0.00 0.00 26.41 24.61 0.00 25.18 0.00 27.49 28.25 0.00 25.85 26.61 0.00 23.29 24.92 28.76 26.64 25.64 22.82 26.02 26.63 0.00 23.31 27.98 32.83 27.23 23.35 0.00 21.14 22.84 27.85 23.33 24.42 20.79 26.43 24.05 0.00 20.87 25.33 29.56 25.08 20.71 34.04 KBS T2 23.74 24.90 27.91 25.70 25.58 23.59 27.81 25.61 28.92 24.14 0.00 32.11 27.36 23.61 31.62 26.13 25.55 28.43 25.61 28.77 24.41 28.11 25.62 31.90 26.57 0.00 0.00 26.92 25.62 34.37 23.31 0.00 27.83 25.53 24.73 22.92 27.42 25.41 0.00 22.74 28.30 31.30 26.28 23.41 0.00 KBS T3 24.12 25.27 0.00 25.67 27.11 23.43 27.44 26.03 33.18 24.52 27.39 0.00 25.27 24.10 0.00 25.14 25.34 30.20 25.91 25.89 25.20 27.67 0.00 0.00 24.92 28.23 0.00 27.19 25.35 0.00 24.33 24.16 29.18 24.49 27.40 23.63 0.00 24.50 29.98 26.51 27.82 0.00 26.63 24.64 34.02 KBS T4 24.14 0.00 31.77 25.82 26.89 22.76 0.00 25.44 0.00 23.29 28.59 31.07 25.93 23.63 0.00 24.55 24.16 29.54 25.63 26.74 23.75 0.00 25.72 0.00 26.20 27.74 32.46 25.46 25.12 0.00 24.82 25.64 29.12 25.03 26.49 25.06 26.98 25.43 33.27 26.01 27.80 30.05 27.32 25.26 0.00 Negative and No Template Controls aceA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NTC 0.00 0.00 0.00 33.52 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 145 Table D. 6 . SmartChip Ct values for xplA assays. Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cycle threshold of 28 and a zero value indicates no amplification Sample PS3 PS4 PS5 Pub1 Pub2 Pub3 Pub4 Pub6 Plasmid Standards 10 1 copies per rxn 0.00 0.00 0.00 27.76 25.89 31.52 27.58 0.00 0.00 29.44 29.35 27.79 27.88 0.00 26.98 29.51 0.00 0.00 0.00 27.95 0.00 0.00 0.00 30.81 10 2 copies per rxn 30.71 26.50 0.00 24.65 23.65 28.09 23.72 0.00 29.08 0.00 0.00 24.76 24.07 0.00 23.45 27.16 0.00 26.25 28.85 0.00 24.25 28.50 24.68 27.24 10 3 copies per rxn 26.00 23.63 24.78 21.59 21.08 26.93 21.37 23.86 26.42 22.77 24.77 21.65 20.90 24.67 21.51 22.62 26.83 23.16 0.00 0.00 21.08 25.87 21.29 23.16 10 4 copies per rxn 20.69 17.64 28.65 17.14 16.61 21.37 16.60 16.94 20.35 18.67 26.90 16.49 16.64 21.66 17.20 16.15 20.55 18.14 26.27 0.00 16.43 20.62 16.87 18.43 10 5 copies per rxn 15.00 13.60 15.23 14.21 13.00 16.51 13.44 11.89 14.81 13.75 15.59 14.00 13.22 16.53 13.73 12.28 15.09 13.56 15.44 0.00 13.20 16.56 13.64 12.11 10 6 copies per rxn 10.88 11.23 10.40 11.93 10.73 14.34 11.09 8.84 11.17 10.53 10.32 11.17 10.10 13.38 10.56 8.62 11.31 10.70 10.45 0.00 10.52 13.66 10.92 9.08 10 7 copies per rxn 6.85 5.98 6.03 7.52 6.41 9.74 6.78 0.00 6.64 6.06 6.42 7.38 6.18 9.73 6.71 0.00 6.75 6.24 6.22 0.00 6.56 9.83 6.94 0.00 Shallow Zone Aquifer MW32 (Pre) 28.60 23.86 24.97 23.65 23.26 27.79 23.02 23.51 0.00 23.98 24.42 0.00 23.78 28.04 23.81 24.24 27.30 23.87 0.00 0.00 25.17 0.00 24.41 23.28 MW32 (Post) 0.00 29.01 30.64 0.00 0.00 0.00 0.00 0.00 0.00 29.32 0.00 0.00 0.00 0.00 0.00 0.00 32.58 27.21 0.00 0.00 0.00 34.17 0.00 0.00 MW62 (Pre) 27.17 24.80 0.00 0.00 28.59 32.19 28.48 0.00 26.60 0.00 24.90 0.00 28.15 0.00 0.00 25.30 27.16 0.00 25.24 28.89 28.44 34.02 28.61 25.04 MW62 (Post) 30.56 0.00 0.00 34.74 0.00 0.00 0.00 30.59 0.00 0.00 0.00 0.00 0.00 0.00 0.00 33.49 30.49 0.00 30.10 0.00 0.00 28.56 0.00 0.00 Perched Zone Aquifer MW48 (Pre) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 24.07 27.33 0.00 0.00 0.00 31.15 0.00 0.00 23.97 0.00 0.00 33.05 28.67 30.30 28.79 26.12 MW48 (Post) 34.72 0.00 0.00 0.00 0.00 0.00 0.00 30.28 32.16 30.70 30.44 0.00 0.00 0.00 0.00 0.00 MW60R (Pre) 28.44 28.29 27.34 0.00 0.00 34.41 34.72 28.69 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 MW60R (Post) 26.32 0.00 26.94 32.10 0.00 32.79 34.23 23.46 0.00 25.81 0.00 0.00 0.00 30.52 0.00 23.67 MW64 (Pre) 29.43 26.98 0.00 0.00 0.00 0.00 34.98 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 28.77 26.85 0.00 0.00 0.00 0.00 33.60 28.91 146 Table D. 6 Sample PS3 PS4 PS5 Pub1 Pub2 Pub3 Pub4 Pub6 MW64 (Post) 28.65 28.63 25.93 0.00 31.12 0.00 0.00 0.00 28.86 28.25 27.27 0.00 0.00 34.52 33.28 0.00 MW66 (Pre) 27.08 24.06 0.00 26.35 0.00 30.84 33.08 0.00 26.86 24.91 0.00 26.79 0.00 32.92 0.00 25.70 28.28 24.74 25.38 28.51 0.00 33.31 0.00 0.00 MW66 (Post) 0.00 27.06 29.63 29.12 28.51 0.00 29.06 28.87 27.89 25.42 0.00 26.70 0.00 32.29 0.00 0.00 MW67 (Pre) 28.81 27.75 26.99 0.00 0.00 29.20 0.00 27.57 0.00 26.32 26.12 33.22 29.89 28.52 0.00 0.00 0.00 26.87 26.55 0.00 0.00 29.96 29.53 27.60 MW67 (Post) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 33.29 0.00 0.00 0.00 0.00 0.00 34.20 Red Cedar River Red Cedar 0.00 23.26 25.44 0.00 0.00 32.00 33.45 26.62 0.00 22.88 24.58 30.21 0.00 30.97 0.00 26.36 Sediment MW71 0.00 30.61 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 34.57 31.90 0.00 0.00 0.00 0.00 0.00 0.00 Agricultural Soils MSU Soil E 0.00 0.00 24.81 0.00 0.00 32.35 0.00 25.54 29.30 25.12 25.32 0.00 0.00 33.56 0.00 0.00 28.69 0.00 25.66 28.19 0.00 0.00 32.18 0.00 MSU Soil F 28.17 0.00 25.61 32.84 0.00 0.00 0.00 26.52 29.32 0.00 26.83 31.10 0.00 34.82 0.00 0.00 0.00 26.50 0.00 0.00 0.00 0.00 0.00 26.78 KBS T1 28.02 0.00 25.44 0.00 0.00 0.00 34.38 26.69 28.73 0.00 25.21 29.95 0.00 30.44 0.00 26.87 27.36 25.44 24.24 32.90 0.00 0.00 0.00 25.23 KBS T2 29.98 0.00 25.99 33.05 0.00 33.90 31.72 0.00 0.00 0.00 25.08 30.33 0.00 30.55 30.16 25.51 0.00 0.00 24.03 29.78 0.00 32.03 0.00 23.92 KBS T3 0.00 26.01 25.63 0.00 0.00 0.00 0.00 0.00 28.70 0.00 25.78 0.00 0.00 0.00 0.00 0.00 27.37 25.05 24.84 0.00 0.00 0.00 0.00 0.00 KBS T4 0.00 0.00 24.90 0.00 33.10 31.56 0.00 0.00 0.00 25.16 24.25 31.24 0.00 0.00 0.00 0.00 26.73 0.00 23.95 0.00 0.00 0.00 0.00 26.24 Negative and No Template Controls aceA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NTC 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 147 Table D. 7 . SmartChip Ct values for the 16S rRNA assay . These results correspond to the 3 chip runs performed: 1) xenB and pnrB , 2) xplA and nfsI , and 3) xenA and diaA . Green text indicates the Ct value falls within the standard curve, red text indicates it falls outside of the standard curve or is above the cy cle threshold of 28 and a zero value indicates no amplification Sample 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 Plasmid Standards 10 1 copies per rxn 27.96 27.12 28.89 27.43 27.81 27.39 28.14 27.48 28.00 10 2 copies per rxn 24.60 25.18 27.57 0.00 25.43 26.31 24.55 25.62 26.97 10 3 copies per rxn 20.92 22.30 22.16 21.13 21.99 22.20 21.23 22.05 22.56 10 4 copies per rxn 18.76 19.42 19.68 18.22 19.75 0.00 18.62 19.64 19.57 10 5 copies per rxn 14.56 14.83 14.94 15.22 15.36 15.24 14.98 15.13 15.07 10 6 copies per rxn 11.91 12.06 12.51 12.28 12.31 12.54 12.16 12.17 12.65 10 7 copies per rxn 8.09 8.39 8.01 8.65 8.46 8.29 8.38 0.00 8.29 10 8 copies per rxn 5.72 5.55 5.34 5.64 5.44 5.58 5.58 5.44 5.57 Shallow Zone Aquifer MW32 (Pre) 13.74 13.78 13.88 13.80 13.90 14.07 13.45 13.67 13.72 MW32 (Post) 10.94 11.08 11.08 10.68 10.92 10.77 11.13 11.04 11.47 MW62 (Pre) 12.21 12.23 12.16 12.20 12.11 12.24 12.05 12.35 12.29 MW62 (Post) 10.29 10.26 10.41 10.33 10.97 10.48 10.77 10.68 10.53 Perched Zone Aquifer MW48 (Pre) 21.61 21.70 20.40 13.41 13.54 13.52 12.39 12.56 12.56 MW48 (Post) 10.50 10.85 10.50 10.57 10.77 10.32 MW60R (Pre) 14.28 14.51 14.43 20.02 20.12 19.98 19.83 20.56 20.28 MW60R (Post) 12.09 11.90 11.91 11.96 12.05 12.01 148 Table D. 7 Sample 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 MW64 (Pre) 15.41 15.43 15.28 18.61 17.36 17.20 16.00 16.21 15.88 MW64 (Post) 10.93 10.96 11.01 11.31 11.60 11.26 MW66 (Pre) 13.35 13.13 13.02 13.45 13.93 13.42 0.00 13.95 13.92 MW66 (Post) 12.39 12.19 12.22 10.60 10.72 10.74 MW67 (Pre) 14.67 14.71 14.66 14.75 14.58 14.72 14.40 14.49 14.41 MW67 (Post) 23.61 23.70 23.74 22.07 22.25 22.28 Red Cedar River Red Cedar 13.32 13.35 13.40 13.32 13.36 13.45 Sediment MW71 19.32 19.50 20.16 18.87 19.56 19.16 19.37 20.18 19.37 Agricultural Soils MSU Soil E 14.12 14.76 14.50 15.31 14.64 14.52 14.82 14.76 14.90 MSU Soil F 14.29 14.32 14.41 14.75 14.98 14.94 15.15 15.00 15.04 KBS T1 14.36 14.26 14.28 14.19 14.97 14.36 13.98 13.66 13.78 KBS T2 14.38 14.32 14.16 14.46 14.51 14.51 14.26 14.46 14.44 KBS T3 14.44 14.54 14.46 14.33 14.48 14.37 14.47 14.68 14.49 KBS T4 14.17 14.37 14.18 14.28 14.40 14.38 14.39 14.46 14.67 Negative and No Template Controls aceA 28.56 29.07 27.86 NTC 28.00 28.56 28.85 149 Table D. 8 . SmartChip standard curve characteristics Gene Assay Plasmid Log( Linear Range) Efficiency (%) R 2 Slope Y - Intercept diaA 1 CP012395 1 to 7 102.6 0.995 - 3.26 29.6 3 CP012395 1 to 7 102.4 0.998 - 3.26 29.7 4 AP009049 2 to 7 78.3 0.986 - 3.98 34.3 nfsI 1 CP001918 2 to 7 80.5 0.991 - 3.90 33.7 2 CP001918 2 to 7 84.6 0.993 - 3.76 31.4 3 CP001918 2 to 7 86.0 0.995 - 3.71 31.5 4 CP001918 1 to 7 87.0 0.988 - 3.68 31.1 5 CP001918 1 to 7 84.1 0.994 - 3.77 31.9 6 CP001918 1 to 7 84.8 0.988 - 3.75 31.9 7 CP001918 2 to 7 76.1 0.985 - 4.07 34.6 8 CP001918 1 to 7 84.9 0.983 - 3.75 31.4 Pub1 M63808 2 to 7 91.9 0.918 - 3.53 32.3 Pub2 M63808 2 to 7 76.2 0.935 - 4.06 33.6 pnrB 1 CP010979 1 to 7 87.0 0.997 - 3.68 31.3 2 MCBI01000011 3 to 7 60.1 0.987 - 4.89 39.9 3 CP010979 3 to 7 84.1 0.991 - 3.77 37.0 4 CP010979 1 to 7 90.6 0.995 - 3.57 30.5 5 CP010979 2 to 7 91.5 0.988 - 3.54 33.7 Pub1 AF532912 2 to 7 91.0 0.968 - 3.56 31.7 Pub2 AF532912 2 to 7 89.9 0.980 - 3.59 31.9 xenA 1 CP004045 2 to 7 81.5 0.989 - 3.86 33.9 2 AP013070 4 to 7 97.3 0.997 - 3.39 36.0 3 AP013070 4 to 7 93.5 0.997 - 3.49 36.9 4 CP004045 3 to 7 90.8 0.995 - 3.57 36.7 5 AP013070 4 to 7 96.1 0.995 - 3.42 30.5 6 CP004045 1 to 7 89.7 0.998 - 3.60 31.7 7 AP013070 4 to 7 91.1 0.997 - 3.55 32.9 8 CP004045 2 to 7 88.5 0.995 - 3.63 33.8 9 FOEO01000004 2 to 7 83.6 0.995 - 3.79 33.1 10 CP004045 2 to 7 88.2 0.997 - 3.64 32.5 11 FOEO01000004 2 to 7 89.4 0.995 - 3.61 31.3 12 CP004045 1 to 7 92.4 0.997 - 3.52 31.3 14 AP013070 4 to 7 93.3 0.997 - 3.49 32.2 15 CP004045 2 to 7 85.8 0.996 - 3.72 33.0 16 CP004045 2 to 7 89.5 0.998 - 3.60 32.9 17 FOEO01000004 2 to 7 87.5 0.995 - 3.66 31.8 Pub1 AF154061 3 to 6 74.8 0.989 - 4.12 32.1 xenB 1 CP014205 2 to 7 76.5 0.995 - 4.05 35.8 2 CP014205 2 to 7 84.1 0.996 - 3.77 33.0 4 CP014205 2 to 7 82.7 0.990 - 3.82 35.6 5 CP014205 1 to 7 88.4 0.990 - 3.64 32.2 6 CP014205 3 to 7 68.1 0.993 - 4.43 38.6 7 CP014205 2 to 7 83.8 0.991 - 3.78 33.0 8 CP014205 2 to 7 74.3 0.996 - 4.14 36.0 9 CP014205 3 to 7 80.6 0.986 - 3.89 36.4 10 CP002620 3 to 7 87.1 0.991 - 3.68 37.2 11 CP014205 1 to 7 93.0 0.986 - 3.50 31.5 14 CP014205 3 to 7 79.1 0.993 - 3.95 37.7 16 CP014205 4 to 7 83.8 0.986 - 3.78 38.1 17 CP002620 3 to 7 82.6 0.995 - 3.82 35.5 19 CP014205 1 to 7 90.1 0.988 - 3.58 31.8 150 Table D. 8 . Gene Assay Plasmid Log( Linear Range) Efficiency (%) R 2 Slope Y - Intercept xenB Pub1 AF154062 3 to 7 68.5 0.990 - 4.42 35.8 xplA 3 CP007257 3 to 7 60.4 0.991 - 4.88 40.3 4 AF449421 2 to 7 75.4 0.993 - 4.10 34.8 5 BAFD01000115 4 to 7 40.2 0.930 - 6.82 52.3 Pub1 AF449421 1 to 7 97.3 0.995 - 3.39 31.3 Pub2 AF449421 1 to 7 94.5 0.994 - 3.46 30.8 Pub3 AF449421 3 to 7 79.1 0.985 - 3.95 37.2 Pub4 AF449421 1 to 7 96.3 0.995 - 3.41 30.9 Pub6 AF449421 _ 300 - 959 2 to 6 61.8 0.984 - 4.79 36.9 16S rRNA 16S_rRNA.1 KX858536 2 to 8 107.0 0.998 - 3.16 30.9 16S_rRNA.2 KX858536 1 to 8 103.4 0.994 - 3.24 31.6 16S_rRNA.3 KX858536 2 to 8 93.0 0.995 - 3.50 33.2 151 Table D. 9 . Gene copies per mL or g of starting material for nfsI false positive amplification. Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 Pub1 Pub2 Shallow Zone Aquifer (gene copies per mL of groundwater) MW32 (Pre) 1.06E+03 N/A N/A N/A N/A 2.78E+02 N/A N/A N/A N/A 1.03E+03 N/A N/A N/A N/A 2.39E+02 N/A N/A N/A N/A 9.99E+02 N/A N/A N/A N/A 2.64E+02 N/A N/A N/A N/A MW32 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW62 (Pre) 1.90E+04 N/A N/A N/A N/A 5.87E+03 N/A N/A N/A N/A 1.85E+04 N/A N/A N/A N/A 3.81E+03 N/A N/A N/A N/A 2.21E+04 N/A N/A N/A N/A N/A N/A N/A N/A N/A MW62 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Perched Zone Aquifer (gene copies per mL of groundwater) MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 9.28E+03 N/A N/A N/A N/A N/A 3.98E+02 N/A N/A N/A 1.49E+04 N/A N/A N/A N/A N/A 7.89E+02 N/A N/A N/A MW48 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Post) 6.15E+03 N/A N/A N/A N/A N/A N/A N/A N/A N/A 3.91E+03 N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Post) N/A N/A N/A N/A N/A N/A 7.50E+03 N/A N/A N/A N/A N/A N/A N/A N/A N/A 7.79E+03 N/A N/A N/A MW66 (Pre) 2.13E+03 N/A N/A N/A N/A N/A 1.75E+03 N/A N/A N/A 1.17E+03 N/A N/A N/A N/A N/A 9.76E+02 N/A N/A N/A 1.21E+03 N/A N/A N/A N/A N/A 1.29E+03 N/A N/A N/A MW66 (Post) N/A N/A N/A N/A N/A N/A 2.81E+02 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.36E+03 N/A N/A N/A MW67 (Pre) 1.18E+03 N/A N/A N/A N/A N/A 4.71E+02 N/A N/A N/A 8.84E+02 N/A N/A N/A N/A N/A 4.16E+02 N/A N/A N/A 1.57E+03 N/A N/A N/A N/A N/A 3.51E+02 N/A N/A N/A MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Red Cedar River (gene copies per mL of river water) Red Cedar N/A N/A N/A N/A N/A N/A 3.51E+03 N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.17E+03 N/A N/A N/A Sediment (gene copies per g of sediment) MW71 N/A N/A N/A N/A N/A N/A 2.32E+05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.21E+05 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soils (gene copies per g of soil) MSU Soil E 1.05E+06 N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.11E+05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.66E+05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 152 Table D. 9 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 Pub1 Pub2 MSU Soil F N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A KBS T1 9.17E+05 N/A N/A N/A N/A N/A 6.66E+05 N/A N/A N/A 4.35E+05 N/A N/A N/A N/A 2.60E+05 3.67E+05 N/A N/A N/A 2.61E+06 N/A N/A N/A N/A 7.16E+05 N/A N/A N/A N/A KBS T2 N/A N/A N/A N/A N/A 1.93E+05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 3.79E+05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.98E+05 N/A N/A N/A N/A KBS T3 6.08E+05 N/A N/A N/A N/A 2.27E+05 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 6.41E+05 N/A N/A N/A 1.68E+06 N/A N/A N/A N/A 2.65E+05 1.10E+06 N/A N/A N/A KBS T4 1.63E+06 N/A N/A N/A N/A 2.94E+05 6.42E+05 N/A N/A N/A N/A N/A N/A N/A N/A 6.28E+05 1.41E+06 N/A N/A N/A 2.47E+06 N/A N/A N/A N/A 6.52E+05 1.98E+06 N/A N/A N/A Table D. 10 . Gene copies per mL or g of starting material for pnrB false positive amplification . Sample PS1 PS2 PS3 PS4 PS5 Pub1 Pub2 Shallow Zone Aquifer (gene copies per mL of groundwater) MW32 (Pre) 1.38E+02 7.25E+03 3.74E+03 8.23E+01 N/A N/A 3.61E+03 2.08E+02 5.62E+03 5.11E+03 N/A N/A N/A 4.19E+03 N/A 6.06E+03 N/A 2.02E+02 N/A N/A 2.62E+03 MW32 (Post) N/A N/A 1.13E+04 N/A N/A N/A N/A N/A N/A 2.48E+04 N/A N/A N/A 5.13E+03 N/A N/A 3.45E+04 N/A N/A N/A 7.65E+03 MW62 (Pre) 8.63E+02 6.06E+04 2.58E+05 N/A N/A N/A N/A 6.76E+02 5.46E+04 2.80E+05 N/A N/A N/A N/A 9.12E+02 7.55E+04 3.38E+05 N/A N/A N/A N/A MW62 (Post) N/A 3.08E+03 N/A N/A N/A N/A N/A N/A 5.44E+04 N/A N/A N/A N/A N/A N/A 2.93E+04 N/A N/A N/A N/A N/A Perched Zone Aquifer (gene copies per mL of groundwater) MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A 9.04E+03 N/A 1.11E+02 N/A N/A 2.74E+03 N/A 1.88E+04 N/A 1.41E+02 N/A N/A 3.13E+03 MW48 (Post) N/A 1.24E+05 N/A N/A N/A N/A N/A N/A 9.93E+04 N/A N/A N/A N/A N/A MW60R (Pre) N/A 3.03E+04 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 8.47E+03 N/A N/A N/A N/A N/A MW60R (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Post) N/A 5.01E+05 N/A N/A N/A N/A N/A N/A 6.69E+05 N/A N/A N/A N/A N/A 153 Table D. 10 Sample PS1 PS2 PS3 PS4 PS5 Pub1 Pub2 MW66 (Pre) N/A 3.80E+03 5.34E+03 N/A N/A N/A 1.93E+03 N/A 4.57E+03 7.10E+03 N/A N/A N/A 1.47E+03 N/A 4.78E+03 N/A N/A N/A N/A 6.95E+02 MW66 (Post) N/A 9.68E+03 N/A N/A N/A N/A N/A N/A 4.67E+04 N/A N/A N/A N/A N/A MW67 (Pre) N/A N/A N/A N/A N/A N/A 9.10E+02 N/A N/A N/A N/A N/A N/A 1.36E+03 N/A N/A N/A N/A N/A N/A 1.22E+03 MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Red Cedar River (gene copies per mL of river water) Red Cedar N/A N/A N/A 5.16E+02 N/A N/A 1.12E+04 N/A N/A N/A 1.03E+03 N/A N/A 1.89E+04 Sediment (gene copies per g of sediment) MW71 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soils (gene copies per g of soil) MSU Soil E N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MSU Soil F N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A KBS T1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A KBS T2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.22E+05 N/A N/A N/A N/A N/A N/A 8.02E+04 N/A N/A N/A KBS T3 1.01E+05 N/A N/A N/A N/A N/A 7.82E+05 1.37E+05 N/A N/A 1.15E+05 N/A N/A N/A 2.42E+05 N/A N/A 2.01E+05 N/A N/A 2.12E+06 KBS T4 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 154 Table D. 11 . Gene copies per mL or g of starting material for xenA Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 Shallow Zone Aquifer (gene copies per mL of groundwater) MW32 (Pre) 3.21 E+ 02 N/A N/A 3.80 E+ 04 N/A 4.18 E+ 02 N/A N/A 8.06 E+ 02 1.20 E+ 03 N/A 8.03 E+ 02 N/A 4.39 E+ 03 1.91 E+ 03 N/A N/A 6.37 E+ 02 N/A N/A 2.78 E+ 04 N/A 6.42 E+ 02 N/A N/A 4.93 E+ 02 7.52 E+ 02 N/A 1.24 E+ 03 N/A 4.44 E+ 03 1.57 E+ 03 N/A N/A 7.43 E+ 02 N/A N/A 4.13 E+ 04 N/A 8.10 E+ 02 N/A N/A 1.24 E+ 03 6.51 E+ 02 N/A 8.36 E+ 02 N/A 4.35 E+ 03 3.36 E+ 03 N/A N/A MW32 (Post) N/A N/A N/A 3.65 E+ 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.10 E+ 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.24 E+ 03 N/A N/A N/A N/A N/A N/A 1.10 E+ 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.46 E+ 03 N/A N/A N/A MW62 (Pre) 2.41 E+ 03 N/A N/A 1.27 E+ 05 N/A 5.41 E+ 02 N/A 9.32 E+ 03 N/A N/A N/A N/A N/A 3.03 E+ 04 N/A N/A N/A 3.99 E+ 03 N/A N/A 1.40 E+ 05 N/A 9.40 E+ 02 N/A 6.62 E+ 03 N/A N/A N/A N/A N/A 2.49 E+ 04 N/A N/A N/A 4.41 E+ 03 N/A N/A 1.83 E+ 05 N/A 1.16 E+ 03 N/A 9.37 E+ 03 N/A N/A N/A N/A N/A 2.55 E+ 04 N/A N/A N/A MW62 (Post) N/A N/A N/A N/A N/A 2.56 E+ 02 N/A N/A N/A N/A N/A N/A N/A 8.67 E+ 02 N/A N/A N/A N/A N/A N/A N/A N/A 2.17 E+ 03 N/A N/A N/A N/A N/A N/A N/A 4.12 E+ 03 N/A N/A N/A N/A N/A N/A N/A N/A 1.52 E+ 03 N/A N/A N/A N/A N/A N/A N/A 3.43 E+ 03 N/A N/A N/A Perched Zone Aquifer (gene copies per mL of groundwater) MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.97 E+ 02 N/A N/A 2.25 E+ 05 N/A 1.83 E+ 03 N/A N/A 2.35 E+ 03 8.66 E+ 02 4.04 E+ 03 3.22 E+ 03 N/A 4.89 E+ 03 N/A N/A N/A 3.12 E+ 02 N/A N/A 5.43 E+ 05 N/A 6.08 E+ 03 N/A N/A 4.29 E+ 03 1.20 E+ 03 7.79 E+ 03 6.94 E+ 03 N/A 5.62 E+ 03 N/A N/A N/A MW48 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 155 Table D. 11 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 MW60R (Post) N/A N/A N/A 1.17 E+ 04 N/A 1.18 E+ 03 N/A 8.87 E+ 02 N/A N/A N/A 2.02 E+ 02 N/A 3.93 E+ 03 N/A N/A N/A N/A N/A N/A 1.27 E+ 04 N/A 5.61 E+ 02 N/A 9.23 E+ 02 N/A N/A N/A 2.79 E+ 02 N/A 2.91 E+ 03 N/A N/A N/A MW64 (Pre) N/A N/A N/A 1.55 E+ 04 N/A N/A N/A 5.31 E+ 02 N/A N/A N/A N/A N/A 3.09 E+ 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 8.58 E+ 02 N/A N/A N/A N/A N/A N/A 1.14 E+ 04 N/A N/A N/A 5.19 E+ 02 N/A N/A N/A N/A N/A 1.95 E+ 03 N/A N/A N/A MW64 (Post) N/A N/A N/A 1.48 E+ 05 N/A 3.20 E+ 04 N/A N/A N/A N/A N/A 1.30 E+ 03 N/A 2.25 E+ 04 N/A N/A N/A N/A N/A N/A 1.69 E+ 05 N/A 3.44 E+ 04 N/A N/A N/A N/A N/A 2.50 E+ 03 N/A 2.71 E+ 04 N/A N/A N/A MW66 (Pre) N/A N/A N/A 4.72 E+ 04 N/A 4.17 E+ 02 N/A N/A 7.41 E+ 02 N/A 6.19 E+ 02 N/A N/A 8.12 E+ 03 N/A N/A N/A N/A N/A N/A 5.06 E+ 04 N/A 4.06 E+ 02 N/A 1.74 E+ 03 5.30 E+ 02 N/A 5.61 E+ 02 6.27 E+ 02 N/A 6.47 E+ 03 8.85 E+ 02 N/A N/A N/A N/A N/A 1.83 E+ 04 N/A 2.14 E+ 02 N/A 1.52 E+ 03 N/A N/A 4.39 E+ 02 1.83 E+ 02 N/A 4.90 E+ 03 4.49 E+ 02 N/A N/A MW66 (Post) N/A N/A N/A 7.92 E+ 03 N/A 3.45 E+ 02 N/A N/A N/A N/A N/A N/A N/A 1.67 E+ 03 N/A N/A N/A N/A N/A N/A 2.04 E+ 04 N/A 1.26 E+ 03 N/A N/A N/A N/A N/A N/A N/A 6.76 E+ 03 N/A N/A N/A MW67 (Pre) 7.95 E+ 02 N/A N/A 6.43 E+ 03 N/A N/A N/A 2.34 E+ 03 N/A N/A N/A 1.50 E+ 03 N/A 2.82 E+ 03 N/A N/A N/A N/A N/A N/A 9.69 E+ 03 N/A 9.26 E+ 02 N/A 4.12 E+ 03 1.39 E+ 03 N/A N/A 2.16 E+ 03 N/A 3.52 E+ 03 N/A N/A N/A 4.72 E+ 02 N/A N/A 1.03 E+ 04 N/A 9.13 E+ 02 N/A 5.33 E+ 03 1.73 E+ 03 N/A N/A 2.67 E+ 03 N/A 2.22 E+ 03 N/A N/A N/A MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 156 Table D. 11 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 Red Cedar River (gene copies per mL of river water) Red Cedar N/A N/A N/A 2.36 E+ 05 N/A 8.20 E+ 02 N/A 4.47 E+ 03 N/A N/A N/A 4.98 E+ 02 N/A 2.19 E+ 04 N/A N/A N/A N/A N/A N/A 3.04 E+ 05 N/A 1.58 E+ 03 N/A 5.99 E+ 03 N/A N/A N/A 3.89 E+ 02 N/A 2.84 E+ 04 N/A N/A N/A Sediment (gene copies per g of sediment) MW71 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soil (gene copies per g of soil) MSU Soil E N/A N/A N/A 2.36 E+ 07 N/A N/A N/A 1.48 E+ 06 N/A N/A N/A 1.69 E+ 05 N/A 7.74 E+ 06 N/A N/A N/A N/A N/A N/A 7.82 E+ 06 N/A 8.77 E+ 04 N/A 5.12 E+ 05 N/A N/A N/A N/A N/A 2.18 E+ 06 N/A N/A N/A N/A N/A N/A 1.00 E+ 07 N/A 1.07 E+ 05 N/A 3.25 E+ 05 N/A N/A N/A 1.45 E+ 05 N/A 2.58 E+ 06 N/A N/A N/A MSU Soil F N/A N/A N/A N/A N/A 1.34 E+ 05 N/A 1.23 E+ 06 N/A N/A N/A N/A N/A 5.85 E+ 06 N/A N/A N/A N/A N/A N/A N/A N/A 5.70 E+ 04 N/A 4.39 E+ 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 3.54 E+ 05 N/A N/A N/A N/A N/A 1.35 E+ 06 N/A N/A N/A KBS T1 N/A N/A N/A 2.17 E+ 07 N/A 3.03 E+ 05 N/A 1.48 E+ 06 N/A N/A N/A 8.60 E+ 04 N/A 8.81 E+ 06 N/A N/A N/A N/A N/A N/A 1.00 E+ 07 N/A 1.51 E+ 05 N/A N/A N/A N/A N/A N/A N/A 3.36 E+ 06 N/A N/A N/A N/A N/A N/A 4.12 E+ 07 N/A N/A N/A 2.12 E+ 06 N/A N/A N/A 2.31 E+ 06 N/A 9.41 E+ 06 N/A N/A N/A KBS T2 N/A N/A N/A 1.04 E+ 07 N/A 1.23 E+ 05 N/A 5.39 E+ 05 N/A N/A N/A N/A N/A 4.27 E+ 06 N/A N/A N/A N/A N/A N/A 2.39 E+ 07 N/A 2.45 E+ 05 N/A 2.16 E+ 06 N/A N/A N/A 1.04 E+ 05 N/A N/A N/A N/A N/A N/A N/A N/A 1.88 E+ 07 N/A 2.27 E+ 05 N/A N/A N/A N/A N/A 8.82 E+ 04 N/A 5.30 E+ 06 N/A N/A N/A 157 Table D. 11 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 KBS T3 N/A N/A N/A N/A N/A N/A N/A 5.96 E+ 05 N/A N/A N/A N/A N/A 3.97 E+ 06 N/A N/A N/A N/A N/A N/A 1.05 E+ 07 N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.33 E+ 06 N/A N/A N/A N/A N/A N/A 6.05 E+ 07 N/A N/A N/A 2.84 E+ 06 N/A N/A N/A N/A N/A 8.75 E+ 06 N/A N/A N/A KBS T4 N/A N/A N/A N/A N/A 2.90 E+ 05 N/A 1.73 E+ 06 N/A N/A N/A N/A N/A 8.81 E+ 06 N/A N/A N/A 6.07 E+ 05 N/A N/A 3.43 E+ 07 N/A 3.18 E+ 05 N/A N/A N/A N/A N/A 1.47 E+ 05 N/A 1.18 E+ 07 1.20 E+ 06 N/A N/A 5.78 E+ 05 N/A N/A 5.49 E+ 07 N/A 5.54 E+ 05 N/A 2.71 E+ 06 N/A N/A N/A 3.85 E+ 05 N/A 1.20 E+ 07 1.05 E+ 06 N/A N/A Table D. 12 . Gene copies per mL or g of starting material for xenB . Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 Shallow Zone Aquifer (gene copies per mL of groundwater) MW32 (Pre) 1.64 E+ 04 3.59 E+ 03 4.23 E+ 03 2.51 E+ 03 2.03 E+ 04 8.35 E+ 03 3.64 E+ 03 N/A 5.59 E+ 03 1.18 E+ 03 1.72 E+ 04 4.25 E+ 04 N/A 5.84 E+ 03 N/A 1.46 E+ 04 4.08 E+ 03 5.35 E+ 03 2.48 E+ 03 1.39 E+ 04 7.35 E+ 03 3.24 E+ 03 6.24 E+ 03 7.28 E+ 03 8.62 E+ 02 1.94 E+ 04 6.86 E+ 04 N/A 4.43 E+ 03 N/A 1.88 E+ 04 3.87 E+ 03 7.79 E+ 03 3.27 E+ 03 1.83 E+ 04 7.45 E+ 03 4.47 E+ 03 6.39 E+ 03 6.00 E+ 03 1.23 E+ 03 2.76 E+ 04 6.50 E+ 04 N/A 4.87 E+ 03 N/A MW32 (Post) N/A N/A N/A 2.70 E+ 02 N/A N/A N/A N/A N/A 3.02 E+ 02 N/A N/A N/A N/A N/A N/A N/A N/A 6.01 E+ 02 N/A N/A N/A N/A N/A 1.34 E+ 03 N/A N/A N/A N/A N/A N/A N/A N/A 6.63 E+ 02 N/A N/A N/A N/A N/A 2.00 E+ 03 N/A N/A N/A N/A N/A MW62 (Pre) 4.02 E+ 04 3.28 E+ 03 N/A 2.33 E+ 03 1.14 E+ 05 8.75 E+ 03 3.53 E+ 03 1.02 E+ 05 4.04 E+ 04 N/A 3.17 E+ 04 N/A N/A 9.14 E+ 02 N/A 3.91 E+ 04 2.45 E+ 03 N/A N/A 1.43 E+ 05 6.51 E+ 03 2.98 E+ 03 8.76 E+ 04 2.45 E+ 04 7.78 E+ 02 4.14 E+ 04 N/A N/A 9.53 E+ 02 N/A 5.02 E+ 04 4.86 E+ 03 N/A 3.75 E+ 03 1.34 E+ 05 9.50 E+ 03 4.41 E+ 03 1.14 E+ 05 3.55 E+ 04 1.69 E+ 03 5.05 E+ 04 N/A N/A 1.24 E+ 03 N/A 158 Table D. 12 Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 MW62 (Post) 1.80 E+ 03 7.15 E+ 02 7.02 E+ 02 3.32 E+ 02 N/A 6.65 E+ 02 N/A N/A N/A 1.39 E+ 02 4.39 E+ 03 N/A N/A 3.46 E+ 02 N/A 3.85 E+ 03 3.97 E+ 03 4.13 E+ 03 2.01 E+ 03 N/A 5.76 E+ 03 N/A N/A N/A 4.63 E+ 02 4.66 E+ 04 N/A N/A 4.57 E+ 03 N/A 5.39 E+ 03 1.86 E+ 03 4.25 E+ 03 2.17 E+ 03 N/A 3.35 E+ 03 N/A N/A N/A 1.03 E+ 03 1.23 E+ 04 N/A N/A 2.85 E+ 03 N/A Perched Zone Aquifer (gene copies per mL of groundwater) MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 3.86 E+ 04 2.11 E+ 04 4.54 E+ 04 4.46 E+ 04 5.70 E+ 04 N/A 2.12 E+ 04 1.01 E+ 05 4.95 E+ 04 1.81 E+ 05 2.53 E+ 05 6.81 E+ 03 4.56 E+ 04 N/A N/A 8.45 E+ 04 4.66 E+ 04 1.03 E+ 05 8.58 E+ 04 1.20 E+ 05 N/A 3.28 E+ 04 1.66 E+ 05 1.20 E+ 05 4.56 E+ 05 6.95 E+ 05 1.09 E+ 04 1.13 E+ 05 N/A MW48 (Post) 5.16 E+ 04 N/A N/A 3.07 E+ 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.22 E+ 04 N/A N/A 1.83 E+ 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Pre) 5.37 E+ 03 1.38 E+ 03 N/A 7.23 E+ 02 N/A 2.51 E+ 03 N/A N/A N/A 2.26 E+ 02 N/A N/A N/A 1.84 E+ 03 N/A 9.72 E+ 02 N/A N/A 1.79 E+ 02 N/A N/A N/A N/A N/A 1.71 E+ 02 N/A N/A N/A 3.60 E+ 02 N/A 1.35 E+ 03 9.41 E+ 02 N/A 3.64 E+ 02 N/A 4.77 E+ 02 N/A N/A N/A 2.52 E+ 02 N/A N/A N/A 3.07 E+ 02 N/A MW60R (Post) 9.12 E+ 03 2.93 E+ 03 N/A 7.46 E+ 02 2.30 E+ 04 1.29 E+ 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A 5.54 E+ 03 2.10 E+ 03 N/A 4.10 E+ 02 7.79 E+ 03 6.48 E+ 02 N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Pre) 4.08 E+ 03 9.29 E+ 02 9.77 E+ 02 7.33 E+ 02 1.08 E+ 04 2.18 E+ 03 1.69 E+ 03 N/A N/A 7.11 E+ 01 9.92 E+ 03 N/A N/A 1.20 E+ 03 N/A 2.03 E+ 03 2.57 E+ 03 9.97 E+ 02 1.30 E+ 03 4.05 E+ 03 1.46 E+ 03 2.20 E+ 03 N/A N/A 1.07 E+ 03 3.68 E+ 03 N/A N/A 1.39 E+ 03 N/A 3.84 E+ 03 9.28 E+ 02 2.09 E+ 03 4.15 E+ 02 1.37 E+ 04 2.17 E+ 03 1.59 E+ 03 N/A N/A N/A 8.72 E+ 03 N/A N/A 1.01 E+ 03 N/A MW64 (Post) 3.18 E+ 04 N/A N/A 1.42 E+ 04 6.85 E+ 04 2.44 E+ 04 N/A N/A N/A 8.43 E+ 03 N/A N/A N/A 1.10 E+ 04 N/A 4.13 E+ 04 N/A N/A 1.71 E+ 04 8.59 E+ 04 3.91 E+ 04 N/A N/A N/A 1.23 E+ 04 N/A N/A N/A 1.33 E+ 04 N/A 159 Table D. 12 Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 MW66 (Pre) 1.37 E+ 04 3.53 E+ 03 5.21 E+ 03 2.70 E+ 03 1.61 E+ 04 5.98 E+ 03 4.54 E+ 03 N/A N/A 8.43 E+ 02 3.84 E+ 04 6.94 E+ 04 N/A 4.75 E+ 03 N/A 1.26 E+ 04 4.04 E+ 03 5.39 E+ 03 2.69 E+ 03 2.07 E+ 04 7.40 E+ 03 4.94 E+ 03 N/A N/A N/A 3.60 E+ 04 8.15 E+ 04 N/A 3.90 E+ 03 N/A 6.05 E+ 03 8.38 E+ 02 1.22 E+ 03 5.96 E+ 02 8.69 E+ 03 1.56 E+ 03 6.41 E+ 02 N/A N/A 1.59 E+ 02 8.41 E+ 03 N/A N/A 5.35 E+ 02 N/A MW66 (Post) 5.22 E+ 03 5.66 E+ 02 1.55 E+ 03 3.69 E+ 02 1.56 E+ 04 2.05 E+ 03 2.13 E+ 03 N/A N/A 4.72 E+ 02 4.42 E+ 03 N/A N/A 1.15 E+ 03 N/A 2.44 E+ 04 3.53 E+ 03 6.51 E+ 03 2.65 E+ 03 7.11 E+ 04 7.84 E+ 03 4.92 E+ 03 N/A N/A 1.26 E+ 03 1.40 E+ 04 N/A N/A 3.22 E+ 03 N/A MW67 (Pre) 1.10 E+ 04 2.98 E+ 03 4.36 E+ 03 2.50 E+ 03 2.64 E+ 04 5.35 E+ 03 6.28 E+ 03 2.89 E+ 04 N/A 5.98 E+ 02 3.45 E+ 04 1.02 E+ 05 N/A 4.96 E+ 03 N/A 1.40 E+ 04 4.04 E+ 03 4.71 E+ 03 2.21 E+ 03 2.85 E+ 04 4.43 E+ 03 3.57 E+ 03 2.54 E+ 04 N/A 5.61 E+ 02 2.42 E+ 04 4.41 E+ 04 N/A 2.84 E+ 03 N/A 1.57 E+ 04 4.94 E+ 03 7.71 E+ 03 3.40 E+ 03 3.79 E+ 04 5.82 E+ 03 7.97 E+ 03 2.85 E+ 04 N/A 1.49 E+ 03 3.21 E+ 04 1.10 E+ 05 N/A 5.50 E+ 03 N/A MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Red Cedar River (gene copies per mL of river water) Red Cedar 3.44 E+ 04 N/A N/A 1.60 E+ 03 N/A 2.83 E+ 03 N/A 1.59 E+ 04 N/A N/A N/A N/A N/A N/A N/A 4.61 E+ 04 N/A N/A 3.19 E+ 03 N/A 6.36 E+ 03 N/A 3.23 E+ 04 N/A N/A N/A N/A N/A N/A N/A Sediment (gene copies per g of sediment) MW71 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soil (gene copies per g of soil) MSU Soil E 1.98 E+ 06 1.23 E+ 06 N/A 7.01 E+ 05 N/A 9.05 E+ 05 N/A N/A N/A N/A N/A N/A N/A 2.25 E+ 05 N/A N/A N/A N/A 1.71 E+ 05 N/A 3.34 E+ 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.37 E+ 06 4.51 E+ 05 N/A 3.09 E+ 05 N/A 4.10 E+ 05 N/A N/A N/A N/A N/A N/A N/A 1.55 E+ 05 N/A 160 Table D. 12 Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 MSU Soil F 3.31 E+ 06 N/A N/A 6.83 E+ 05 4.19 E+ 06 9.09 E+ 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.23 E+ 06 N/A N/A 2.35 E+ 05 N/A 5.10 E+ 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 3.00 E+ 06 N/A N/A 3.03 E+ 05 3.56 E+ 06 5.18 E+ 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A KBS T1 4.87 E+ 06 1.06 E+ 06 N/A N/A 3.92 E+ 06 1.21 E+ 06 N/A 5.36 E+ 06 N/A 9.99 E+ 04 N/A N/A N/A 2.04 E+ 05 N/A 7.82 E+ 06 8.72 E+ 05 N/A 2.05 E+ 05 5.12 E+ 06 3.13 E+ 06 1.60 E+ 06 N/A N/A 1.37 E+ 06 N/A N/A N/A 1.44 E+ 06 N/A 4.89 E+ 07 5.73 E+ 06 N/A 3.09 E+ 06 1.78 E+ 07 1.99 E+ 07 2.34 E+ 06 1.68 E+ 07 N/A 1.25 E+ 07 N/A N/A N/A 1.45 E+ 07 N/A KBS T2 5.78 E+ 06 8.40 E+ 05 6.31 E+ 05 3.55 E+ 05 5.04 E+ 06 1.87 E+ 06 5.61 E+ 05 3.44 E+ 06 N/A 7.55 E+ 05 N/A N/A N/A 1.16 E+ 06 N/A 2.08 E+ 06 7.94 E+ 05 N/A 5.29 E+ 05 N/A 1.59 E+ 06 N/A 4.82 E+ 06 N/A 2.14 E+ 05 N/A N/A N/A 4.50 E+ 05 N/A 9.27 E+ 06 N/A 8.36 E+ 05 5.00 E+ 05 9.91 E+ 06 3.55 E+ 06 8.79 E+ 05 4.90 E+ 06 N/A 2.39 E+ 06 N/A N/A N/A 1.67 E+ 06 N/A KBS T3 4.37 E+ 06 6.31 E+ 05 N/A 3.41 E+ 05 N/A 1.93 E+ 06 6.50 E+ 05 N/A N/A 5.51 E+ 05 N/A N/A N/A 7.99 E+ 05 N/A 2.82 E+ 06 6.96 E+ 05 N/A 3.37 E+ 05 N/A 7.60 E+ 05 6.56 E+ 05 N/A N/A 4.89 E+ 05 N/A N/A N/A 4.11 E+ 05 N/A 6.74 E+ 06 2.15 E+ 06 N/A 1.25 E+ 06 N/A 2.97 E+ 06 N/A N/A N/A 2.59 E+ 05 N/A N/A N/A 9.85 E+ 05 N/A KBS T4 7.10 E+ 06 N/A N/A 5.10 E+ 05 N/A 4.78 E+ 06 N/A 5.91 E+ 06 N/A 2.04 E+ 06 N/A N/A N/A 1.78 E+ 06 N/A 6.28 E+ 06 2.29 E+ 06 N/A 6.44 E+ 05 N/A 2.93 E+ 06 N/A 5.59 E+ 06 N/A 3.35 E+ 05 N/A N/A N/A 7.61 E+ 05 N/A 5.75 E+ 06 9.88 E+ 05 N/A 1.01 E+ 06 N/A 1.41 E+ 06 N/A 7.10 E+ 06 N/A 4.07 E+ 05 N/A N/A N/A 7.48 E+ 05 N/A 161 Table D. 13 . Gene copies per mL or g of starting material for xplA false positive amplification . Sample PS3 PS4 PS5 Pub1 Pub2 Pub3 Pub4 Pub6 Shallow Zone Aquifer (gene copies per mL of groundwater) MW32 (Pre) N/A 2.51E+03 5.42E+04 N/A 7.83E+02 N/A 1.09E+03 3.24E+03 N/A 2.34E+03 6.52E+04 N/A 5.57E+02 N/A 6.39E+02 2.28E+03 N/A 2.49E+03 N/A N/A 2.20E+02 N/A 4.25E+02 3.62E+03 MW32 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW62 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.49E+05 N/A N/A N/A N/A 6.17E+03 N/A N/A 3.29E+05 N/A N/A N/A N/A 1.03E+04 MW62 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Perched Zone Aquifer (gene copies per mL of groundwater) MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A 1.14E+04 N/A N/A N/A N/A N/A N/A N/A 1.19E+04 N/A N/A N/A N/A N/A N/A N/A MW48 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Post) N/A N/A N/A N/A N/A N/A N/A 8.13E+03 N/A N/A N/A N/A N/A N/A N/A 5.73E+03 MW64 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Post) N/A N/A 5.14E+05 N/A N/A N/A N/A N/A N/A N/A 4.38E+05 N/A N/A N/A N/A N/A MW66 (Pre) N/A 2.24E+03 N/A 1.50E+02 N/A N/A N/A N/A N/A 1.39E+03 N/A 1.11E+02 N/A N/A N/A N/A N/A 1.53E+03 N/A N/A N/A N/A N/A N/A MW66 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW67 (Pre) N/A N/A 2.74E+04 N/A N/A N/A N/A N/A N/A N/A 3.67E+04 N/A N/A N/A N/A N/A N/A N/A 3.09E+04 N/A N/A N/A N/A N/A MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Red Cedar River (gene copies per mL of river water) Red Cedar N/A 1.75E+04 2.31E+05 N/A N/A N/A N/A 3.63E+03 N/A 2.32E+04 3.30E+05 N/A N/A N/A N/A 4.39E+03 Sediment (gene copies per g of sediment) MW71 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soils (gene copies per g of soil) MSU Soil E N/A N/A 8.15E+07 N/A N/A N/A N/A N/A N/A N/A 4.50E+07 N/A N/A N/A N/A N/A N/A N/A 3.98E+07 N/A N/A N/A N/A N/A 162 Table D. 13 Sample PS3 PS4 PS5 Pub1 Pub2 Pub3 Pub4 Pub6 MSU Soil F N/A N/A 5.39E+07 N/A N/A N/A N/A 9.42E+05 N/A N/A 2.69E+07 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 6.03E+05 KBS T1 N/A N/A 6.31E+07 N/A N/A N/A N/A 9.56E+05 N/A N/A 5.96E+07 N/A N/A N/A N/A 7.66E+05 N/A N/A 1.52E+08 N/A N/A N/A N/A 3.11E+06 KBS T2 N/A N/A 4.36E+07 N/A N/A N/A N/A N/A N/A N/A 8.33E+07 N/A N/A N/A N/A 1.98E+06 N/A N/A 1.07E+08 N/A N/A N/A N/A 3.81E+06 KBS T3 N/A 8.02E+05 4.63E+07 N/A N/A N/A N/A N/A N/A N/A 5.07E+07 N/A N/A N/A N/A N/A N/A 2.39E+06 1.05E+08 N/A N/A N/A N/A N/A KBS T4 N/A N/A 9.73E+07 N/A N/A N/A N/A N/A N/A N/A 1.35E+08 N/A N/A N/A N/A N/A N/A N/A 1.60E+08 N/A N/A N/A N/A N/A Table D. 14 . Gene copies per mL or g of starting material for 16S rRNA amplification or false positive amplification Sample 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 Shallow Zone Aquifer (gene copies per mL of groundwater) MW32 (Pre) 1.35E+06 1.63E+06 1.69E+06 1.29E+06 1.49E+06 1.50E+06 1.67E+06 1.76E+06 1.88E+06 MW32 (Post) 2.26E+07 2.42E+07 2.33E+07 7.03E+07 6.94E+07 7.32E+07 6.39E+07 8.09E+07 5.88E+07 MW62 (Pre) 2.07E+07 2.46E+07 2.65E+07 1.86E+07 2.40E+07 2.24E+07 3.08E+07 3.00E+07 3.21E+07 MW62 (Post) 1.67E+07 1.98E+07 1.66E+07 9.59E+07 7.13E+07 9.44E+07 4.00E+07 5.00E+07 5.21E+07 Perched Zone Aquifer (gene copies per mL of groundwater) MW48 (Pre) 4.40E+03 5.87E+03 2.34E+04 1.72E+06 1.93E+06 2.15E+06 3.61E+06 3.87E+06 4.03E+06 MW48 (Post) 2.82E+08 2.58E+08 3.08E+08 3.15E+08 3.21E+08 4.10E+08 MW60R (Pre) 9.13E+05 9.71E+05 1.18E+06 1.40E+04 1.81E+04 3.06E+04 1.61E+04 1.33E+04 2.51E+04 MW60R (Post) 1.10E+07 1.52E+07 1.51E+07 9.40E+06 1.06E+07 1.10E+07 MW64 (Pre) 4.00E+05 5.04E+05 6.73E+05 4.12E+04 1.35E+05 2.01E+05 4.94E+05 5.50E+05 8.64E+05 MW64 (Post) 1.38E+08 1.59E+08 1.47E+08 1.40E+08 1.35E+08 1.67E+08 163 Table D. 14 Sample 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 MW66 (Pre) 1.79E+06 2.59E+06 2.97E+06 1.67E+06 1.47E+06 2.29E+06 N/A 1.44E+06 1.65E+06 MW66 (Post) 3.62E+06 5.04E+06 5.04E+06 2.88E+07 3.11E+07 2.90E+07 MW67 (Pre) 6.85E+05 8.41E+05 1.02E+06 6.46E+05 9.25E+05 9.75E+05 8.14E+05 9.61E+05 1.17E+06 MW67 (Post) 1.03E+03 1.43E+03 2.60E+03 2.99E+03 3.77E+03 6.42E+03 Red Cedar River (gene copies per mL of river water) Red Cedar 9.13E+06 1.10E+07 1.16E+07 9.78E+06 1.17E+07 1.20E+07 Sediment (gene copies per g of sediment) MW71 2.15E+07 2.60E+07 2.53E+07 2.90E+07 2.41E+07 4.73E+07 2.07E+07 1.60E+07 4.25E+07 Agricultural Soils (gene copies per g of soil) MSU Soil E 1.46E+09 1.15E+09 1.60E+09 4.03E+08 8.27E+08 1.04E+09 5.73E+08 7.55E+08 8.04E+08 MSU Soil F 1.12E+09 1.37E+09 1.48E+09 6.00E+08 6.44E+08 7.84E+08 4.37E+08 6.14E+08 7.09E+08 KBS T1 1.17E+09 1.58E+09 1.78E+09 1.16E+09 8.33E+08 1.47E+09 2.49E+09 3.89E+09 3.97E+09 KBS T2 9.65E+08 1.26E+09 1.60E+09 1.28E+09 1.55E+09 1.79E+09 1.33E+09 1.44E+09 1.68E+09 KBS T3 8.68E+08 1.02E+09 1.24E+09 1.08E+09 1.22E+09 1.51E+09 1.47E+09 1.59E+09 2.11E+09 KBS T4 1.73E+09 1.88E+09 2.45E+09 1.79E+09 2.05E+09 2.39E+09 1.77E+09 2.10E+09 2.11E+09 164 Table D. 15 . Gene copies per 16S rRNA gene copy for nfsI positive amplification. Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 Pub1 Pub2 Shallow Zone Aquifer MW32 (Pre) 6.53E - 04 N/A N/A N/A N/A 1.71E - 04 N/A N/A N/A N/A 6.89E - 04 N/A N/A N/A N/A 1.60E - 04 N/A N/A N/A N/A 5.67E - 04 N/A N/A N/A N/A 1.50E - 04 N/A N/A N/A N/A MW32 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW62 (Pre) 7.72E - 04 N/A N/A N/A N/A 2.38E - 04 N/A N/A N/A N/A 7.71E - 04 N/A N/A N/A N/A 1.59E - 04 N/A N/A N/A N/A 7.37E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A MW62 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Perched Zone Aquifer MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.80E - 03 N/A N/A N/A N/A N/A 2.06E - 04 N/A N/A N/A 3.86E - 03 N/A N/A N/A N/A N/A 2.04E - 04 N/A N/A N/A MW48 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Post) 4.06E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 3.67E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Post) N/A N/A N/A N/A N/A N/A 4.73E - 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 5.78E - 05 N/A N/A N/A MW66 (Pre) 8.20E - 04 N/A N/A N/A N/A N/A 6.75E - 04 N/A N/A N/A 7.97E - 04 N/A N/A N/A N/A N/A 6.65E - 04 N/A N/A N/A 8.43E - 04 N/A N/A N/A N/A N/A 8.95E - 04 N/A N/A N/A MW66 (Post) N/A N/A N/A N/A N/A N/A 5.57E - 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.36E - 05 N/A N/A N/A MW67 (Pre) 1.40E - 03 N/A N/A N/A N/A N/A 5.59E - 04 N/A N/A N/A 9.55E - 04 N/A N/A N/A N/A N/A 4.50E - 04 N/A N/A N/A 1.64E - 03 N/A N/A N/A N/A N/A 3.66E - 04 N/A N/A N/A MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Red Cedar River Red Cedar N/A N/A N/A N/A N/A N/A 3.18E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 3.56E - 04 N/A N/A N/A Sediment MW71 N/A N/A N/A N/A N/A N/A 8.93E - 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A 9.20E - 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soils MSU Soil E 9.11E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.97E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 6.17E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 165 Table D. 15 . Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 Pub1 Pub2 MSU Soil F N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A KBS T1 5.81E - 04 N/A N/A N/A N/A N/A 4.22E - 04 N/A N/A N/A 5.22E - 04 N/A N/A N/A N/A 3.13E - 04 4.40E - 04 N/A N/A N/A 6.72E - 04 N/A N/A N/A N/A 1.84E - 04 N/A N/A N/A N/A KBS T2 N/A N/A N/A N/A N/A 1.53E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.45E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.08E - 04 N/A N/A N/A N/A KBS T3 5.99E - 04 N/A N/A N/A N/A 2.24E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 5.24E - 04 N/A N/A N/A 1.05E - 03 N/A N/A N/A N/A 1.66E - 04 6.90E - 04 N/A N/A N/A KBS T4 8.65E - 04 N/A N/A N/A N/A 1.56E - 04 3.41E - 04 N/A N/A N/A N/A N/A N/A N/A N/A 3.06E - 04 6.87E - 04 N/A N/A N/A 1.18E - 03 N/A N/A N/A N/A 3.10E - 04 9.41E - 04 N/A N/A N/A Table D. 16 . Gene copies per 16S rRNA gene copy for pnrB positive amplification. Sample PS1 PS2 PS3 PS4 PS5 Pub1 Pub2 Shallow Zone Aquifer MW32 (Pre) 1.02E - 04 5.37E - 03 2.77E - 03 6.10E - 05 N/A N/A 2.67E - 03 1.62E - 04 4.35E - 03 3.96E - 03 N/A N/A N/A 3.24E - 03 N/A 3.63E - 03 N/A 1.21E - 04 N/A N/A 1.57E - 03 MW32 (Post) N/A N/A 4.98E - 04 N/A N/A N/A N/A N/A N/A 3.53E - 04 N/A N/A N/A 7.29E - 05 N/A N/A 5.41E - 04 N/A N/A N/A 1.20E - 04 MW62 (Pre) 4.16E - 05 2.92E - 03 1.25E - 02 N/A N/A N/A N/A 3.64E - 05 2.94E - 03 1.51E - 02 N/A N/A N/A N/A 2.96E - 05 2.45E - 03 1.10E - 02 N/A N/A N/A N/A MW62 (Post) N/A 1.85E - 04 N/A N/A N/A N/A N/A N/A 5.68E - 04 N/A N/A N/A N/A N/A N/A 7.33E - 04 N/A N/A N/A N/A N/A Perched Zone Aquifer MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A 5.25E - 03 N/A 6.45E - 05 N/A N/A 1.59E - 03 N/A 5.21E - 03 N/A 3.90E - 05 N/A N/A 8.67E - 04 MW48 (Post) N/A 4.39E - 04 N/A N/A N/A N/A N/A N/A 3.15E - 04 N/A N/A N/A N/A N/A MW60R (Pre) N/A 3.32E - 02 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 5.25E - 01 N/A N/A N/A N/A N/A MW60R (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Post) N/A 3.64E - 03 N/A N/A N/A N/A N/A N/A 4.79E - 03 N/A N/A N/A N/A N/A 166 Table D. 16 Sample PS1 PS2 PS3 PS4 PS5 Pub1 Pub2 MW66 (Pre) N/A 2.12E - 03 2.98E - 03 N/A N/A N/A 1.08E - 03 N/A 2.74E - 03 4.26E - 03 N/A N/A N/A 8.81E - 04 N/A N/A N/A N/A N/A N/A N/A MW66 (Post) N/A 2.68E - 03 N/A N/A N/A N/A N/A N/A 1.62E - 03 N/A N/A N/A N/A N/A MW67 (Pre) N/A N/A N/A N/A N/A N/A 1.33E - 03 N/A N/A N/A N/A N/A N/A 2.11E - 03 N/A N/A N/A N/A N/A N/A 1.50E - 03 MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Red Cedar River Red Cedar N/A N/A N/A 5.65E - 05 N/A N/A 1.23E - 03 N/A N/A N/A 1.05E - 04 N/A N/A 1.93E - 03 Sediment MW71 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soils MSU Soil E N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MSU Soil F N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A KBS T1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A KBS T2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 9.58E - 05 N/A N/A N/A N/A N/A N/A 6.03E - 05 N/A N/A N/A KBS T3 1.16E - 04 N/A N/A N/A N/A N/A 9.00E - 04 1.27E - 04 N/A N/A 1.06E - 04 N/A N/A N/A 1.65E - 04 N/A N/A 1.37E - 04 N/A N/A 1.44E - 03 KBS T4 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 167 Table D. 17 . Gene copies per 16S rRNA gene copy for xenA Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 Shallow Zone Aquifer MW32 (Pre) 1.90 E - 04 N/A N/A 2.25 E - 02 N/A 2.47 E - 04 N/A N/A 4.78 E - 04 7.09 E - 04 N/A 4.76 E - 04 N/A 2.60 E - 03 1.13 E - 03 N/A N/A 4.26 E - 04 N/A N/A 1.86 E - 02 N/A 4.29 E - 04 N/A N/A 3.29 E - 04 5.02 E - 04 N/A 8.26 E - 04 N/A 2.97 E - 03 1.05 E - 03 N/A N/A 3.96 E - 04 N/A N/A 2.20 E - 02 N/A 4.32 E - 04 N/A N/A 6.60 E - 04 3.47 E - 04 N/A 4.45 E - 04 N/A 2.32 E - 03 1.79 E - 03 N/A N/A MW32 (Post) N/A N/A N/A 1.57 E - 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.51 E - 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A 3.06 E - 05 N/A N/A N/A N/A N/A N/A 1.87 E - 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.19 E - 05 N/A N/A N/A MW62 (Pre) 9.09 E - 05 N/A N/A 4.78 E - 03 N/A 2.04 E - 05 N/A 3.52 E - 04 N/A N/A N/A N/A N/A 1.14 E - 03 N/A N/A N/A 1.78 E - 04 N/A N/A 6.22 E - 03 N/A 4.19 E - 05 N/A 2.95 E - 04 N/A N/A N/A N/A N/A 1.11 E - 03 N/A N/A N/A 1.37 E - 04 N/A N/A 5.69 E - 03 N/A 3.61 E - 05 N/A 2.92 E - 04 N/A N/A N/A N/A N/A 7.96 E - 04 N/A N/A N/A MW62 (Post) N/A N/A N/A N/A N/A 1.54 E - 05 N/A N/A N/A N/A N/A N/A N/A 5.23 E - 05 N/A N/A N/A N/A N/A N/A N/A N/A 2.29 E - 05 N/A N/A N/A N/A N/A N/A N/A 4.37 E - 05 N/A N/A N/A N/A N/A N/A N/A N/A 2.91 E - 05 N/A N/A N/A N/A N/A N/A N/A 6.59 E - 05 N/A N/A N/A Perched Zone Aquifer MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.38 E - 04 N/A N/A 1.05 E - 01 N/A 8.51 E - 04 N/A N/A 1.10 E - 03 4.04 E - 04 1.89 E - 03 1.50 E - 03 N/A 2.28 E - 03 N/A N/A N/A 7.75 E - 05 N/A N/A 1.35 E - 01 N/A 1.51 E - 03 N/A N/A 1.06 E - 03 2.97 E - 04 1.93 E - 03 1.72 E - 03 N/A 1.39 E - 03 N/A N/A N/A MW48 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 168 Table D. 17 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 MW60R (Post) N/A N/A N/A 7.73 E - 04 N/A 7.81 E - 05 N/A 5.88 E - 05 N/A N/A N/A 1.34 E - 05 N/A 2.61 E - 04 N/A N/A N/A N/A N/A N/A 1.15 E - 03 N/A 5.08 E - 05 N/A 8.36 E - 05 N/A N/A N/A 2.53 E - 05 N/A 2.63 E - 04 N/A N/A N/A MW64 (Pre) N/A N/A N/A 2.30 E - 02 N/A N/A N/A 7.89 E - 04 N/A N/A N/A N/A N/A 4.59 E - 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.26 E - 03 N/A N/A N/A N/A N/A N/A 1.32 E - 02 N/A N/A N/A 6.01 E - 04 N/A N/A N/A N/A N/A 2.25 E - 03 N/A N/A N/A MW64 (Post) N/A N/A N/A 1.01 E - 03 N/A 2.18 E - 04 N/A N/A N/A N/A N/A 8.87 E - 06 N/A 1.53 E - 04 N/A N/A N/A N/A N/A N/A 1.01 E - 03 N/A 2.07 E - 04 N/A N/A N/A N/A N/A 1.50 E - 05 N/A 1.62 E - 04 N/A N/A N/A MW66 (Pre) N/A N/A N/A 1.59 E - 02 N/A 1.40 E - 04 N/A N/A 2.49 E - 04 N/A 2.08 E - 04 N/A N/A 2.73 E - 03 N/A N/A N/A N/A N/A N/A 2.21 E - 02 N/A 1.77 E - 04 N/A 7.59 E - 04 2.31 E - 04 N/A 2.45 E - 04 2.73 E - 04 N/A 2.82 E - 03 3.86 E - 04 N/A N/A N/A N/A N/A 1.11 E - 02 N/A 1.30 E - 04 N/A 9.22 E - 04 N/A N/A 2.67 E - 04 1.11 E - 04 N/A 2.98 E - 03 2.73 E - 04 N/A N/A MW66 (Post) N/A N/A N/A 1.57 E - 03 N/A 6.84 E - 05 N/A N/A N/A N/A N/A N/A N/A 3.31 E - 04 N/A N/A N/A N/A N/A N/A 7.02 E - 04 N/A 4.33 E - 05 N/A N/A N/A N/A N/A N/A N/A 2.33 E - 04 N/A N/A N/A MW67 (Pre) 7.83 E - 04 N/A N/A 6.33 E - 03 N/A N/A N/A 2.31 E - 03 N/A N/A N/A 1.48 E - 03 N/A 2.77 E - 03 N/A N/A N/A N/A N/A N/A 9.94 E - 03 N/A 9.50 E - 04 N/A 4.23 E - 03 1.42 E - 03 N/A N/A 2.21 E - 03 N/A 3.61 E - 03 N/A N/A N/A 4.05 E - 04 N/A N/A 8.88 E - 03 N/A 7.83 E - 04 N/A 4.57 E - 03 1.49 E - 03 N/A N/A 2.29 E - 03 N/A 1.91 E - 03 N/A N/A N/A MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 169 Table D. 17 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 Red Cedar River Red Cedar N/A N/A N/A 2.03 E - 02 N/A 7.06 E - 05 N/A 3.85 E - 04 N/A N/A N/A 4.29 E - 05 N/A 1.88 E - 03 N/A N/A N/A N/A N/A N/A 2.55 E - 02 N/A 1.32 E - 04 N/A 5.01 E - 04 N/A N/A N/A 3.25 E - 05 N/A 2.38 E - 03 N/A N/A N/A Sediment MW71 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soil MSU Soil E N/A N/A N/A 1.47 E - 02 N/A N/A N/A 9.21 E - 04 N/A N/A N/A 1.06 E - 04 N/A 4.83 E - 03 N/A N/A N/A N/A N/A N/A 7.53 E - 03 N/A 8.45 E - 05 N/A 4.94 E - 04 N/A N/A N/A N/A N/A 2.10 E - 03 N/A N/A N/A N/A N/A N/A 1.25 E - 02 N/A 1.33 E - 04 N/A 4.04 E - 04 N/A N/A N/A 1.80 E - 04 N/A 3.21 E - 03 N/A N/A N/A MSU Soil F N/A N/A N/A N/A N/A 9.07 E - 05 N/A 8.33 E - 04 N/A N/A N/A N/A N/A 3.97 E - 03 N/A N/A N/A N/A N/A N/A N/A N/A 7.27 E - 05 N/A 5.60 E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 4.99 E - 04 N/A N/A N/A N/A N/A 1.90 E - 03 N/A N/A N/A KBS T1 N/A N/A N/A 1.22 E - 02 N/A 1.71 E - 04 N/A 8.35 E - 04 N/A N/A N/A 4.84 E - 05 N/A 4.96 E - 03 N/A N/A N/A N/A N/A N/A 6.84 E - 03 N/A 1.03 E - 04 N/A N/A N/A N/A N/A N/A N/A 2.29 E - 03 N/A N/A N/A N/A N/A N/A 1.04 E - 02 N/A N/A N/A 5.33 E - 04 N/A N/A N/A 5.82 E - 04 N/A 2.37 E - 03 N/A N/A N/A KBS T2 N/A N/A N/A 6.49 E - 03 N/A 7.70 E - 05 N/A 3.38 E - 04 N/A N/A N/A N/A N/A 2.67 E - 03 N/A N/A N/A N/A N/A N/A 1.34 E - 02 N/A 1.37 E - 04 N/A 1.21 E - 03 N/A N/A N/A 5.84 E - 05 N/A N/A N/A N/A N/A N/A N/A N/A 1.12 E - 02 N/A 1.35 E - 04 N/A N/A N/A N/A N/A 5.23 E - 05 N/A 3.15 E - 03 N/A N/A N/A 170 Table D. 17 Sample PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS12 PS14 PS15 PS16 PS17 Pub1 KBS T3 N/A N/A N/A N/A N/A N/A N/A 4.81 E - 04 N/A N/A N/A N/A N/A 3.20 E - 03 N/A N/A N/A N/A N/A N/A 6.95 E - 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.86 E - 03 N/A N/A N/A N/A N/A N/A 2.87 E - 02 N/A N/A N/A 1.35 E - 03 N/A N/A N/A N/A N/A 4.15 E - 03 N/A N/A N/A KBS T4 N/A N/A N/A N/A N/A 1.18 E - 04 N/A 7.07 E - 04 N/A N/A N/A N/A N/A 3.60 E - 03 N/A N/A N/A 2.54 E - 04 N/A N/A 1.43 E - 02 N/A 1.33 E - 04 N/A N/A N/A N/A N/A 6.16 E - 05 N/A 4.94 E - 03 5.00 E - 04 N/A N/A 2.74 E - 04 N/A N/A 2.60 E - 02 N/A 2.63 E - 04 N/A 1.29 E - 03 N/A N/A N/A 1.83 E - 04 N/A 5.69 E - 03 4.98 E - 04 N/A N/A Table D. 18 . Gene copies per 16S rRNA gene copy for xenB Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 Shallow Zone Aquifer MW32 (Pre) 1.22 E - 02 2.66 E - 03 3.13 E - 03 1.86 E - 03 1.50 E - 02 6.19 E - 03 2.70 E - 03 N/A 4.14 E - 03 8.75 E - 04 1.27 E - 02 3.15 E - 02 N/A 4.33 E - 03 N/A 1.13 E - 02 3.16 E - 03 4.14 E - 03 1.92 E - 03 1.08 E - 02 5.70 E - 03 2.51 E - 03 4.83 E - 03 5.64 E - 03 6.68 E - 04 1.50 E - 02 5.32 E - 02 N/A 3.43 E - 03 N/A 1.13 E - 02 2.31 E - 03 4.66 E - 03 1.95 E - 03 1.10 E - 02 4.46 E - 03 2.68 E - 03 3.82 E - 03 3.59 E - 03 7.38 E - 04 1.65 E - 02 3.89 E - 02 N/A 2.92 E - 03 N/A MW32 (Post) N/A N/A N/A 1.20 E - 05 N/A N/A N/A N/A N/A 1.34 E - 05 N/A N/A N/A N/A N/A N/A N/A N/A 8.55 E - 06 N/A N/A N/A N/A N/A 1.91 E - 05 N/A N/A N/A N/A N/A N/A N/A N/A 1.04 E - 05 N/A N/A N/A N/A N/A 3.14 E - 05 N/A N/A N/A N/A N/A MW62 (Pre) 1.94 E - 03 1.58 E - 04 N/A 1.12 E - 04 5.50 E - 03 4.22 E - 04 1.70 E - 04 4.90 E - 03 1.95 E - 03 N/A 1.53 E - 03 N/A N/A 4.40 E - 05 N/A 2.10 E - 03 1.32 E - 04 N/A N/A 7.68 E - 03 3.50 E - 04 1.60 E - 04 4.71 E - 03 1.32 E - 03 4.19 E - 05 2.23 E - 03 N/A N/A 5.13 E - 05 N/A 1.63 E - 03 1.58 E - 04 N/A 1.22 E - 04 4.36 E - 03 3.08 E - 04 1.43 E - 04 3.71 E - 03 1.15 E - 03 5.50 E - 05 1.64 E - 03 N/A N/A 4.03 E - 05 N/A 171 Table D. 18 Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 MW62 (Post) 1.08 E - 04 4.29 E - 05 4.21 E - 05 1.99 E - 05 N/A 3.99 E - 05 N/A N/A N/A 8.34 E - 06 2.63 E - 04 N/A N/A 2.07 E - 05 N/A 4.02 E - 05 4.14 E - 05 4.31 E - 05 2.10 E - 05 N/A 6.00 E - 05 N/A N/A N/A 4.83 E - 06 4.86 E - 04 N/A N/A 4.77 E - 05 N/A 1.35 E - 04 4.66 E - 05 1.06 E - 04 5.42 E - 05 N/A 8.39 E - 05 N/A N/A N/A 2.57 E - 05 3.07 E - 04 N/A N/A 7.13 E - 05 N/A Perched Zone Aquifer MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.24 E - 02 1.23 E - 02 2.64 E - 02 2.59 E - 02 3.31 E - 02 N/A 1.23 E - 02 5.89 E - 02 2.88 E - 02 1.05 E - 01 1.47 E - 01 3.95 E - 03 2.65 E - 02 N/A N/A 2.34 E - 02 1.29 E - 02 2.85 E - 02 2.38 E - 02 3.33 E - 02 N/A 9.08 E - 03 4.59 E - 02 3.31 E - 02 1.26 E - 01 1.93 E - 01 3.01 E - 03 3.14 E - 02 N/A MW48 (Post) 1.83 E - 04 N/A N/A 1.09 E - 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.34 E - 04 N/A N/A 5.82 E - 06 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Pre) 5.88 E - 03 1.52 E - 03 N/A 7.92 E - 04 N/A 2.75 E - 03 N/A N/A N/A 2.48 E - 04 N/A N/A N/A 2.01 E - 03 N/A 6.96 E - 02 N/A N/A 1.28 E - 02 N/A N/A N/A N/A N/A 1.22 E - 02 N/A N/A N/A 2.58 E - 02 N/A 8.35 E - 02 5.84 E - 02 N/A 2.26 E - 02 N/A 2.96 E - 02 N/A N/A N/A 1.57 E - 02 N/A N/A N/A 1.90 E - 02 N/A MW60R (Post) 8.28 E - 04 2.67 E - 04 N/A 6.78 E - 05 2.09 E - 03 1.17 E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 5.89 E - 04 2.23 E - 04 N/A 4.36 E - 05 8.29 E - 04 6.89 E - 05 N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Pre) 1.02 E - 02 2.33 E - 03 2.44 E - 03 1.83 E - 03 2.71 E - 02 5.45 E - 03 4.22 E - 03 N/A N/A 1.78 E - 04 2.48 E - 02 N/A N/A 3.01 E - 03 N/A 4.92 E - 02 6.24 E - 02 2.42 E - 02 3.17 E - 02 9.82 E - 02 3.55 E - 02 5.34 E - 02 N/A N/A 2.60 E - 02 8.94 E - 02 N/A N/A 3.38 E - 02 N/A 7.76 E - 03 1.88 E - 03 4.22 E - 03 8.40 E - 04 2.78 E - 02 4.40 E - 03 3.21 E - 03 N/A N/A N/A 1.76 E - 02 N/A N/A 2.03 E - 03 N/A MW64 (Post) 2.31 E - 04 N/A N/A 1.03 E - 04 4.98 E - 04 1.78 E - 04 N/A N/A N/A 6.13 E - 05 N/A N/A N/A 7.97 E - 05 N/A 2.96 E - 04 N/A N/A 1.23 E - 04 6.16 E - 04 2.80 E - 04 N/A N/A N/A 8.84 E - 05 N/A N/A N/A 9.54 E - 05 N/A 172 Table D. 18 Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 MW66 (Pre) 7.64 E - 03 1.98 E - 03 2.91 E - 03 1.51 E - 03 9.02 E - 03 3.35 E - 03 2.54 E - 03 N/A N/A 4.71 E - 04 2.15 E - 02 3.88 E - 02 N/A 2.66 E - 03 N/A 7.57 E - 03 2.43 E - 03 3.23 E - 03 1.61 E - 03 1.25 E - 02 4.44 E - 03 2.96 E - 03 N/A N/A N/A 2.16 E - 02 4.89 E - 02 N/A 2.34 E - 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW66 (Post) 1.44 E - 03 1.57 E - 04 4.30 E - 04 1.02 E - 04 4.31 E - 03 5.68 E - 04 5.89 E - 04 N/A N/A 1.30 E - 04 1.22 E - 03 N/A N/A 3.16 E - 04 N/A 8.45 E - 04 1.22 E - 04 2.26 E - 04 9.18 E - 05 2.47 E - 03 2.72 E - 04 1.71 E - 04 N/A N/A 4.38 E - 05 4.84 E - 04 N/A N/A 1.12 E - 04 N/A MW67 (Pre) 1.61 E - 02 4.36 E - 03 6.37 E - 03 3.65 E - 03 3.86 E - 02 7.81 E - 03 9.16 E - 03 4.22 E - 02 N/A 8.73 E - 04 5.04 E - 02 1.49 E - 01 N/A 7.24 E - 03 N/A 2.17 E - 02 6.25 E - 03 7.29 E - 03 3.42 E - 03 4.41 E - 02 6.86 E - 03 5.53 E - 03 3.94 E - 02 N/A 8.69 E - 04 3.75 E - 02 6.83 E - 02 N/A 4.40 E - 03 N/A 1.92 E - 02 6.07 E - 03 9.47 E - 03 4.18 E - 03 4.66 E - 02 7.15 E - 03 9.78 E - 03 3.50 E - 02 N/A 1.83 E - 03 3.94 E - 02 1.35 E - 01 N/A 6.75 E - 03 N/A MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Red Cedar River Red Cedar 3.77 E - 03 N/A N/A 1.75 E - 04 N/A 3.10 E - 04 N/A 1.74 E - 03 N/A N/A N/A N/A N/A N/A N/A 4.71 E - 03 N/A N/A 3.27 E - 04 N/A 6.50 E - 04 N/A 3.30 E - 03 N/A N/A N/A N/A N/A N/A N/A Sediment MW71 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soil MSU Soil E 1.36 E - 03 8.48 E - 04 N/A 4.82 E - 04 N/A 6.22 E - 04 N/A N/A N/A N/A N/A N/A N/A 1.55 E - 04 N/A N/A N/A N/A 4.23 E - 04 N/A 8.28 E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.38 E - 03 7.87 E - 04 N/A 5.39 E - 04 N/A 7.16 E - 04 N/A N/A N/A N/A N/A N/A N/A 2.71 E - 04 N/A 173 Table D. 18 Sample PS1 PS2 PS4 PS5 PS6 PS7 PS8 PS9 PS10 PS11 PS14 PS16 PS17 PS19 Pub1 MSU Soil F 2.96 E - 03 N/A N/A 6.10 E - 04 3.74 E - 03 8.11 E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 2.05 E - 03 N/A N/A 3.92 E - 04 N/A 8.49 E - 04 N/A N/A N/A N/A N/A N/A N/A N/A N/A 6.85 E - 03 N/A N/A 6.92 E - 04 8.15 E - 03 1.18 E - 03 N/A N/A N/A N/A N/A N/A N/A N/A N/A KBS T1 4.15 E - 03 9.01 E - 04 N/A N/A 3.35 E - 03 1.03 E - 03 N/A 4.57 E - 03 N/A 8.53 E - 05 N/A N/A N/A 1.74 E - 04 N/A 6.76 E - 03 7.53 E - 04 N/A 1.77 E - 04 4.42 E - 03 2.71 E - 03 1.38 E - 03 N/A N/A 1.18 E - 03 N/A N/A N/A 1.24 E - 03 N/A 1.97 E - 02 2.30 E - 03 N/A 1.24 E - 03 7.16 E - 03 8.01 E - 03 9.42 E - 04 6.77 E - 03 N/A 5.03 E - 03 N/A N/A N/A 5.85 E - 03 N/A KBS T2 5.99 E - 03 8.71 E - 04 6.54 E - 04 3.68 E - 04 5.23 E - 03 1.94 E - 03 5.82 E - 04 3.57 E - 03 N/A 7.82 E - 04 N/A N/A N/A 1.21 E - 03 N/A 1.63 E - 03 6.21 E - 04 N/A 4.14 E - 04 N/A 1.25 E - 03 N/A 3.78 E - 03 N/A 1.67 E - 04 N/A N/A N/A 3.52 E - 04 N/A 6.97 E - 03 N/A 6.28 E - 04 3.76 E - 04 7.45 E - 03 2.67 E - 03 6.61 E - 04 3.68 E - 03 N/A 1.80 E - 03 N/A N/A N/A 1.26 E - 03 N/A KBS T3 5.03 E - 03 7.27 E - 04 N/A 3.93 E - 04 N/A 2.23 E - 03 7.49 E - 04 N/A N/A 6.35 E - 04 N/A N/A N/A 9.20 E - 04 N/A 2.61 E - 03 6.44 E - 04 N/A 3.12 E - 04 N/A 7.03 E - 04 6.07 E - 04 N/A N/A 4.52 E - 04 N/A N/A N/A 3.81 E - 04 N/A 4.59 E - 03 1.46 E - 03 N/A 8.52 E - 04 N/A 2.02 E - 03 N/A N/A N/A 1.76 E - 04 N/A N/A N/A 6.70 E - 04 N/A KBS T4 4.10 E - 03 N/A N/A 2.95 E - 04 N/A 2.76 E - 03 N/A 3.42 E - 03 N/A 1.18 E - 03 N/A N/A N/A 1.03 E - 03 N/A 3.51 E - 03 1.28 E - 03 N/A 3.60 E - 04 N/A 1.64 E - 03 N/A 3.13 E - 03 N/A 1.87 E - 04 N/A N/A N/A 4.25 E - 04 N/A 3.25 E - 03 5.58 E - 04 N/A 5.68 E - 04 N/A 7.95 E - 04 N/A 4.01 E - 03 N/A 2.30 E - 04 N/A N/A N/A 4.22 E - 04 N/A 174 Table D. 19 . Gene copies per 16S rRNA gene copy for xplA positive amplification. Sample PS3 PS4 PS5 Pub1 Pub2 Pub3 Pub4 Pub6 Shallow Zone Aquifer MW32 (Pre) N/A 1.54E - 03 3.33E - 02 N/A 4.81E - 04 N/A 6.67E - 04 1.99E - 03 N/A 1.57E - 03 4.37E - 02 N/A 3.73E - 04 N/A 4.29E - 04 1.53E - 03 N/A 1.41E - 03 N/A N/A 1.25E - 04 N/A 2.41E - 04 2.06E - 03 MW32 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW62 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.04E - 02 N/A N/A N/A N/A 2.57E - 04 N/A N/A 1.10E - 02 N/A N/A N/A N/A 3.44E - 04 MW62 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Perched Zone Aquifer MW48 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A 5.89E - 03 N/A N/A N/A N/A N/A N/A N/A 3.08E - 03 N/A N/A N/A N/A N/A N/A N/A MW48 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW60R (Post) N/A N/A N/A N/A N/A N/A N/A 5.36E - 04 N/A N/A N/A N/A N/A N/A N/A 5.38E - 04 MW64 (Pre) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW64 (Post) N/A N/A 3.24E - 03 N/A N/A N/A N/A N/A N/A N/A 3.25E - 03 N/A N/A N/A N/A N/A MW66 (Pre) N/A 8.65E - 04 N/A 5.79E - 05 N/A N/A N/A N/A N/A 9.50E - 04 N/A 7.59E - 05 N/A N/A N/A N/A N/A 1.06E - 03 N/A N/A N/A N/A N/A N/A MW66 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A MW67 (Pre) N/A N/A 3.25E - 02 N/A N/A N/A N/A N/A N/A N/A 3.97E - 02 N/A N/A N/A N/A N/A N/A N/A 3.22E - 02 N/A N/A N/A N/A N/A MW67 (Post) N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Red Cedar River Red Cedar N/A 1.59E - 03 2.09E - 02 N/A N/A N/A N/A 3.29E - 04 N/A 1.99E - 03 2.82E - 02 N/A N/A N/A N/A 3.75E - 04 Sediment MW71 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Agricultural Soils MSU Soil E N/A N/A 7.07E - 02 N/A N/A N/A N/A N/A N/A N/A 5.44E - 02 N/A N/A N/A N/A N/A N/A N/A 5.28E - 02 N/A N/A N/A N/A N/A 175 Table D. 19 Sample PS3 PS4 PS5 Pub1 Pub2 Pub3 Pub4 Pub6 MSU Soil F N/A N/A 3.94E - 02 N/A N/A N/A N/A 6.88E - 04 N/A N/A 4.17E - 02 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 9.83E - 04 KBS T1 N/A N/A 4.00E - 02 N/A N/A N/A N/A 6.06E - 04 N/A N/A 7.16E - 02 N/A N/A N/A N/A 9.20E - 04 N/A N/A 3.92E - 02 N/A N/A N/A N/A 8.01E - 04 KBS T2 N/A N/A 3.45E - 02 N/A N/A N/A N/A N/A N/A N/A 5.39E - 02 N/A N/A N/A N/A 1.28E - 03 N/A N/A 7.43E - 02 N/A N/A N/A N/A 2.65E - 03 KBS T3 N/A 7.90E - 04 4.56E - 02 N/A N/A N/A N/A N/A N/A N/A 4.15E - 02 N/A N/A N/A N/A N/A N/A 1.50E - 03 6.58E - 02 N/A N/A N/A N/A N/A KBS T4 N/A N/A 5.16E - 02 N/A N/A N/A N/A N/A N/A N/A 6.59E - 02 N/A N/A N/A N/A N/A N/A N/A 7.62E - 02 N/A N/A N/A N/A N/A 176 APPENDIX E Groundwater Pre - and Post - Biostimulation Results 177 Figure E. 1 . Average log gene copies per mL of groundwater in shallow zone aquifer well MW62. Error bars represent the standard deviation and an asterisk (*) denotes a statistically significant difference (p<0.05) between the pre - and post - biostimulation levels. 0 1 2 3 4 5 6 7 8 9 nfsI_PS1 nfsI_PS6 pnrB_PS1 pnrB_PS2 pnrB_PS3 pnrB_PS4 pnrB_Pub2 xenA_PS1 xenA_PS4 xenA_PS6 xenA_PS9 xenA_PS10 xenA_PS12 xenA_PS15 xenA_PS16 xenB_PS1 xenB_PS2 xenB_PS4 xenB_PS5 xenB_PS6 xenB_PS7 xenB_PS8 xenB_PS9 xenB_PS10 xenB_PS11 xenB_PS14 xenB_PS16 xenB_PS19 xplA_PS4 xplA_PS5 xplA_Pub2 xplA_Pub4 xplA_Pub6 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 Log Gene Copies per mL Assay MW32 Pre-biostimulation Post-biostimulation * 178 Figure E . 2 . Average log gene copies per mL of groundwater in shallow zone aquifer well MW62. Error bars represent the standard deviation and an asterisk (*) denotes a statistically significant difference (p<0.05) between the pre - and po st - biostimulation levels. Figure E. 3 . Average log gene copies per mL of groundwater in perched zone aquifer well MW48. Error bars represent the standard deviation. 0 1 2 3 4 5 6 7 8 9 nfsI_PS1 nfsI_PS6 pnrB_PS1 pnrB_PS2 pnrB_PS3 xenA_PS1 xenA_PS4 xenA_PS6 xenA_PS8 xenA_PS15 xenB_PS1 xenB_PS2 xenB_PS4 xenB_PS5 xenB_PS6 xenB_PS7 xenB_PS8 xenB_PS9 xenB_PS10 xenB_PS11 xenB_PS14 xenB_PS19 xplA_PS5 xplA_Pub6 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 Log Gene Copies per mL Assay MW62 Pre-biostimulation Post-biostimulation 0 1 2 3 4 5 6 7 8 9 10 nfsI_PS1 nfsI_PS7 pnrB_PS2 pnrB_PS4 pnrB_Pub2 xenA_PS1 xenA_PS4 xenA_PS6 xenA_PS9 xenA_PS10 xenA_PS11 xenA_PS12 xenA_PS15 xenB_PS1 xenB_PS2 xenB_PS4 xenB_PS5 xenB_PS6 xenB_PS7 xenB_PS9 xenB_PS10 xenB_PS11 xenB_PS14 xenB_PS16 xenB_PS17 xenB_PS19 xplA_PS3 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 Log Gene Copies per mL Assay MW48 Pre-biostimulation Post-biostimulation * * * 179 Figure E. 4 . Average log gene copies per mL of groundwater in perched zone aquifer well MW60R. Error bars represent the standard deviation. Figure E. 5 . Average log gene copies per mL of groundwater in perched zone aquifer well MW64. Error bars represent the standard deviation. 0 1 2 3 4 5 6 7 8 nfsI_PS1 pnrB_PS2 xenA_PS4 xenA_PS6 xenA_PS8 xenA_PS12 xenA_PS15 xenB_PS1 xenB_PS2 xenB_PS5 xenB_PS6 xenB_PS7 xenB_PS11 xenB_PS19 xplA_Pub6 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 Log Gene Copies per mL Assay MW60R Pre-biostimulation Post-biostimulation 0 1 2 3 4 5 6 7 8 9 nfsI_PS7 pnrB_PS2 xenA_PS4 xenA_PS6 xenA_PS8 xenA_PS12 xenA_PS15 xenB_PS1 xenB_PS2 xenB_PS4 xenB_PS5 xenB_PS6 xenB_PS7 xenB_PS8 xenB_PS11 xenB_PS14 xenB_PS19 xplA_PS5 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 Log Gene Copies per mL Assay MW64 Pre-biostimulation Post-biostimulation 180 Figure E. 6 . Average log gene copies per mL of groundwater in perched zone aquifer well MW66. Error bars represent the standard deviation. Figure E. 7 . Average l og gene copies per mL of groundwater in perched zone aquifer well MW67. Error bars represent the standard deviation. 0 1 2 3 4 5 6 7 8 9 nfsI_PS1 nfsI_PS7 pnrB_PS2 pnrB_PS3 pnrB_Pub2 xenA_PS4 xenA_PS6 xenA_PS8 xenA_PS9 xenA_PS11 xenA_PS12 xenA_PS15 xenA_PS16 xenB_PS1 xenB_PS2 xenB_PS4 xenB_PS5 xenB_PS6 xenB_PS7 xenB_PS8 xenB_PS11 xenB_PS14 xenB_PS16 xenB_PS19 xplA_PS4 xplA_Pub1 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 Log Gene Copies per mL Assay MW66 Pre-biostimulation Post-biostimulation 0 1 2 3 4 5 6 7 nfsI_PS1 nfsI_PS7 pnrB_Pub2 xenA_PS1 xenA_PS4 xenA_PS6 xenA_PS8 xenA_PS9 xenA_PS12 xenA_PS15 xenB_PS1 xenB_PS2 xenB_PS4 xenB_PS5 xenB_PS6 xenB_PS7 xenB_PS8 xenB_PS9 xenB_PS11 xenB_PS14 xenB_PS16 xenB_PS19 xplA_PS5 16S_rRNA.1 16S_rRNA.2 16S_rRNA.3 Log Gene Copies per mL Assay MW67 Pre-biostimulation Post-biostimulation 181 APPENDIX F Statistical Analysis 182 The pre - and post - biostimulation gene copies per mL concentrations were tested for statist ically significant difference in individual wells and for each aquifer (MW32 and MW62 samples were combined for the shallow zone aquifer and MW48, MW60R, MW64, MW66, and MW67 for the perched zone aquifer). Due to the small sample sizes (n<30), normal distr ibution could not be assumed to use the parametric statistical tests. Therefore, the Shapiro - Wilk test was used to test each sample group, pre - and post - biostimulation, for normal distribution. This test requires at least 3 data points so any assay that di d not yield at least 3 detections in either the pre - or post - biostimulation samples was assumed to not meet the normal distribution requirement for the parametric tests. For assays that both the pre - and post - biostimulation sample groups had normal distrib - sample t - - test for those with unequal variance (p 0.05). For assays where either the pre - or post - biostimulation sample group did not follow the normal distribution (p 0.05), the Brown - Forsythe test was used to test for equal variance. The significant difference between the medians was then evaluated with the Mann - Whitney U test for those with equal variance (p>0.05). The significant difference could not be determined for samples that did not follow the normal distribution and had unequal variance. The results from this analysis are rep orted in Tables F.1 through F.7 for the individual wells and Tables F.8 and F.9 for the shallow and perched zone aquifer, respectively. 183 Table F. 1 . P - values for statistical tests with the MW32 gene copies per mL results. - values in bold indicate a Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test Null Hypothesis The sample distribution is normal 1 2 1 2 1 2 1 2 Median 1 =Median 2 Data Group Pre - biostimulation Post - biostimulation Both Both Both Both Both pnrB_PS3 N / A 8.21E - 01 N/A N/A N/A 2.67E - 01 2.00E - 01 pnrB_Pub2 7.13E - 01 N / A N/A N/A N/A 1.40E - 01 2.00E - 01 xenA_PS4 4.58E - 01 1.05E - 02 N/A N/A N/A 4.59E - 01 4.00E - 01 xenA_PS15 9.29E - 01 N / A N/A N/A N/A 3.05E - 02 N/A xenB_PS5 6.58E - 02 2.83E - 01 3.69E - 01 1.38E - 03 N/A N/A N/A xenB_PS11 2.47E - 01 7.55E - 01 1.07E - 01 8.18E - 01 N/A N/A N/A 16S _rRNA .1 2.76E - 01 2.38E - 01 2.70E - 04 N/A 7.67E - 02 N/A N/A 16S _rRNA .2 9.99E - 01 3.69E - 01 1.03E - 04 N/A 8.22E - 02 N/A N/A 16S _rRNA .3 9.90E - 01 5.46E - 01 2.40E - 04 N/A 7.76E - 02 N/A N/A Table F. 2 . P - values for statistical tests with the MW 6 2 gene - values in bold indicate a Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test Null Hypothesis The sample distribution is normal 1 2 1 2 1 2 1 2 Median 1 =Median 2 Data Group Pre - biostimulation Post - biostimulation Both Both Both Both Both pnrB_PS2 5.37E - 01 9.75E - 01 2.99E - 01 9.74E - 02 N/A N/A N/A xenA_PS6 6.80E - 01 6.50E - 01 1.89E - 01 5.03E - 01 N/A N/A N/A xenA_PS15 2.05E - 01 3.87E - 01 5.06E - 01 2.54E - 04 N/A N/A N/A xenB_PS1 1.69E - 01 8.41E - 01 1.60E - 01 4.32E - 04 N/A N/A N/A xenB_PS2 6.55E - 01 6.78E - 01 7.06E - 01 3.20E - 01 N/A N/A N/A xenB_PS5 N / A 1.50E - 01 N/A N/A N/A 8.96E - 01 2.00E - 01 xenB_PS7 4.62E - 01 9.37E - 01 5.40E - 01 4.41E - 02 N/A N/A N/A xenB_PS11 N / A 7.02E - 01 N/A N/A N/A 5.01E - 01 4.00E - 01 xenB_PS14 9.60E - 01 3.37E - 01 2.97E - 01 2.25E - 01 N/A N/A N/A xenB_PS19 2.09E - 01 7.96E - 01 1.68E - 02 N/A 3.33E - 01 N/A N/A 16S _rRNA .1 3.17E - 01 5.55E - 01 5.37E - 02 3.13E - 01 N/A N/A N/A 16S _rRNA .2 1.86E - 01 8.10E - 01 3.53E - 02 N/A 2.98E - 01 N/A N/A 16S _rRNA .3 8.27E - 01 9.04E - 01 3.37E - 02 N/A 3.47E - 01 N/A N/A 184 Table F. 3 . P - values for statistical tests with the MW 48 - values in bold indicate a Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test Null Hypothesis The sample distribution is normal 1 2 1 2 1 2 1 2 Median 1 =Median 2 Data Group Pre - biostimulation Post - biostimulation Both Both Both Both Both pnrB_PS2 N / A N / A N / A N / A N / A 6.28E - 32 N/A xenB_PS5 N / A N / A N / A N / A N / A 1.68E - 32 N/A 16S _rRNA .1 9.48E - 01 N / A N / A N / A N / A 2.76E - 04 N/A 16S _rRNA .2 9.98E - 01 N / A N / A N / A N / A 4.66E - 05 N/A 16S _rRNA .3 9.36E - 01 N / A N / A N / A N / A 1.19E - 05 N/A Table F. 4 . P - values for statistical tests with the MW 60R - values in bold indicate a Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test Null Hypothesis The sample distribution is normal 1 2 1 2 1 2 1 2 Median 1 =Median 2 Data Group Pre - biostimulation Post - biostimulation Both Both Both Both Both xenB_PS1 1.46E - 01 N / A N / A N / A N / A 8.57E - 01 2.00E - 01 xenB_PS2 N / A N / A N / A N / A N / A 8.30E - 32 N/A xenB_PS5 6.51E - 01 N / A N / A N / A N / A 9.29E - 01 4.00E - 01 xenB_PS7 N / A N / A N / A N / A N / A 3.30E - 32 N/A 16S _rRNA .1 3.96E - 03 N / A N / A N / A N / A 2.80E - 01 2.00E - 01 16S _rRNA .2 8.43E - 03 N / A N / A N / A N / A 1.79E - 02 N/A 16S _rRNA .3 7.87E - 03 N / A N / A N / A N / A 4.54E - 02 N/A 185 Table F. 5 . P - values for statistical tests with the MW6 4 - values in bold indicate a Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test Null Hypothesis The sample distribution is normal 1 2 1 2 1 2 1 2 Median 1 =Median 2 Data Group Pre - biostimulation Post - biostimulation Both Both Both Both Both xenA_PS4 NA NA N / A N / A N / A 1.17E - 32 N/A xenA_PS15 9.72E - 01 NA N / A N / A N / A 4.95E - 02 N/A xenB_PS1 2.12E - 01 NA N / A N / A N / A 1.12E - 02 N/A xenB_PS5 6.89E - 01 NA N / A N / A N / A 1.20E - 02 N/A xenB_PS6 5.65E - 01 NA N / A N / A N / A 1.20E - 01 2.00E - 01 xenB_PS7 1.32E - 02 NA N / A N / A N / A 1.75E - 04 N/A xenB_PS11 NA NA N / A N / A N / A 6.13E - 33 N/A xenB_PS19 9.72E - 01 NA N / A N / A N / A 1.07E - 03 N/A 16S _rRNA .1 3.81E - 01 NA N / A N / A N / A 8.55E - 03 N/A 16S _rRNA .2 1.97E - 01 NA N / A N / A N / A 4.51E - 06 N/A 16S _rRNA .3 5.43E - 01 NA N / A N / A N / A 1.38E - 05 N/A Table F. 6 . P - values for statistical tests with the MW6 6 - values in bold indicate a Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test Null Hypothesis The sample distribution is normal 1 2 1 2 1 2 1 2 Median 1 =Median 2 Data Group Pre - biostimulation Post - biostimulation Both Both Both Both Both nfsI_PS7 7.91E - 01 N / A N / A N / A N / A 2.08E - 01 8.00E - 01 pnrB_PS2 3.88E - 01 N / A N / A N / A N / A 9.54E - 06 N/A xenA_PS4 1.86E - 01 N / A N / A N / A N / A 7.25E - 01 4.00E - 01 xenA_PS6 9.22E - 02 N / A N / A N / A N / A 1.68E - 02 N/A xenA_PS15 9.68E - 01 N / A N / A N / A N / A 1.23E - 01 8.00E - 01 xenB_PS1 2.44E - 01 N / A N / A N / A N / A 7.54E - 02 1.00E+00 xenB_PS2 2.84E - 01 N / A N / A N / A N / A 7.24E - 01 4.00E - 01 xenB_PS4 7.30E - 02 N / A N / A N / A N / A 5.64E - 01 8.00E - 01 xenB_PS5 1.32E - 02 N / A N / A N / A N / A 6.61E - 01 4.00E - 01 xenB_PS6 7.44E - 01 N / A N / A N / A N / A 3.43E - 03 N/A xenB_PS7 4.48E - 01 N / A N / A N / A N / A 6.15E - 01 8.00E - 01 xenB_PS8 1.59E - 01 N / A N / A N / A N / A 9.84E - 01 1.00E+00 186 Table F. 6 Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test xenB_PS11 N / A N / A N / A N / A N / A 1.94E - 30 N/A xenB_PS14 1.42E - 01 N / A N / A N / A N / A 6.76E - 01 4.00E - 01 xenB_PS19 3.68E - 01 N / A N / A N / A N / A 7.96E - 01 8.00E - 01 16S _rRNA .1 N / A N / A N / A N / A N / A 1.10E - 32 N/A 16S _rRNA .2 3.99E - 02 N / A N / A N / A N / A 1.19E - 04 N/A 16S _rRNA .3 9.73E - 01 N / A N / A N / A N / A 3.31E - 05 N/A Table F. 7 . P - values for statistical tests with the MW6 7 - values in bold indicate a Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test Null Hypothesis The sample distribution is normal 1 2 1 2 1 2 1 2 Median 1 =Median 2 Data Group Pre - biostimulation Post - biostimulation Both Both Both Both Both 16S _rRNA .1 4.30E - 01 N / A N/ A N/ A N/ A 3.45E - 01 2.00E - 01 16S _rRNA .2 5.65E - 01 N / A N/ A N/ A N/ A 3.05E - 01 2.00E - 01 16S _rRNA .3 3.87E - 01 N / A N/ A N/ A N/ A 3.65E - 01 2.00E - 01 Table F. 8 . P - values for statistical tests with the shallow zone aquifer gene copies per mL results - values in bold indicate a significant difference (p 0.05). Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test Null Hypothesis The sample distribution is normal 1 2 1 2 1 2 1 2 Median 1 =Median 2 Data Group Pre - biostimulation Post - biostimulation Both Both Both Both Both pnrB_PS2 6.62E - 02 9.75E - 01 7.37E - 01 7.88E - 01 N / A N/A N/A pnrB_PS3 9.76E - 02 8.21E - 01 9.41E - 03 N / A 9.87E - 02 N/A N/A pnrB_Pub2 7.13E - 01 N/A N/A N/A N/A 1.40E - 01 2.00E - 01 xenA_PS4 2.03E - 01 1.05E - 02 N/A N/A N/A 1.58E - 01 7.14E - 01 xenA_PS6 9.37E - 01 6.50E - 01 3.64E - 02 N / A 4.23E - 01 N/A N/A xenA_PS15 2.88E - 02 9.09E - 01 N/A N/A N/A 3.03E - 06 N / A xenB_PS1 1.96E - 01 8.41E - 01 2.22E - 02 N / A 7.63E - 03 N/A N/A xenB_PS2 9.90E - 01 6.78E - 01 2.36E - 01 9.83E - 02 N/A N/A N/A 187 Table F. 8. Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U test xenB_PS4 5.94E - 01 5.66E - 02 8.99E - 01 1.53E - 01 N/A N/A N/A xenB_PS5 2.12E - 01 4.99E - 02 N/A N/A N/A 6.68E - 01 4.33E - 03 xenB_PS7 9.26E - 01 9.37E - 01 1.56E - 01 4.72E - 03 N/A N/A N/A xenB_PS11 5.86E - 01 5.72E - 01 2.00E - 01 4.67E - 01 N/A N/A N/A xenB_PS14 7.15E - 01 3.37E - 01 3.59E - 01 4.01E - 01 N/A N/A N/A xenB_PS19 8.81E - 02 7.96E - 01 9.32E - 01 7.81E - 01 N/A N/A N/A Table F. 9 . P - values for statistical tests with the perched zone aquifer gene copies per mL results - values in bold indicate a significant difference (p 0.05). Test Shapiro - Wilk Bartlett's test Student t - test Welch's t - test Brown - Forsythe test Mann - Whitney U tes t Null Hypothesis The sample distribution is normal 1 2 1 2 1 2 1 2 Median 1 =Median 2 Data Group Pre - biostimulation Post - biostimulation Both Both Both Both Both nfsI_PS1 9.12E - 04 N/A N/A N/A N/A 6.39E - 01 4.00E - 01 nfsI_PS7 1.32E - 01 1.20E - 01 9.32E - 05 N/A 1.82E - 01 N/A N/A pnrB_PS2 4.00E - 02 8.04E - 02 N/A N/A N/A 5.62E - 02 4.66E - 03 xenA_PS4 3.45E - 05 8.57E - 03 N/A N/A N/A 6.64E - 01 9.58E - 01 xenA_PS6 1.76E - 03 3.30E - 03 N/A N/A N/A 1.40E - 01 3.66E - 01 xenA_PS8 3.02E - 01 N/A N/A N/A N/A 2.12E - 01 5.00E - 01 xenA_PS12 2.29E - 01 3.39E - 01 2.15E - 01 2.78E - 01 N/A N/A N/A xenA_PS15 9.07E - 01 5.69E - 02 6.47E - 05 N/A 1.96E - 01 N/A N/A xenB_PS1 1.04E - 01 2.87E - 01 8.03E - 04 N/A 2.22E - 02 N/A N/A xenB_PS2 1.04E - 05 7.44E - 01 N/A N/A N/A 4.55E - 01 4.12E - 01 xenB_PS4 5.61E - 05 N/A N/A N/A N/A 6.34E - 01 1.00E+00 xenB_PS5 3.04E - 06 3.58E - 03 N/A N/A N/A 4.98E - 01 7.64E - 01 xenB_PS6 2.41E - 02 2.05E - 01 N/A N/A N/A 8.07E - 02 4.04E - 01 xenB_PS7 1.18E - 05 6.85E - 02 N/A N/A N/A 8.10E - 01 9.66E - 01 xenB_PS8 6.96E - 01 N/A N/A N/A N/A 5.65E - 01 1.00E+00 xenB_PS11 1.20E - 05 3.69E - 01 N/A N/A N/A 6.11E - 01 2.62E - 01 xenB_PS14 9.09E - 06 N/A N/A N/A N/A 5.45E - 01 3.08E - 01 xenB_PS19 3.67E - 06 4.10E - 01 N/A N/A N/A 6.38E - 01 4.42E - 01 xplA_PS5 7.40E - 01 N/A N/A N/A N/A 5.35E - 04 N/A 188 REFERENCES 189 REFERENCES 1. 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