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MMMHE ll THBHS <2 o ~51.) vb LIBRARY Michigan State University This is to certify that the dissertation entitled CHARACTERIZATION OF GENES INVOLVED IN THE DEGRADATION OF CARBON TETRACHLORIDE BY PSEUDOMONAS STUTZERI STRAIN KC presented by Lycely del Carmen Sepfilveda-Torres has been accepted towards fulfillment of the requirements for Ph.D . degree in Microbiology \ I 3 Major professor Date é//Z/0fl MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DARLBUgE 9 DATE DUE DATE DUE ' H 9 L PU. 11/00 Wm.w5-p.14 CHARACTERIZATION OF GENES INVOLVED IN THE DEGRADATION OF CARBON TETRACHLORIDE BY PSEUDOMONAS S T U T ZERI STRAIN KC Lycely del Carmen Septilveda-Torres AN ABSTRACT OF A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 2000 P581 seer fem 51w: [EU-Ci for I met? abili sequ fund ler b}‘ ax Caibc ABSTRACT Pseudomonas stutzeri strain KC is a denitrifying aquifer isolate that produces and secretes pyridine-2,6-bis(thiocarboxylic acid), a compound that chelates copper to fortuitously transform carbon tetrachloride without producing chloroform. Although P. stutzeri strain KC has been successfully used for full-scale bioremediation of carbon tetrachloride contaminated sites, no information was known about the genes responsible for the carbon tetrachloride degradation capacity. The present dissertation describes the methods used for the generation of four P. stutzeri strain KC mutants with a reduced ability to degrade carbon tetrachloride. The DNA interrupted in the mutants was sequenced and analyzed using various tools, allowing the assignment of possible gene functions. The information obtained from the aforementioned studies was combined with information about other genes involved in carbon tetrachloride dehalogenation mutated by another research group, in order to propose a possible biosynthesis pathway for the carbon tetrachloride dehalogenation agent pyridine-2,6-bis(thiocarboxylic acid). Copyright Lycely del Carmen Sepulveda-Torres 2000 To my family: Papi, Mami, Jorgito, Tony, Coca, Zulma, Yelitza, Yarelis and Elliot. Thank you for being the wind beneath my wings. Para mi familia: Papi, Mami, Jorgito, Tony, Coca, Zulma, Yelitza, Yarelis y Elliot. Gracias por ser mi fuente de fortaleza e inspiracion. ACKNOWLEDGMENTS It is my desire to show appreciation and give credit to the people who made possible the fulfillment of my doctoral dream. I would like to thank my advisor, Dr. Craig Criddle, for supporting me in my studies to the best of his abilities. I feel very fortunate for having a special relationship with my advisor; one that only a few graduate students experience during their graduate careers. You are my supporting mentor, an enthusiastic colleague and a good friend. Thank you for believing in me, even in the moments when I lost faith in myself. I anticipate having a life-long professional relationship with you. It is my desire to give a special recognition to Dr. James Tiedje, my co-adviser. Thank you very much for making me your protégé since I came to Michigan State University (MSU) for the first time as a Center for Microbial Ecology (CME) summer intern. Thank you for your letter of support for the National Science Foundation pre-doctoral fellowship, for making me the first Puerto Rican woman to be part of the CME and for been my dedicated co-adviser. It has been a great honor working with you. I am one of the few graduate students who have had the opportunity of working with one of the best scientists of their time. I admire you for your scientific achievements and for been such a gracious human being. I am sure that the National Academy of Science will soon give you the recognition you deserve. Special thanks to my guidance committee members: Dr. Patrick Oriel, Dr. Robert Brubaker and Dr. Loren Snyder for their helpful criticism and advice. I am especially grateful to Dr. Frans deBruijn, Dr. Michael Dybas and Dr. Joan Broderick for providing good guidance when I needed it the most. Thank you all for sharing your knowledge with me. I wish you success in your future endeavors. I would like to express my gratitude to the graduate students, post-doctoral fellows, faculty and staff of the Department of Microbiology, the CME and the Environmental Engineering Program for lending a helping hand every time I needed help or advice; and for providing a friendly learning environment. Thanks to Mr. Jorge Rodrigues, Dr. Anne Milcamp, Dr. Sabine Rech, Dr. K. Padmanabhan and Dr. Ann Gustafson for helpful discussions about my work and for providing help whenever I asked. My deepest appreciation to Dr. Ronald Crawford, Dr. Thomas Lewis, Mr. Marc Cortese, Mr. Jon Sebat and the rest of the Crawford group working on Pseudomonas stutzeri strain KC. Thank you very much for sharing my passion about this bacterium and for keeping me informed about your findings. I congratulate you on your well-deserved accomplishments in this project. Special thanks to the National Science Foundation for a pre-doctoral fellowship and to the CME and the Institute for Environmental Toxicology for providing financial support for my studies. vi as] ach pm! pray I am indebted to Dr. and Mrs. Stuart Sleight and the rest of the Sleight family for “adopting” me into their family. Thank you very much for providing me a place that I could truly call home away from home. You made my six years away from my homeland and my loved ones less difficult to bear. Thanks to Maria Gutierrez for reminding me that life is more beautiful when seen through the eyes of a child. I would like to recognize the dear friends I met at Michigan State University: Carmen Medina, Vladimir Ferrer, Dr. Olga Hemandez-Patino, Hector Ayala, Veronica Griintzig and Dr. Paul R. Martin for helping me keep a warm heart during the cold Michigan winters. I would also like to acknowledge my lifelong friends Leslie Strutton, Melissa Colon and Ivelisse Torres for their support and encouragement across the distance. I would like to express my deepest gratitude to my parents for their unconditional love and support. Thank you for all the years of sacrifice in order to provide my brothers and I the best education you could afford. Thank you for believing in me and for seeing in my achievements the fulfillment of your own truncated dreams. I love you and I am very proud of you. I would be nothing without you. Thanks to my brothers Jorge Rafael and Juan Antonio, my extended family, and countless neighbors and friends for their love, prayers and encouragement. vii for La hel SIL] Special thanks to my husband Elliot for his endless love and support. Even though our status changed during the past five years; one thing remained the same: you were there to share my triumphs and defeats with me. Thank you very much for being my soul mate, my confidant, my lover and my best friend. Your love brings out the best of me. I look forward to the rest of our journey together. I would like to recognize Pseudomonas stutzeri strain KC for being such a fascinating and intriguing organism. You have been the protagonist of my scientific melodrama, but my interest in discovering you was stronger than the frustrations you caused me. I am sure that you will be the subject of study for many other Ph.D. students. Lastly, I would like to thank the Lord for putting the right people on my path, in order to help me accomplish the goals I have set for myself and for showing me His love in every step I take. viii TABLE OF CONTENTS LIST OF TABLES .................................................................................. xii LIST OF FIGURES ................................................................................ xiv CHAPTER 1 INTRODUCTION: PSEUDOMONAS STUTZERI STRAIN KC AND THE DEGRADATION OF CARBON TETRACHLORIDE ............... 1 Carbon Tetrachloride: Characteristics and Problems ................. 2 Mechanisms of Carbon Tetrachloride Transformation ............... 4 Pseudomonas stutzeri strain KC ......................................... 8 The Discovery of Pyridine-2,6-bis(thiocarboxylic acid) as a Carbon Tetrachloride Degrading Compound ................... 15 Outline of this Dissertation ............................................. 23 References ................................................................. 24 CHAPTER 2 GENERATION AND INITIAL CHARACTERIZATION OF PSEUDOMONAS STUTZERI STRAIN KC MUTANTS WITH IMPAIRED ABILITY TO DEGRADE CARBON TETRACHLORIDE ..................................................... 30 Abstract .................................................................... 3 1 Introduction ................................................................ 3 1 Materials and Methods ................................................... 32 Results ..................................................................... 33 Discussion ................................................................. 35 References ................................................................. 35 APP CHAPTER 3 CHAPTER 4 APPENDIX A APPENDIX B SEQUENCE AND ANALYSIS OF THE GENES INTERRUPTED IN FOUR PSEUDOMONAS S T UYZERI STRAIN KC MUTANTS WITH IMPAIRED ABILITY TO DEGRADE CARBON TETRACHLORIDE .................... 37 Abstract .................................................................... 38 Introduction ................................................................ 39 Materials and Methods ................................................... 41 Results ...................................................................... 53 Discussion ................................................................. 60 References ................................................................. 86 RECOMMENDATIONS FOR FUTURE RESEARCH. . . . . . . ......98 PHYLOGENY AND TAXONOMY OF PSEUDOMONAS STUTZERI STRAIN KC ............................................... 106 Summary ................................................................. 109 Introduction .............................................................. 1 10 Methods .................................................................. 1 12 Results ..................................................................... l 19 Discussion ................................................................ 135 Acknowledgements ..................................................... 1 39 References ................................................................ 140 SEQUENCE OF A 8,274 BASE PAIR EC 0R1 FRAGMENT MUTATED IN FOUR PSEUDOMONAS S TUT ZERI STRAIN KC TRANSPOSITIONAL MUTANTS WITH IMPAIRED ABILITY TO DEGRADE CARBON TETRACHLORIDE. . ....l48 APPENDIX C MOTIF INFORMATION ABOUT PROTEINS ASSOCIATED TO THE CARBON TETRACHLORIDE DEGRADATION CAPACITY OF PSEUDOMONAS ST UT ZERI STRAIN KC. . ..156 xi Ta Ta Ia Ial Tat Tab Tab Tab} Tabl Tabl. Tab]. Table 2.1 Table 2.2 Table 3.1 Table 3.2 Table 3.3 Table 3.4 Table 3.5 Table A.1 Table A2 Table A3 Table A4 Table A5 Table C.1 LIST OF TABLES Results of the 14CT microtiter plate assay and the gas chromatography assay Petri plate assay of secreted factor production by wild-type and recombinant KC colonies grown in the presence of Pseudomonas fluorescens and l4C-carbon tetrachloride. List of strains and plasmids used in this study. Internet-based programs used for DNA and protein analyses. Physical characteristics of the open reading frames encoded in the P. stutzeri strain KC 8.3 kb EcoRI fragment interrupted in the mutants impaired in CCl4 degradation. Putative functions of open reading frames found in the P. stutzeri strain KC 8.3 kb EcoRI fragment interrupted in the mutants impaired in CC14 degradation. Degradation of CCl4 by strains KC657, KC1896, KC2753 and KC3164 when the supernatant from wild type strain KC culture capable of degrading CCl4 is provided. Bacterial strains used in this study. Substrate utilization by strain KC and various Pseudomonas stutzeri, P. balearica and P. putida strains Antibiotic susceptibility test for strain KC and various Pseudomonas stutzeri, P. balearica and P. putida strains Cellular fatty acid composition of strain KC and several Pseudomonas stutzeri, P. balearica and P. putida strains DNA-DNA similarity results for strain KC and several Pseudomonas strains Transmembrane domains for proteins identified by Sepulveda and by Lewis, as predicted by TMHMM, HMMTOP, SOSUI and TMPred. xii Ia Ia Tal Ial Tat Tab Tab Tabl Tabl Table C.2 Table C.3 Table 04 Table C.5 Table C.6 Table C.7 Table C.8 Table C.9 Table C. 10 Table C.11 Table C.12 Table C.13 Table C.14 Transmembrane domains for proteins identified by Sepulveda and by Lewis, as predicted by PSort, DAS and TopPred2. Leader peptides for proteins identified by Sepulveda and by Lewis, as predicted by SignalP, PSort, and SPScan. Information about some motifs found by BLOCKS in ORF-2435. Information about a motif found by BLOCKS in ORF-3626. Information about a motif found by PROSITE Pattern in ORF-4099. Information about some motifs found by BLOCKS and PRINTS in ORF- 4460. Information about some motifs found by BLOCKS and PROSITE pattern in ORF-6289. Information about a motif found by Pfam in ORF-K. Information about some motifs found by BLOCKS in ORF-L. Information about some motifs found by PROSITE pattern and BLOCKS in ORF-M. Information about some motifs found by BLOCKS in ORF-N. Information about some motifs found in ORF-O by PROSITE pattern and BLOCKS. Information about some motifs found in ORF-P by BLOCKS. xiii Fi Fig Figure 1.1 Figure 1.2 Figure 1.3 Figure 2.1 Figure 2.2 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 Figure 3.9 LIST OF FIGURES Chemical structure of pyridine-2,6-bis(thiocarboxylic acid) (PDTC). Mechanism for iron chelation by pyridine-2,6-bis(thiocarboxylic acid) as proposed by Ockels et a]. Proposed pathway for the degradation of CCl4 by Cu-PDTC. Luciferase activity, expressed as relative light units divided by OD600, during growth of strain KC300, strain KC657, strain KC1896, and strain KC2753 in Simulated Groundwater Medium containing 1, 5, 10 and 20 11M Fe3+. Luciferase activity, expressed as relative light units divided by OD600, during growth of strain KC300, strain KC657, strain KC1896, strain KC2753 and strain KC3164in tryptic soy broth. Cloning and sequencing of genes mutated in P. stutzeri strain KC. Mapping of the transposition insertion points for transpositional mutants KC657, KC1896, KC2753 and KC3164. Open reading frames found in the 8,274 bp EcoRI fragment mutated in KC657, KC1896, KC2753 and KC3164. Stem-loop secondary structure observed between positions 2,295 and 2,331. Organization of 16 open reading frames in a 25.7 kb fragment capable of restoring the CCl4 degradation capacity in strain CTN]. Organization of the DNA region between ORF-1009 and ORF-2435. Pathway leading to bacterial cell wall precursors and L-lysine from L-aspartate. Overview of the proposed synthesis pathway for PDTC. Proposed sequence of events for the synthesis of PDTC in Pseudomonas stutzeri strain KC. xiv Figure A.1 Figure A.2 Figure A.3 Figure A.4 Simplified phenogram based on the UPGMA analysis or normalized BOX, REP and ERIC fingerprinting of strain KC and Pseudomonas stutzeri strains. Boostrap parsimony tree obtained when the 16S rDNA gene of strain KC is compaired to some sequences available in the Ribosomal Database Project. BamHI restriction digests of the 16S rRNA gene amplified by PCR using the Pseudomonas stutzeri-specific primers fpsl 58 and rp31271. Dendrogram depicting phylogenetic relationships among strain KC, several P. stutzeri strains and type strains of other Pseudomonas species, as estimated by comparing the ITS sequence. XV CHAPTER 1 INTRODUCTION: PSEUDOMONAS S T U T ZERI S T RAIN KC AND THE DEGRADATION OF CARBON TETRACHLORIDE CC AI pm (Sit €31.15 persi C61. Carbon Tetrachloride: Characteristics and Problems Carbon tetrachloride (CC14) is a non-polar and nonflammable, colorless solvent. It was widely used in the past in the processing of nuclear fuel, in dry cleaning operations, in the manufacture of fire extinguishers, refrigerants, aerosols, and chlorinated organic compounds and as an extractant, a fumigant and a metal degreaser (Verschueren, 1983). An estimated 2.3 million kg/year of CCl4 were discharged during manufacture and processing, and approximately 27.2 million kg/year were released as solvent emissions (Sittig, 1985). The widespread use and inadequate disposal of carbon tetrachloride caused the contamination of many groundwater supplies. Carbon tetrachloride is very persistent in groundwater due to its resistance to hydrolysis. The estimated half-life of CCl4 in aqueous solutions is 7,000 years (V ogel et al., 1987a). Carbon tetrachloride contamination represents a hazard to groundwater reservoirs. A study performed in the 1980’s by the United States Environmental Protection Agency (U .S. E.P.A.) indicated that 29 of 113 (25%) public water supply systems tested have a concentration of up to 400 ug CCl4/L; exceeding the 5 pg CCl4/L established standard (Sittig, 1985). One of the best characterized sites highly contaminated with carbon tetrachloride is the Hanford site in southwestern Washington (Last & Rohay, 1991; Skeen et al., 1994). The Hanford site is located in an area of approximately 1,500 km2 that was selected in the 1940’s by the US. government for the production of nuclear materials to be used by the United States in World War 11. Carbon tetrachloride contaminated solutions were discarded in liquid waste disposal facilities during the plutonium recovery em‘iro. processes. An estimated 1,000 metric tons of carbon tetrachloride were generated over a 20-year period. Carbon tetrachloride permeated through the vadose zone, contaminating at least 7 km2 with concentrations over 1,000 times the US. EPA. standard for drinking water. The US. Department of Energy is conducting bioremediation experiments in order to remove carbon tetrachloride from the Hanford site and protect the Colombia River located only miles away. The degradation of CC14 is of special interest, not only for its adverse contributions to the environment, but also for the hazards presented to human health. Carbon tetrachloride is highly toxic and is a suspected carcinogen. It can adversely affect different organs including the eyes, kidneys and liver. Excessive exposure can affect the gastrointestinal tract, while acute exposure can cause serious malfunctioning of the liver that may eventually cause death (Sittig, 1985; Vogel & McCarty, 1987b). The manufacture of CC14 was prohibited under the terms of the amended Montreal protocol because CCl4 is also an ozone-depleting agent (Programme, 1994). Even though CC14 is now released at a much-reduced rate, the CC14 still present in water reservoirs represents an environmental and a health hazard. Therefore, the decontamination of CCl4 polluted sites is of great significance. Me revie Tatar. A con a Inch Th6 for reducn'o CleClmn mchlom degradaz' Cell may 51.2mm, Mechanisms of Carbon Tetrachloride Transformation Several bacteria can transform carbon tetrachloride to different dechlorination states, depending on their metabolic capabilities and the culture conditions. This section of the dissertation provides an overview of CCl4 degradation mechanisms. More extensive reviews of the CCl4 degradation pathways are available elsewhere (Hashsham, 1996; Tatara, 1996). A common step in the transformation of CCl4 is the addition of a single electron, yielding a trichloromethyl radical and a chlorine ion, as shown in reaction 1.1. CCl4 + e_ —'> 'CCI3 + CI. (1.1) The formation of the trichloromethyl radical is believed to be the rate limiting step in the reductions of alkyl halides (Bakac & Espenson, 1985; Wade & Castro, 1973). The electron source can be anything from reduced metals to enzymes and co-factors. The trichloromethyl radical is highly reactive and will further react to produce a variety of degradation products. The trichloromethyl radical can also react directly with insoluble cell materials to form permanent covalent bonds. The latter process accounts for a significant portion of the transformed CCl4 products in biological systems (Ahr et al., 1980). h} drc PTOCC: H-‘drofle The trichloromethyl radical can dimerize to produce hexachloroethane as described in reaction 1.2. The formation of hexachloroethane from carbon tetrachloride was first observed in 1969 when oral administration of CCl4 to rabbits caused exhalation of hexachloroethane (Fowler, 1969). 2[°CC13] —-——§ C2Cl6 (1.2) Chloroform (CCl3) is one of the most common products of CC14 transformation. Chloroform can be produced from CCl4 via the trichloromethyl radical by 2 mechanisms: (i) removal of a hydrogen atom from lipids or proteins (Luke et al., 1987), (ii) hydrogenation of the trichloromethyl radical using reducing power from a cometabolic process. These reactions are labeled 1.3 and 1.4, respectively. -CC13+RH —' CHC13+-R (1.3) ~CC13 + H+ + e“ —* CHC13 (1.4) Hydrogenation of the trichloromethyl radical using reducing power from a cometabolic process has been reported in many microbial systems (see Hashsham, 1996; Tatara, 1996 for extensive lists). Several microbial consortia or isolates can form chloroform from CCl4. Further reductions to dichloromethane and methylchloride are also possible in highly reduced environments such as methanogenic and sulfate respiring microcosms (V ogel et al., 1987a). Nevertheless, as the degree of chlorination decreases from CC14 to methylci & 31ch ditTeren‘: (Butler. COCIIZ}'1 Chloro' With a is alsc Chloro' (Built Carbo ESChg al., 1 diChI. “ans (Km- Carbt Garb. {mic Ime1 methylchloride, the reaction becomes less energetically and kinetically favorable (Vogel & McCarty, 1987b). Chloroform can also be produced from the reaction of CC14 with different biologically active compounds like glutathione, cysteine and ascorbic acid (Butler, 1961), and bacterial transition metal — coenzyme pairs including vitamin B1 z-Co, coenzyme F 439-Ni and hematin-Fe (Gantzer & Wackett, 1991). Chloroform is not a desirable compound because it is highly recalcitrant and persistent with a half life of hydrolysis at room temperature of 1,850 years (Jeffers et al., 1989). It is also highly toxic and a suspected carcinogen (Sittig, 1985). The formation of chloroform from CC14 was first reported in 1961 in the breath of animals given CC14 (Butler, 1961). Carbon disulfide (CSz) was reported as a minor CCl4 transformation product of Escherichia coli cells grown with fumarate as the source of carbon and energy (Criddle et al., 1990a), and a major transformation product in anaerobic enrichments grown in dichloromethane (Hashsham et al., 1995). Carbon disulfide is a major product of CCl4 transformation in abiotic systems containing a high concentration of bisulfide ion (HS—) (Kriegman-King & Reinhard, 1992). If the hydroxide ion is also present in the system, carbon disulfide can be hydrolyzed to carbon dioxide yielding the net formation of carbon dioxide from carbon tetrachloride. Adewuyi and Carmichael (1987) proposed the following sequence of reactions for the formation of carbon dioxide from CCl4 via a CS; intermediate (reactions 1.5 to 1.8). The in: ykklt Reinha the prc final p1 If [he 1 ablOIlc p‘eIQXV CCI4 + 2113‘ + 211* _, C82 + 4HC1 (1.5) CSZ + OH- ——> CSZOH_ (slow) (1.6) CSZOH' + OH— -———> CSOZH‘ + HS- (1.7) CSOZH_ + OH_ ——> CO3HT + HS_ (1.8) The interactions between the trichloromethyl radical and various sulfur containing ions to yield thiophosgene (CCle) and C02 from CC14 was smdied by Kriegman-King and Reinhard (1992). As seen in reactions 1.9 to 1.11, trichloromethanethiolate (CC13S—) is the proposed common intermediate. A two step reaction will then produce C02 as the final product (reactions 1.12 and 1.13). ~CC13 + HS‘ _. CC13S" + ~11 (1.9) oCCl3 + 32032‘ + e— —> CC13S_ + 3032‘ (1.10) ~CC13 + 3.2“ + e— —> CC13S_ + 3,.-.2‘ (1.11) CCI3S_ ——> celzs + or (1 . 12) CClzs + 2011‘ —> co2 + 2113‘ (1.13) If the trichloromethyl radical is produced under aerobic conditions in mammalian or abiotic systems, molecular oxygen reacts with the trichloromethyl radical to give a peroxy radical as demonstrated in reaction 1.14. The peroxy radical is eventually trans inten therr (Iaufo. transformed to C02 by a series of reactions where phosgene (CCle) is a major intermediate (Kubik & Anders, 1980; Shah et al., 1979). Reactions 1.15 to 1.21 depict the main steps in this process, starting with a reduced organic compound (RH). ~CC13 + oz ——-> £01300 (1.14) -CC13OO + RH _. °CC13OOH + -R (1.15) ~CCI3OOH + H20 —» CCIZO + H202 + HCl (1.16) 2(-CCI3OOH) —> new + 02 (1.17) ~CC13O ———> CCIZO + -c1 (1.18) ~CC13O + 11* + e‘ _, CCI3OH (1.19) CC13OH —> CCIZO + HCl (1.20) CCIZO + H20 __, co2 + 2HC1 (1.21) Pseudomonas stutzeri strain KC Pseudomonas stutzeri strain KC (ATCC deposit number 55595, DSM deposit number 7136) is a denitrifying bacterium originally isolated from an aquifer in Seal Beach, California. It can rapidly degrade CC14 to C02 (~ 50%), a cell associated fraction (~5%), formate (~20%) and unidentified nonvolatile compounds (~ 25%), when CC14 is provided under iron-limiting conditions (Criddle et al., 1990b; Dybas et al., 1995; Lewis & Crawford, 1993). Pseudomonas stutzeri strain KC was the first isolated bacterium chlc rates ICSCI abou EVen under CODIGI only a Shaui Inotfle dUfing swan.) mime ( “enrul biotech. blOaUgr injected 3000) 1110 Tea: capable of degrading CC14 under anoxic conditions with minimal accumulation of chloroform. It is also unique in its ability to degrade CCl4 faster than the degradation rates observed in the reduced methanogenic microcosms that favor dehalogenation reactions (Krone & Thauer, 1992). Appendix A provides more detailed information about the phylogeny and taxonomy of Pseudomonas stutzeri strain KC. Even though the CCl4 degradation capacity of P. stutzeri strain KC is not fully understood, this organism has proven to be beneficial for the remediation of CCl4 contaminated sites because the CCl4 dechlorination reaction is rapid, with half-lives of only a few minutes (Tatara et al., 1995) and occurs without accumulation of chloroform. Strain KC attaches to aquifer sediments, but it can also exist in a free-swimming highly motile form that is chemotactic towards nitrate, and it can sustain dechlorination activity during migration (Witt et al., 1999a; Witt et al., 1999b). Emerson (1999) reported that strain KC reproducibly forms complex colony patterns on agar motility plates containing nitrite or nitrate. Five other species of pseudomonads tested under identical conditions were unable to form such patterns. Strain KC has also assumed great significance for biotechnology because of its use in one of the first full-scale field aquifer bioaugmentation applications. Large volumes of strain KC were grown on-site and injected into a CCl4—contaminated aquifer in Schoolcrafi, Michigan (Hyndman et al., 2000). The resulting biocurtain for CC14 degradation has now been maintained for over two years, with efficient removal of CCl4. The mad L'm'x CC 14 the C close} of im. the tra 1993 ). associz secrete Culture; C0ndillt System Secreted mleOllo tranngr (1]., 1999 The pair 511 ”grout approx l T The CCl4 degradation capacity of strain KC remained a mystery for many years. Efforts made by the Criddle group at Michigan State University and the Crawford group at the University of Idaho, helped elucidate the conditions that allowed the rapid degradation of CCl4 by P. stutzeri strain KC. Results of the first experiments performed to characterize the CCl4 degradation capacity of strain KC indicated that the degradation of CCl4 is closely related to the amount of trace elements available in the culture medium. Addition of iron and cobalt inhibit the transformation, but trace amounts of copper are needed for the transformation to occur (Criddle et al., 1990b; Lewis & Crawford, 1993; Tatara et al., 1993). The degradation of CCl4 by P. stutzeri strain KC requires cell-membrane associated reducing power that is independent of the electron transport chain and a small secreted supernatant factor that is produced when the bacterium is grown in iron limiting cultures (Tatara, 1996). This secreted factor is produced under aerobic and anaerobic conditions, but the transformation of CC14 only occurs when oxygen is removed from the system (Tatara et al., 1993). Carbon tetrachloride transformation also occurs when the secreted factor is combined with actively respiring bacteria, yeast cells, or aquifer microflora (Tatara et al., 1993; Tatara et al., 1995). The secreted factor can be transported without retardation through aquifer sediments and agar (Sepulveda-Torres et al., 1999; Tatara et al., 1995). The pattern of growth of P. stutzeri strain KC and the production of the secreted factor suggested that the factor is involved in trace-nutrient delivery. A very rapid initial phase of growth is observed in which no secreted factor production is detected. However, at approximately 12 hours of growth, the rate of total protein production slows and the 10 seen due linki; no(:4 19901 Hansf secont limitir. (Cndd known secrete: 1995).. can deg that Sid: Uiohnad EXpen' m intermci. (I 995) r formate form a [‘r meg”) secreted factor production accelerates. This pattern suggests that the factor is produced due to the limitation of a required nutrient or metal (Tatara et al., 1993). Observations linking the iron scavenging hypothesis to CCl4 transformation included the following: (i) no CCl4 transformation occurs if iron is present in the cell-culture medium (Criddle et al., 1990b), (ii) addition of iron to grown cultures of P. stutzeri strain KC inhibited CCI4 transformation, possibly by competing for a binding site (Tatara et al., 1993), (iii) the second order rate coefficient for CCl4 transformation increased for cells grown in iron- limiting media (Tatara et al., 1993), (iv) CC14 transformation was also inhibited by cobalt (Criddle el al., 1990b; Lewis & Crawford, 1993; Lewis & Crawford, 1999a) which is known to form stable complexes with siderophores (Ekkehardt et al., 1990), (v) the secreted factor had an apparent molecular weight of less than 500 Daltons (Dybas et al., 1995), which correlates with the molecular weights of siderophores, (vi) other cell types can degrade CCl4 when the secreted factor is provided (Tatara et al., 1995). It is known that siderophores that are produced by one microorganism can be utilized by others (Hohnadel & Meyer, 1988). Experiments with l4CC14 were performed to determine the identity of the degradation intermediates of the strain KC mediated CCl4 dehalogenation process. Dybas et a1. (1995) reported the formation of radioactive formate from l4CCl4. The formation of formate requires a two-electron transfer to the CC14 molecule. The first electron will form a trichloromethyl radical (reaction 1.1). The addition of a second electron results in the formation of a dichlorocarbene radical (reaction 1.22) that can subsequently be hyd. Carl Lewis electrc Static : hydrolyzed to formate and carbon monoxide, as illustrated in reactions 1.23 and 1.24. Carbon monoxide formation was not tested in the Dybas et al. report. ~ccl3 + e' __, :cc12 + or (1.22) :CClz + 21120 —> (311202 + 2HC1 (1.23) :CCl2 + H20 —> co + 2HC1 (1.24) Lewis and Crawford (1995, 1999a) postulated that CCl4 degradation occurs via a one- electron reduction pathway. They detected phosgene in l4CC14 experiments performed in static serum bottles containing an oxygenic atmosphere. Molecular oxygen reacts to the trichloromethyl radical to produce a peroxy radical (reaction 1.25) that could further react to produce phosgene as indicated in reactions 1.26 to 1.29). Phosgene is a highly reactive molecule that readily hydrolyzes to C02 in aqueous solutions (reaction 1.30). The formation of phosgene from CCl4 was proven by the addition of trapping agents such as N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) or cysteine to the culture medium, followed by the subsequent identification of the corresponding condensation products (reactions 1.31 and 1.32). ~CC13+02 —> ~OOCCI3 (1.25) [-OOCCl3] ————> 2cc13o-+o2 (1.26) CCl30' I COClz +Cl° (1.27) 12 ThiOph Thioph, (macho) [hiOPhos (DRIED, CC13O- + H+ + e” —> CC13OH (1.28) CC13OH _> COC12+HC1 (1.29) COC12+H20 -——> C02+HC1 (1.30) 0 Foo— +NH3-C—CH2—SH +COC12 —> NH S +2HC1 (1.31) I E f H C00" (cysteine) o _ H II _ O—CHz-CHz-U-CHz-CHz-fi-O + COClz —> (HEPES) o o 0 ll 0 fl—CHz—CHZ—s—O‘ +2HC1 (1-32) \ /\__/ 1|) Thiophosgene was identified in similar experiments performed under anoxic conditions. Thiophosgene is formed when the trichloromethyl radical reacts with a sulfur species (reactions 1.33 —- 1.34). It hydrolyzes to produce C02 (reaction 1.35). The formation of thiophosgene from CC14 was proven by the addition of N,N’-dimethylethylenediamine (DMED) to the culture medium, followed by the subsequent identification of the corresponding condensation product (reaction 1.36). ~CC13+RS_ -—> ‘scc13 (1.33) 13 Lewis CC14. “pone, Excess1 them 10 on). .m Dybas accUrnu] heron, Usuan). medialu‘ 'scc13 ——> CSC12+C1‘ (1.34) CSC12+H20 ——> C02+HC1 (1.35) CH3—NH—CH2—CH2-NH—CH3 +c:sc12 —> (DMED) S N N Lewis and Crawford (1999a) also looked for the formation of radioactive formate from MCC14. 14C-formate accounted for approximately 5% of the total l4CCl4, as previously reported by Dybas et al. (1995). The addition of formate at more than 300-fold molar excess to the CC14 addition had no significant effect on the total C02 production allowing them to conclude that formate is indeed a final product of the CC14 degradation pathway of P. stutzeri strain KC. This result confirms that the two-electron pathway proposed by Dybas er al. occurs to some extent. The Crawford group observed chloroform accumulation in their cultures. Nevertheless, chloroform corresponds to less than 4% of the total carbon in all their cultures, indicating that the trichloromethyl radical is not usually involved in hydrogen abstraction reactions in the CC14 dehalogenation processes mediated by P. stutzeri strain KC. 14 The Degl The s reeen (F1 gm secret el al.. from t ability lTiChIOI PDTC. degrade Showed result is that in); Partially indicmC inaCIlVa The Discovery of Pyridine-2,6-bis(thiocarboxylic acid) as a Carbon Tetrachloride Degrading Compound The secreted factor responsible for the CC14 degradation capacity of P. stutzeri KC was recently identified as pyridine-2,6-bis(thiocarboxylic acid) (PDTC) (CAS # 69945-42-2) (Figure 1.1) (Lee et al., 1999). This compound was originally described as a molecule secreted by Pseudomonas putida DSM 3601 grown under iron-limited conditions (Ockels et al., 1978). In assays using Ti(III) citrate, both synthetic PDTC and PDTC isolated from the supernatant of strain KC cells grown under iron-limiting conditions, had the ability to degrade CCl4. Other chlorinated compounds such as chloroform, trichloromonofluoromethane (CC13F) or 1,1,1-trichloroacetic acid did not react with PDTC. Similar results were seen in assays when PDTC was added to cells that do not degrade CCl4 on their own (Lee et al., 1999). PDTC coupled to iron or copper also showed CCl4 degradation capacity when added to living cells or Ti(III) citrate. This result is consistent with the previous observation made by Dybas et al. (1995) showing that iron addition at the reaction level did not inhibit the CCl4 degradation capacity of partially purified secreted factor. The experiments performed by Lee et al. (1999) also indicate that PDTC can be used as a catalyst with a limited turnover, suggesting inactivation upon reaction with CC14. PDTC cobah Holm. PTEVio O\\ | //O c 7 c H—S / N \s—H Figure 1.1 Chemical structure of pyridine-2,6-bis(thiocarboxylic acid) (PDTC). PDTC is known to form a variety of complexes with transition metals including iron, cobalt, nickel and palladium (Espinet et al., 1994; Hildebrand et al., 1984; Kruger & Holm, 1990). The redox potential of Fe-PDTC and Ni-PDTC complexes have been previously described (Hildebrand et al., 1984; Kruger & Holm, 1990). Figure 1.2 illustrates the change in oxidation state of the iron center in Fe-PDTC complexes. 16 Flgu propt L"Pot ( 199< pTC‘ge PD“ a bag- \ \ 2 l + F e3+ O I O / /O \ / / O \\C N C/ ——> \C N C / / \ S/ l \s __:S S‘_ . 3+ ............ , ZFe i"; s" I ‘~~s \ / __ __ C N C / \ 2— 0 / \ \o I \ I / 0 / O __ __ \ '/ >2 N c< S l ...... S oxidation ...... Fe3+_,.v"°'.. 3"“ I ‘ ‘~S reduction \C C/ N / \ O / l \ \O / Figure 1.2 Mechanism for iron chelation by pryridine-2,6-bis(thiocarboxylic acid) as pr0posed by Ockels er a1. (1978). Adapted from Lee et al.(1999). Upon the discovery of PDTC as the CC14 dechlorination agent, Lewis and collaborators (1999b) studied the CCl4 degradation capacity of synthetic PDTC - metal complexes and presented their work in a preliminary report. Free acid PDTC as well as F e-PDTC, Cu- PDTC and Ni-PDTC conferred the CCl4 degradation capacity to P. stutzeri ATCC 17588, a bacterium incapable of degrading CCl4 on its own. On the other hand, Co-PDTC was 17 ina inh 19c abll PI) “3“ Res sho Wht‘ 0th: redt PTOL cs: The EluC The form) radiC inactive. This result is consistent with previous reports showing cobalt-mediated inhibition of the CC14 degradation pathway (Criddle et al., 1990b; Lewis & Crawford, 1993). When Nags was used as a reductant, only the free acid PDTC and Cu-PDTC were able to remove CCl4 from the medium. When no reductant was added, 1 molecule of Cu- PDTC reacted with 2 molecules of CCl4 whereas the removal mediated by the free-acid was less efficient and more erratic. Results of experiments with l4CCl4 in the presence of PDTC with no added reductants showed a carbon distribution similar to the one observed in experiments performed with whole cultures: 70% C02, 20% volatile material, 10% non-volatile products. On the other hand, more volatile compounds were observed when NaZS was added as a reductant. Carbon disulfide (C82) and carbonyl sulfide (COS) were identified as volatile products obtained from the PDTC-mediated degradation of l3CC14 and the production of CS; was directly correlated to the amount of NaSz added to the reaction mixture. The mechanism of the Cu-PDTC mediated CCl4 degradation process has not been elucidated and the possible mechanism provided in this section is based on speculation. The data available to date suggest that copper (probably Cu”) binds to PDTC, allowing the activation of the electron-dense sulfur to donate an electron to CC14, causing the formation of a trichloromethyl radical and a pyridine-2,6-bis(thiocarboxylate) thyil radical (Lewis, 2000) (Figure 1.3). The thyil radical may be stabilized with resonance on 18 the that tricl sulfu thioc dipie Other intact the copper atom and the function of Cu may be restricted to provide the right conditions that would allow sulfur activation (Broderick, 2000). If the thyil radical and the trichloromethyl radical are in close proximity, they may recombine forming a sulfur- bound trichloromethyl complex that eventually produces phosgene and thiophosgene. Phosgene and thiophosgene are unstable intermediates that hydrolyze to C02 (Figure 1.3A). If trichloromethylthiol is an intermediate of the CC14 degradation pathway, the sulfur-carbon bonds of PDTC are destroyed, forming a carboxyl group. If both thiocarboxylic acid groups are converted into carboxyl groups, PDTC is transformed into dipicolinic acid, a compound that is unable to catalyze the degradation of CC14. On the other hand, if trichloromethanol is produced instead, the thiocarboxylic bond remains intact, allowing the possible regeneration of PDTC. The trichloromethyl radical is highly reactive and may interact with other compounds available in the medium and may lead to the production of CD; as indicated in Figure 1.3C. Of special interest is the lack of chloroform formation during the PDTC-mediated degradation of CC14. As indicated in a previous section of this chapter, chloroform may be produced from the trichloromethyl radical by extracting a hydrogen atom from lipids or proteins or by reacting with a proton and an electron (equations 1.3 and 1.4). If the trichloromethyl and the pyridine-2,6-bis(thiocarboxylate) thyil radicals are formed simultaneously, as suggested in Figure 1.3, these radicals may recombine immediately to form the sulfur-bound trichloromethyl group (Figure 1.3A), decreasing the possibility of chloroform formation. These assumptions suggest that the reactions presented in Figure 1.3C would account only for a minimal fraction of the C02 produced. 19 If the exterr diehlo immet first id al. (1C. dichlor The pa POWer CC14 (1 Prepare] donate: eXlema Center, degrad. are int degrad; degrad; If the trichloromethyl radical receives a second electron (perhaps from copper or from an external electron donor), it would trigger the elimination of chloride and the formation of dichlorocarbene (Figure 1.38). If dichlorocarbene is formed at some extent, it immediately reacts with water to produce carbon monoxide and formate. Formate was first identified as a final product of the strain KC-mediated CC14 degradation by Dybas et al. (1995) and was later confirmed by Lewis and Crawford (1999a), suggesting that dichlorocarbene formation may occur at some extent. The pathway presented in Figure 1.3 does not account for the role of external reducing power in the degradation of CC14. Tatara (1996) demonstrated that the degradation of CC14 was enhanced when the reaction occurred in the presence of cell membrane preparations amended with NADH + H+. He also demonstrated that the electrons donated to CCl4 did not come from the electron transport chain. A possible role of external reducing power may be to maintain the proper oxidation state of the metal center. More experiments are needed in order to elucidate how Cu-PDTC catalyzes the degradation of CCl4, what are the intermediates of the process and what external factors are involved in the degradation pathway. The discovery of Cu-PDTC as the CCl4 degrading agent is only another step in the elucidation of the fascinating CCl4 degradation capacity of Pseudomonas stutzeri stain KC. 20 Figure 1.3 Proposed pathway for the degradation of CC14 by Cu-PDTC. A) Formation of C02 via the condensation of a trichloromethyl radical and a thyil radical. B) Formation of formate and carbon monoxide from a dichlorocarbene intermediate. C) Formation of C02 by the interaction of the trichloromethyl radical with unknown compounds. Final products identified in the dechlorination pathway are indicated by solid boxes. Known pathway intermediates are indicated by dashed boxes. Both sulfurs present in PDTC can theoretically participate in the reactions presented in branch A. Only one cycle is presented for the benefit of simplicity. This figure was modified from (Tatara, 1996) and (Lewis, 2000). 21 11C) I \ CI 0 I \ O I \ O _ O O \\ / // + CC14 C‘ I + \\ / // \\ / // C N c [I ~c—c1 c N C c N c I I H I lCl I I I I I 2+ I 8 —Cu —s s —Cul+—s- s —Cu — S a _ ‘\ ’4 trichloromethyl e -' ........... _| H20 9 / radlcal pyridine-2,6-bis(thiocarboxylate) ' o - - thyil radical i II E l i C i ; / \ I I C1 C1 ! 2 HCI \ : hos ene I O I 0 A I P g : / N sulfur-bound trichloromethyl group C02 Cl / :C t H 0 \ C1 2 CI dichlorocarbene O | \ 0 | .— \\ / C// H s — C — Cl c N l I I1+ I 1 CI 3 _Cu “'0" trichloromethvlthiol OR CI 0 | \ HO— -CI 0 )C + \\ / // 2 H20 c1 C N C HCI , I l 1+ I tr1chloromethanol 3 —Cu —3H k HCI HCI V; 2 HCI I """"""" _‘ : O I .............. _ CO V ! II I ! s I I /C\ i I I I O 5 CI CI : 2 H20 i / i II phosgene . I CI El I C ;__. ________ I ; thiophosgene : / \OH I ' H . ______________ : “20 w C02 formate HCI H23 + 2 HCI 22 OutIiI Outline of This Dissertation The primary objective of this research project was to characterize the genes that confer the CC14 degradation capacity to Pseudomonas stutzeri strain KC. Chapter 2 describes how mutants unable to degrade CC14 were created by transposon mutagenesis. It also provides some information about phenotypic characterization of the aforementioned mutants. Chapter 3 provides an exhaustive genetic analysis of the genes mutated in the P. stutzeri strain KC mutants described in Chapter 2. Information about other genes involved in CC14 degradation, mutated independently by another research group, is also provided. The information available about all the genes was used to propose a pathway for PDTC biosynthesis. Lastly, Chapter 4 provides suggestions for future research that may enhance our knowledge of the physiological role of PDTC and how it could be used for effective remediation of CC14 contaminated sites. Experiments for the elucidation of how Pseudomonas stutzeri strain KC controls PDTC synthesis are also provided. 23 Ade Ahr, Baka- Brode Butler Criddl Cfiddl Dl'bas. REFERENCES Adewuyi, Y. G. & Carmichael, G. R. (1987). Kinetics of hydrolysis and oxidation of carbon disulfide by hydrogen peroxyde in alkaline medium and application to carbonyl sulfide. Environ Sci Technol 21, 170-177. Ahr, H. J., King, L. J., Nastainczyk, W. & Ullrich, V. (1980). The mechanism of chloroform and carbon monoxide formation from tetrachloromethane by microsomal cytochrome P-450. Biochem Pharmachol 29, 2855-2861. Bakac, A. & Espenson, J. H. (1985). Kinetics and mechanism of the alkylnickel formation in one-electron reductions of alkyl halides and hydroperoxides by a macrocyclic nickel (1) complex. J Am Chem Soc 108, 713-719. Broderick, J. (2000). Personal communication, Michigan State University. Butler, T. C. (1961). Reduction of carbon tetrachloride in vivo and reduction of carbon tetrachloride and chloroform in vitro by tissue constituents. J Pharmaco Exp Theory 134, 311. Criddle, C. S., DeWitt, J. t. & McCarthy, P. L. (1990a). Reductive dehalogenation of carbon tetrachloride by Escherichia coli K-12. Appl Environ Microbial 56, 3247- 3254. Criddle, C. S., DeWitt, J. T., Grbic-Galic, D. & McCarthy, P. L. (1990b). Transfromation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions. Appl Environ Microbiol 56, 3240-3246. Dybas, M. J., Tatara, G. M. & Criddle, C. S. (1995). Localization and chracterization of the carbon tetrachloride transformation activity of Pseudomonas sp. strain KC. Appl Envrion Microbiol 61, 758-762. 24 Ekkehardt, H. F., McMurry, T. J., Hugi, A. & Raymond, K. N. (1990). Coordination chemistry of microbial iron transport: structural and spectroscopic characterization of diastereometric Cr(IIl) and Co(III) complexes of desferriferrithiocin. J Am Chem Soc 112, 1854-1860. Emerson, D. (1999). Complex pattern formation by Pseudomanas strain KC in response to nitrate and nitrite. Microbiology 145, 633—641. Espinet, P., Lorenzo, C. & Miguel, J. A. (1994). Palladium complexes with the tridentate dianionic ligand pyridine-2,6-bis(thiocarboxylate), PDTC. Crystal structure of (n-Bu4N)[Pd(pdtc)Br]. J Am Chem Soc 33, 2052-2055. Fowler, S. J. L. (1969). carbon tetrachloride transformation in the rabbit. Br J Pharm 37, 733-737. Gantzer, C. J. & Wackett, L. P. (1991). Reductive dechlorination catalyzed by bacterial transition-metal coenzymes. Environ Sci Technol 25, 715-722. Hashsham, S. A., Scholze, R. & Freedman, D. L. (1995). Cobalamin-enhanced anaerobic biotransformation of carbon tetrachloride. Environ Sci Technol 29, 2856-2863. Hashsham, S. A. (1996). Cobalamin-enhanced anaerobic biotransformation of carbon tetrachloride. Doctoral Dissertation, University of Illinois, Urbana-Champaign. Hildebrand, U., Lex, J., Taraz, K., Winkler, S., Ockels, W. & Budzikiewicz, H. (1984). Untersuchungen zum redox-system bis(pyridin-2,6-dicarbothioato)- ferrat(II)/-ferrat(lll). Z Naturforsch (B) 39B, 1607-1613. Hohnadel, D. & Meyer, J. M. (1988). Specificity of pyoverdin-mediated iron uptake among fluorescent Pseudomonas strains. J Bacteriol 170, 4865-4873. 25 Hym Jeffel Krleg Krone Kubik L331. I Hyndman, D. W., Dybas, M. J., Fomey, L., Heine, R., Mayotte, T., Phanikumar, M. I S., Tatara, G., Tiedje, J., Voice, T., Wallace, R., Wiggert, D., Zhao, X. & Criddle, C. S. (2000). Hydraulic characterization and design of a full scale biocurtain. Ground Water In Press. Jeffers, P. M., Ward, L. M., Woytowitch, L. M. & Wolfe, N. L. (1989). Homogeneous hydrolysis rate constants for selected chlorinated methanes, ethanes, ethenes, and propanes. Environ Sci Tech 23, 965-969. Kriegman-King, M. R. & Reinhard, M. (1992). Transformation of carbon tetrachloride in the presence of sulfide, biotite, and vermiculite. Environ Sci T echnol 26, 2198- 2206. Krone, U. E. & Thauer, R. K. (1992). Dehalogenation of tiichlorofluoromethane (CF C- 11) by Methanosarcina barkeri. FEMS Microbiol Lett 90, 210-214. Kruger, H. J. & Holm, R. H. (1990). Stabilization of trivalent nickel in tetragonal NiS4N2 and NiN6 environments: synthesis, structures, redox potentials, and observations related to [NiFe]-hydrogenases. J Am Chem Soc 112, 2955-2963. Kubik, V. L. & Anders, M. W. (1980). Methabolism of carbon tetrachloride to phosgene. Life Sciences 26, 2151-2155. Last, G. & Rohay, V. (1991). Carbon tetrachloride contamination, 200 West Area, Hanford Site. PNL-SA-19564, Pacific Northwest Laboratory, Richland, Washington. Lee, C. H., Lewis, T. A., Paszczynski, A. & Crawford, R. L. (1999). Identification of an extracellular catalyst of carbon tetrachloride dehalogenation from 26 Lew Leu1 Lewi LCWis L°“is Luke, Pseudomonas stutzeri strain KC as pyridine-2,6-bis(thiocarboxylate). Biochem Biophys Res Commun 261, 562-566. Lewis, T. A. & Crawford, R. L. (1993). Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC. Appl Environ Microbiol 59, 163 5-1641. Lewis, T. A. & Crawford, R. L. (1995). Transformation of carbon tetrachloride via sulfur and oxygen substitution by Pseudomonas sp. strain KC. J Bacteriol 177, 2204-2208. Lewis, T. A. & Crawford, R. L. (1999a). Chemical studies of carbon tetrachloride transformation by Pseudomonas stutzeri strain KC. In Novel Approaches for Bioremediation of Organic Pollution (ed. R. F ass, Y. Flashner and S. Reuveny), pp. 1-1 1. Plenum Publishers, New York. Lewis, T. A., Paszczynski, A., Lee, C. H. & Crawford, R. L. (1999b). The dechlorination agent for Pseudomonas stutzeri strain KC toward carbon tetrachloride is pyridine-2,6-bis(thiocarboxylic acid). In Pseudomonas '99, Maui, Phnwafi. Lewis, T. (2000). Personal communication, University of Vermont. Luke, B. T., Loew, G. H. & McClean, A. D. (1987). Theorethical investigations of theanaerobic reduction of halogenated alkanes by cytochrome p450. 1. Structures, inversion barriers, and heats of formation of halomethyl radical. J Am Chem Soc 109, 1307-1317. 27 Ockels, W., Romer, A. & Budzikiewicz, H. (1978). An F e(II) complex pryridine-2,6-di- (monothiocarboxylic acid) - a novel bacterial metabolic product. Tetrahedron Lett 36, 3341-3342. United Nations Environmental Programme (1994). Adjustment to the Montreal Protocol on substances that deplete the ozone layer. In Principles of International Environmental Law. : 2A. Documents in International Environmental Law (ed. P. Sands, R. Tarasofsky and M. Weiss), pp. 208-223. Manchester University Press, Manchester. Sepulveda-Torres, L. del C., Rajendran, N., Dybas, M. J. & Criddle, C. S. (1999). Generation and initial characterization of Pseudomonas stutzeri KC mutants with impaired ability to degrade carbon tetrachloride. Arch Microbiol 171, 424-429. Shah, H., Hartman, S. P. & Weinhous, S. (1979). Formation of carbonyl chloride in carbon tetrachloride metabolism by rat liver in vitro. Cancer Research 39, 3942- 3947. Sittig, M. (1985). Handbook of Toxic and Hazardous Chemicals and Carcinogens. Noyes Publications, New York. Skeen, R., Amos, K. & Petersen, J. (1994). Influece of nitrate concentration on carbon tetrachloride transformation by a Ddenitrifying microbial consortium. Water Res 28, 2433-2438. Tatara, G. M., Dybas, M. J. & Criddle, C. S. (1993). Effects of medium and trace elements on kinetics of carbon tetrachloride transforamtion by Pseudomonas sp. strain KC. Appl Environ Microbiol 59, 2126-2131. 28 Tatara, G. M., Dybas, M. J. & Criddle, C. S. (1995). Biofactor mediated transformation of carbon tetrachloride by diverse cell types. In Bioremediation of Chlorinated Solvents, vol. 3(4) (ed. R. E. Hinchee, A. Leeson and L. Semprini), pp. 69-76. Battelle Press, Columbus, Ohio. Tatara, G. M. (1996). Physiology of carbon tetrachloride transformation by Pseudomonas stutzeri KC. Doctoral Dissertation, Michigan State University. Verschueren, K. (1983). Handbook of Environmental Data on Organic Chemicals, pp. 341-344. Van Nostrand Reinhold, New York. Vogel, T. M., Criddle, C. S. & McCarty, P. L. (1987a). Transformations of halogenated aliphatic compunds. Environ Sci Technol 21, 722-726. Vogel, T. M. & McCarty, P. L. (1987b). Abiotc and biotic transformations of 1,1,]- trichloroethane aliphatic compounds. Environ Sci T echnol 21, 1208-1213. Wade, R. S. & Castro, C. E. (1973). Oxidation of iron (II) porpyrins by alkyl halides. J Am Chem Soc 95, 226-230. Witt, M. E., Dybas, M. J., C, W. D. & Criddle, C. S. (1999b). Use of bioaugmentation for continuous removal of carbon tetrachloride in model aquifer columns. J Environ Eng Sci 16, 475-485. Witt, M. E., Dybas, M. J., Worden, R. M. & Criddle, C. S. (1999a). Motiltity- enhanced bioremediation of carbon tetrachloride-contaminated aquifer sediments. Environ Sci Technol 33, 295 8-2964. 29 CHAPTER 2 GENERATION AND INITIAL CHARACTERIZATION OF PSEUDOMONAS S T U T ZERI KC MUTANTS WITH IMPAIRED ABILITY TO DEGRADE CARBON TETRACHLORIDE Published in Archives of Microbiology (1999) 171: 424-429 Printed with permission of the publisher 30 Lycel Nara; Craig Gen Race: v1 Arch Microbiol (1999) 171 :424-429 0 Springer-Verlsg 1999 ()RI(iI.\',»\I. I’.\I’I:I{ Lycely Del C. Septilveda-Torm Narayanan Rajendran - Michael J. Dybas Craig S. Criddle Generation and initial characterization of Pseudomonas stutzeri Kc mutants with impaired ability to degrade carbon tetrachloride Received: 23 November 1998 / Accepted: 15 March 1999 Abstract Under iron-limiting conditions. Pseudomonas stutzeri KC secretes a small but as yet unidentified factor that transforms carbon tetrachloride (CI') to C02 and non- volatile products when activated by reduction at cell membranes. Pseudomonas fluorescens and other cell types activate the factor. Triparental mating was used to generate kanamycin-resistant luxzz’I‘nS recombinants of strain KC. Recombinant: were streaked onto the surface of agar medium plugs in microtiter plates and were then screened for carbon tetrachloride degradation by exposing the plates to gaseous "C-carbon tetrachloride. Cl“ re- combinants generated nonvolatile l"C-labeletl products. but four CI" recombinants did not generate significant nonvolatile l‘C-labeled products and had lost the ability to degrade carbon tetrachloride. When colonies of P. fluo- rescens were grown next to colonies of CT‘ recombinants and were exposed to gaseous l‘C-carbon tetrachloride. "C—labeled products accumulated around the P. fluo- rescens colonies. indicating that the factor secreted by CI‘t colonies had diffused through the agar and become activated. When P. fluorescens was grown next to CI‘ colonies, little carbon tetrachloride transformation was observed, indicating a lack of active factor. Expression of Ira reporter genes in three of the CT" mutants was regu- lated by added iron and was induced under the same iron- L. De! C. Sepulveda-Torres Department of Microbiology. Michigan State University. East Lansing. MI 48824. USA L. Del C. Sepulveda-Torres - M. l. Dybas - C. S. Criddle Center for Microbial Ecology, Michigan State University. East Lansing. MI 48824. USA N. Rajendran - C. S. Criddle Department of Civil and Environmental Engineering. Michigan State University, East Lansing. MI 48824. USA C. S. Criddle (8) Department of Civil and Environmental Engineering. Stanford University. Stanford. CA 94305-4020. USA e-mail: criddle®ce.stanford.edu. Tel.: +1-650-723-9032, Fax: +1-650-725-9474 31 limiting conditions that induce carbon tetrachloride trans- formation in the wild-type. Key words hansposon mutagenesis - Carbon tetrachloride - Biotransformation - Biodegradation - Luciferase - Pseudomonas stutzeri KC - Reporter genes - Mutants Abbreviation 67' Carbon tetrachloride - Km Kanamycin - LB Luna broth - RLU Relative light units - Rf Rifampicin - TSB Tryptic soy broth Introduction Carbon tetrachloride (CI') is a suspected carcinogen that also causes acute liver toxicity in animals (Sittig 1985). Its production has been banned under the terms of the amended Montreal protocol because it is an ozone-deplet- ing agent (United Nations Environmental Programme 1994). Most denitrifying organisms that degrade carbon tetrachloride do so slowly with the accumulation of chlo- roform. a compound that can be even more persistent than carbon tetrachloride (Egli et al. 1988; Semprini et al. 1992). Pseudomonas stutzeri strain KC is an aquifer isolate that transforms carbon tetrachloride to carbon dioxide (Criddle et a1. 1990: Lewis and Crawford 1993; Tatara et al. 1993), formate (Dybas et al. 1995), and other non- volatile products without the formation of chloroform (Criddle et a1. 1990; Lewis and Crawford 1993: Tatara et al. 1993). Rapid carbon tetrachloride transformation re- quires a small (500 Da) factor secreted by exponential- stage strain KC cells grown under Fe’t-limiting condi- tions, together with actively growing cells capable of re- generating the secreted factor. The transformation occurs only under anoxic conditions, although the factor is pro- duced under both oxic and denitrifying conditions (Dybas et a1. 1995). Organisms that usually do not degrade carbon tetrachloride are able to do so when the factor is provided (Tatara et al. 1995). Of special interest are the genes re- quired for production. secretion. and activation of the fac- tor. To identify these genes. methods are needed for rapid and efficient screening of large numbers of mutants. Most of the microorganisms that degrade volatile. halogenated hydrocarbons produce C02 and nonvolatile products (Jain and Criddle 1995). The nonvolatile prod- ucts accumulate or are utilized for growth (Nielsen 1990; Chaudhry and Chapalamadugu 1991; Fetzner and Lingens 1994; Jain and Criddle 1995). When I‘C-labeled hydro- carbons are degraded. nonvolatile "C-labeled products are often generated (Wackett and Gibson 1983; Lewis and Crawford 1993; Sepiilveda et al. 1997). To obtain mutants with impaired ability to degrade carbon tenachloride. we screened a large number of P. stutzeri KC lux::Tn5 mu- tants for failure to produce nonvolatile l‘C-labeled prod- ucts from l‘C-carbon tetrachloride. Mutants were then characterized for (to: expression by assaying luciferase under different growth conditions. Materials and mounds Strains and plasmids P. stutzeri strain KC (DSM 7136. ATCC 55595). isolated from an aquifer in Seal Beach (Calif. USA) (Criddle et al. 1990). was maintained on nutrient agar. Pseudomonas fluorescens (ATCC 13525) was obtained from the Department of Microbiology. Michigan State University. Rifampicm-resistant (Rf‘) P. stutzeri KC strains were selected in our laboratory on nutrient agar plates containing rifampicin (100 ug/ml) from a set of spontaneous Rt“ strain KC mutants. An Rf“ strain KC isolate was used in the tri- parental matings described below in order to counterselect for the Escherichia coli donor and helper strains. One Rf“ strain. KC137. was selected for subsequent studies because its carbon tetrachlo- ride degradation capability matched that of the wild-type. All Pseudomonas strains were grown at 20—25 °C with constant shak- ing (150 rpm). 8. call DHSa containing pRL1063a. a kanamycin- resistant (Km‘) transposon delivery plasmid for a luxAB::Tn5 con- struct (Wolk et al. 1991). and E. coli containing the KmR helper plasmid pRK2013 (Ditta et al. 1980) were grown in Luria Broth medium (Sambrook et al. 1989) with 35 ug Kin/ml at 37°C and with shaking at 200 rpm. Chemicals and media Nonradioactive carbon tetrachloride (99% pure) was purchased from Aldrich Chemical (Milwaukee. Wis. USA). l‘C-carbon tetra- chloride (> 99% pure; 250 uCi. specific activity of 4.3 mCi/mmol) purchased from NEN Dupont Research Products (Boston. Mass.. USA) was dissolved in iso-octane to a concentration of 0.136 urnol/ul (1.4 uCi/ul) and was stored at -20 °C. Precipitate-free. iron-free Simulated Groundwater Medium (SGM) was prepared as described by Dybas et al. (1995) with some modifications. The modified medium used half of the sodium hydroxide and potassium phosphate specified in the origi- nal procedure. and Fe-free TN2 trace element solution (Criddle et al. 1990) instead of SGM trace element solution. The medium was prepared in acid-washed glassware. adjusted to an initial pH of 8.2. autoclaved at 121 °C for 25 min. transferred to a laboratory bench for quiescent settling of precipitate. and decanted after 24 h. The precrpitate-free decanted medium was reautoclaved for 25 min at 121°C and cooled before use. Precipitate-free SGM contained 23 mM acetate. 19 mM nitrate. and 0.1 M phosphate as determined using a Dionex model 2000i-SP ion-chromatography system (Sun- nyvale. Calif. USA). No iron was detected usrng a Perkin Elmer (Norwalk. Conn. USA) model 31. ”1 graphite furnace atomic 3b- sorpuon spectroscopy system. 425 Anoxic medium D adjusted to an initial pH of 8.0 was prepared in acid-washed glassware as previously described (Criddle et al. 1990). Noble agar. nutrient agar. nutrient broth. and trypuc soy broth (TSB) were obtained from Difco (Detroit. Mich.. USA). Transposon mutagenesis Triparental matings (Simon et al. 1983) were used to mobilize plasmid pRLlO63a into Rf“ P. stutzeri KC137. E. coli DHSa con- taining the donor plasmid pRLlO63a and E. colt DH5a containing the helper plasmid pRK2013 were grown overnight in LB medium supplemented with 35 ug Kin/ml at 37 C and with shaking at 200 rpm. These cultures were transferred to fresh LB without an- tibiotics using a 4% (by vol.) inoculum and were then grown at the same temperature and shaking speed for 4 h (final cell concentra- tion. ~10” cfu/ml). Cultures of the recipient strain KC137 were grown under aerobic conditions for 16 h in TSB or LB containing 35 ug Rf/ml. or under denitrifying conditions for 2 days in medium D containing 35 ug Rf/ml (final cell concentration. ~10“ cfu/ml). All cultures were washed twice and then resuspended to the origi- nal volume using sterile 0.9% NaCl solution. Mixes were prepared by combining 100 111 of E. cali DHSa (pRL1063). 100 ill of E. coli DHSa (pRK2013). and 500 Ill of strain KC137 in sterile micro- centrifuge tubes. Individual strains and 100 til/100 1.11 pairwise mixes were included as controls to ensure that KmRIRt‘ double mutants were not present in the mating plates. Fifty microliters of each mixture was spotted on 13-min filters (containing pores of 0.45 um in diameter: Millipore. Bedford. Mass. USA) on nutrient agar plates. The plates were incubated at 37 °C for 10 h and then at room temperature for 10 h. The filters were transferred to micro- centrifuge tubes containing 1 ml of a sterile 0.9% NaCl solution. and the bacteria grown on the filter were resuspended in the buffer. Resuspended cells were distributed in ZOO-til aliquots onto nutrient agar plates containing 50 ug Kin/ml and 50 ug Rflml and were then incubated at room temperature for 3 days. Microtiter plate assay for "0an tetrachloride degradation Sterile microtiter plates (96 wells) were filled with 200 |.l.l of me- dium D (pl-l 8) containing 15 g Noble agar 1‘1 and 25 ug Kin/ml. Recombinants obtained by triparental mating were streaked on the agar surfaces of the microtiter plate wells using sterile toothpicks. Inoculated microtiter plates were transferred to a 3.8-I steel paint can (Freund Can Company. Chicago. 111.. USA) containing clean glass marbles to minimize the gas volume within the can (working vol. - 1.9 1). Air in the can was replaced by a 95% nitrogen/5% hydrogen mixture by passing the open can through the interlock of an anaerobic glove box (Coy Laboratories. Detroit. Mich.. USA). A sealed 12 x 32-mm glass vial containing 1.4 umol (14 uCi) of “Carbon tetrachloride was attached to the inner wall of the con- tainer. The septum of the vial was punctured with a needle. releas- ing "C-carbon tetrachloride into the can. and the can was immedi- ately sealed and removed from the glove box for incubation. Plates inside the steel can were exposed to vapor phase l‘C-carbon tetra- chloride t’or 5 days at 20°C and then were vented overnight in a chemical hood. Agar plugs from the microtiter plates were trans- ferred to 10 ml of Safety-solve scrntillation cocktail (RPI, Prospect. 111.. USA) and were assayed for nonvolatile radioactivity for 3 min with a 1500 Tri-carb liquid scintillation counter (Packard Instrument. Downers Grove. 111.. USA). The recombinants that did not produce significant I‘C-labeled nonvolatile products In the microtiter plate assay were analyzed by gas chromatography (GC) to confirm loss of carbon tetrachloride degradation capabilities. Recombinants were grown overnight in 5 ml TSB containing 35 ug Kin/ml. These cultures were used as a 1% inoculum (by vol.) for 10 ml of anoxic medium D (pH 8) vials containing 35 ug Kin/ml and 350 ng of sterile carbon tetrachloride. Cultures were incubated upside down for a week under anoxic conditions at 20°C and with shaking at 150 rpm. The carbon tetra- :hloiide remaining 1n the vials was f"strayed by CC '15 described by Tatara et al. (1993). 426 Table 1 Results of the "CT microtiter plate assay and the Strain gas chromatography assay Noninoculated controls Wild-type Pseudomonas stutzeri KC Recombinants capable of degrading CT in both assays KC300 ‘Average 2 one standard devi- ation for seven replicates KC657 I’Average a: one standard devi- KC1896 ation for nine replicates 7 ‘ Average :e one standard devi- £2812: ation for triplicates Recombinants with impaired ability to degrade CT in both assays dpm per well in % CT degraded after 1 week microtiter plate assay in gas chromatography assay“ 82 t 15' 9 :i; 7 2218:2437“ IOOs: 0 1.416 100 a: 0 465 28 a: 26 51 l 18 :r: 8 291 28 a: 8 459 21 a: 13 Petri plate assay for secreted factor production By itself. the secreted factor produced by strain KC does not reli- ably transform carbon tetrachloride; however. reliable transforma- tion is observed when it is combined with viable whole cells (Dy- bas et al. 1995). Tatara (1996) has demonstrated that carbon tetra- chloride transformation also occurs when the secreted factor is added to crude cell membrane preparations supplemented with NADH. Decreased transformation rates were observed when the factor was added to crude cell membranes lacking N ADH. with lit- tle or no transformation when the secreted factor was added to cy- toplasmic fractions. Both cell membranes and NADH were re- quired for maximum activation. Of interest is the fact that P. fluo- rescens and many other cell types can mediate activation of the se- creted factor (Tatara et al. 1995). In order to determrne if the CT' mutants were impaired for secreted factor production. secretion. or activation. CT‘ mutants and cells of P. fluorescens were grown on Petri plates containing solid medium D (pH 8). Colonies of C1“ mutants and P. fluorescens were grown on Petri plates in an alter- nating “checkerboard" layout with each colony separated by 1.5 cm from adjacent colonies of the other organism. Petn plates were incubated following the same protocol used for the microtiter plates. After the incubation period. l-cm2 agar squares including and surrounding each colony were transferred to 10 ml scintillation cocktail and were counted by the liquid seintillation counter. Expression of lux genes in strain KC mutants CT“ and selected CT‘ recombinants were grown in 5 ml TSB con- taining 35 ug Kin/ml for 24 h at 25 °C and with shaking at 150 rpm. These cultures were used as 0.5% inocula (v/v) for 25-ml cultures of TSB or Fe-free. precrpitate-free SGM containing 35 ug Km/ml supplemented with 0. 5. 10. or 20 uM Fe” as Fem-1.60.); - 121110. Cultures were grown for 29 h at 25°C and with shaking at 150 rpm. and then were used as 0.5% inocula (v/v) for 100-ml cul- tures containing the same antibiotic and Fe” concentrations. Growth of these cultures was monitored over a 48-h period by periodically removrng l-ml aliquots and measuring optical densuy at 600 nm. The light emission assay used to detect lucrferase acuVity was performed on a Berthold Lumat LB 9501 1uminometer(EG&G Wal- lac. Gaithersburg. Md.. USA) by combining 5-ul culture aliquots with 50 til of a solution containing 20 mg bovine serum albumin/ml and 1 ul N-decyl aldehydelml. Samples were vortexed for 30 s and assayed for light emission (relative light units) for l min. Results Generation of mutants with impaired ability to degrade carbon tetrachloride Three thousand five hundred recombinants were obtained from 44 independent triparental mating events. Most re- combinants accumulated roughly the same level of I‘C-la— beled nonvolatile products in the agar plugs as wild-type cells (1.500-3.000 dpm). Only 38 recombinants accumu- lated fewer than 1.000 dpm: 4 were auxotrophs that could not grow in the defined medium used for the GC assay; 30 grew poorly in solid medium D. but grew and de- graded carbon tetrachloride in liquid medium; and 4 were classified as putative CT ' mutants based on their growth in liquid and solid media and on their inability to degrade significant CT in either assay. A PCR probe specific to strain KC was used to confirm that the putative CT' mu- tants were strain KC cells (Dybas et a1. 1998). Table 1 summarizes results for noninoculated controls. the wild- type. one of the recombinants that retained the ability to degrade carbon tetrachloride (strain KC300). and the four mutants that failed to transform carbon tetrachloride in ei- ther assay (C'I‘ mutants). Although the CI“ mutants were only tested once in the microtiter plate assay. the results of the GC assay provided statistically significant proof that these mutants were impaired in carbon tetrachloride degra- dation. Table 2 Petri plate assay of secreted factor production by wild- type and recombinant KC colonies grown in the presence of Pseudomonas fluorescens and l‘C- carbon tetrachloride. P. fluo- rescens was grown alone or in an alternating checkerboard config- uration with wild-type KC. KC300. KC657, KC1896, KC2753. or KC3164. All colonies were separated by 1.5 cm. Radioactivity is reported as dismtegrations per minute per square centimeter of agar for eight replicates Incubation condition dpm/cmz (avg : sd) No-cell control 42 3: 5 P. fluorescens alone 198 :i: 35 P. fluorescens-o-wfld-type strain KC 1.583 a: 335 P. fluorescens+autoclaved wild-type strain KC 215 a: 32 P. fluorescens+KC300 (CT’) 1.905 :i: 179 P. fluorescens+KC657 (CT‘) 95 a: 13 P. fluorescens+KC1896 (CT ‘) 84 a: 13 P. fluorescens+KC2753 (CT') 1 14 :i: 9 P. ,,"luorescens+KC3l64 (CT‘) 102 : 14 1.4E+05 A KC300 news 8 1.0m 8 3.05m 3 6.02m 4.05m 7.03m o.oa+oo o to 20 30 4o 50 Time (h) roams. “a,” B K0657 some!“ 3 8 roams; 3 3.03.031 2.oa+osi 1.050051 0.03m: o 10 20 so 40 so Turn: (1:) 2.0E+05 c KC1896 1.6E+05 g 1.2E+05 c: Q roam :3 53 4.08m o.oa+oo o 10 20 30 40 so Time (h) 3.05m: D 2.sa+os KC2753 3 tom: :3 1.51905 ‘2 3 l.OE+05 53 5.08404 (tom-00 o 10 20 so 40 so Tune (11) Fig. l Luciferase activity. expressed as relative light units divided by OD“. during growth of A strain KC 300. B strain KC 657. C strain KC 1896. and D strain KC 2753 in Simulated Groundwa- ter Medium containing 1 D. 5 A. 10 A. and 20 0 uM Fe”. Due to the low values of OD”, prior to 12 h of growth and in 0 uM Fe”. this ratio was only computed for samples after 12 h of growth and for Fe” concentrations > 0 uM. Plotted values are the average of two independently grown cultures. Data is not shown for strain KC 3164. which exhibited very low lia expression and no clear pattern with increasing iron concentration (similar to strain KC 300) 34 427 Evidence that the CT ‘ mutants were impaired in secreted factor production Table 2 summarizes the results of experiments using an agar-based assay to detect secreted factor production. When P. fluorescens was grown in the presence of wild- type strain KC or strain KC lux: :Tn5 recombinants capa- ble of degrading carbon tetrachloride (such as strain KC300), the radioactivity that accumulated in the agar plugs around the P. fluorescens colonies was more than sevenfold the amount that accumulated when R fluo- rescens was grown alone (Table 2). When P. fluorescens was grown in the presence of any of the putative CI“ mu- tants, the nonvolatile radioactivity was only twice that of noninoculated controls. This indicates that the four puta- tive CI“ mutants either do not produce significant levels of CIT-degrading factor or secrete a largely inactive form of it. Expression of lax genes in CT ' mutants There was no significant difference between the time course for growth of lax: :Tn5 recombinants that were impaired in carbon tetrachloride degradation (e.g.. strain KC657) and the time course for growth of strain KC300 and many other recombinants that retained carbon tetra- chloride degradation activity. The maximum optical den- sity was 0.8 for SGM cultures and 1.6 for TSB cultures af- ter 20 h (data not shown). However. for all of the recom- binants except strain KC3164. addition of Fe‘3 resulted in dramatically reduced luciferase activity (Fig. 1). This ef- fect was not observed with CT * recombinants such as strain KC300 (Fig. l A). Complex media supported low levels of lux expression in the recombinants (Fig.2). but defined mineral medium supported high levels of expres- sion in three of the four CT‘ mutants (Fig. lB—D). For cells grown in SGM containing only 1 uM Fe”. maximal lux expression was sevenfold the level achieved by cells grown in TSB. In contrast. the CT ’ recombinant strain 2.5E+05 2.0E+05 1.5E+05 l .OE+05 rim/00..., 5.03004 0.0E+00 20 30 40 50 Time (h) Fig.2 Luciferase activity. expressed as relative light units divided by ODwo. during growth of strain KC 300 0. strain KC 657 I. strain KC 1896 0. strain KC 2753 O. and strain KC 3164 A in tryptic soy broth. Plotted values rre the average of two indepen- dently grown cultures 428 KC300 had strong lax expression in SGM regardless of Fe” concentration (Fig. 1A). and expression in TSB was only 1.7-fold the level achieved during growth in SGM. Discussion The l‘C-carbon tetrachloride assay was a reliable and effi- cient means of rapidly screening large numbers of recom- binants for carbon tetrachloride degradation. GC analysis confirmed that four mutants were impaired in carbon tetrachloride degradation. GC confirmation was important because some recombinants grew poorly on agar. giving artificially low counts. It is also important because a mu- tant might degrade carbon tetrachloride without generat- ing nonvolatile products. and such a mutant would be in- correctly classified as CI". An interesting result was the finding that the factor se- creted by strain KC could diffuse through agar and still transform carbon tetrachloride when “activated" by viable whole cells such as colonies of P. fluorescens (Table 2). Elevated levels of nonvolatile "C-labeled products were obtained for P. fluorescent colonies adjacent to colonies of wild-type strains KC or KC300. Low levels of non- volatile "C-labeled products were obtained for P. fluo- rescens colonies adjacent to colonies of CT“ mutants (strains KC657, KC1896. KC2753. and KC 3164). This result indicates that the mutants were defective in produc- tion and/or secretion of the secreted factor. The lax: :Tn5 construct on pRL1063a generates a pro- moterless lux transcriptional fusion. Therefore. lux is ex- pressed only when the native promoter of the interrupted gene is activated. For three of the four CT‘ mutants (strains KC657, KC1896. and KC2753). lux expression studies confirmed that the genes required for carbon tetra- chloride degradation are expressed under Fe”-limiting conditions in the late exponential phase of growth (Fig. 1). Mutant KC3164 did not show any significant luciferase expression because the transposon inserted in the wrong orientation with respect to the native promoter. as indi- cated by subsequent DNA sequence analysis (data not shown). The Fe”-dependent response of the CT“ transfor- mants was not an artifact introduced by the transposon. CI" mutant KC300 did not show significant differences in lux expression when grown at different iron concentra- tions (Figs. 1 A. 2). presumably because the transposon in- serted itself in a gene that is not regulated by Fe” avail- ability. For the putative C'I" mutants. light emission was greater for cultures grown in defined rrtineral medium SGM (Fig. 1) than for cultures grown in complex medium TSB (Fig.2). In contrast. light emission for the CT‘ mu- tant KC300 was greater in complex media (Figs. 1 A. 2). Further details related to the identity of the genes inter- rupted in the CI" strains will be provided in a separate publication. The conditions that induce luciferase expression in the CI" strains correspond to conditions that induce secreted factor production in the wildvtype strain (Tatara 1996). Expression of the [rut genes was inversely correlated to the amount of Fe” added for CI" mutants (Fig. 1). This pattern was consistent with previous reports indicating that Fe” addition stimulated cell growth but reduced car- bon tetrachloride degradation (Criddle et al. 1990; Tatara et a1. 1995). This is the first report of mutants with impaired ability to degrade carbon tetrachloride. Initial characterization in- dicates that these mutants either do not produce the car- bon-tetrachloride-degrading factor or they produce a non- functional form of it. The observed interaction of the se- creted factor with other cell types and its apparent ability to obtain reducing equivalents thereby suggest a possible role in cell/cell communication. On the other hand, its known regulation by ferric iron and cobalt suggests a pos- sible role in trace metal acquisition. perhaps not unlike that recently reported for the cytochrome secreted by Geobacrer sulfiirreducens (Seeliger et a1. 1998). Further studies are under way to characterize the genes inacti- vated by the tux: :Tn5 transposon in the strain KC mu- tants. Sequencing and identification of the interrupted genes is expected to provide insight into the identity and physiological role of the carbon-tetrachloride-transform- ing secreted factor and its mechanism of u'anst‘ormation. Acknowledgment L. Sepulveda-Torres thanks the National Sci- ence Foundation for a predoctoral fellowship. We thank F. DeBruijn and A. Milcamps for prowding the lux: :Tn5 delivery plasmids and for their helpful advice. Special thanks to M. Tomashow and A. Gustafson for providing assistance with the luciferase expression assays. We are grateful to L. Dybas and .1. Nguyen for helping with the ion chromatography work and the atomic absorption deter-inma- tion. respecrrvely. This work was supported by the National Science Foundation Center for Microbial Ecology (grant no. BlR-9120006) and by the NIEHS Superfund Basic Research Program of the Insti- tute for Environmental Toxicology (grant no. 580491 1). References Chaudhry GR. Chapalamadugu S (1991) Biodegradation of halo- genated organic compounds. Microbiol Rev 55 : 59-79 Criddle CS. DeWitt JT. Grbic-Galic D. McCarty PL( 1990) Trans~ formation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions. Appl Environ Microbiol 56:3240—3246 Ditta G. Stanfield S. Corbin D. Helinski DR (1980) Broad host range cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium mellloti. Proc Natl Acad Sci USA 77:7347-7351 Dybas M1. Tatara GM. Criddle CS (1995) Localization and char- acterization of the carbon tetrachloride transformation activity of Pseudomonas sp strain KC. Appl Environ Microbiol 61: 758-762 Dybas MJ. et al (1998) Pilobscale evaluation of bioaugmentation for in-riru remediation of a carbon tetrachloride-contaminated aquifer. Environ Sci Technol 32:3598-3611 Egli C.. Tschan T. Scholtz R. Cook AM. Leismger T (1988) Trans- formation of tetrachloromethane to dichloromethane and car- bon dioxide by Acetobacrertum woodi'i'. Appl Environ Micro- biol 54:2819-2823 Fetzner S. Lingens F (1994) Bacterial dehalogenases: biochem- istry. genetics. and biotechnological applications. Microbiol Rev 58 : 641-685 Iain M. Criddle CS (1995) Metabolism and cometabolism of halo. genated C-1 and C-2 hydrocarbons. In: Singh VP (Cd) Bio- transformatonst microbial transtormations of health risk com- pounds. Elsevier. Amsterdam. The Netherlands. pp 65-102 Lewis TA. Crawford RL (1993) Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bac- terium Pseudomonas sp. strain KC. Appl Environ Microbiol 59: 1635-1641 Lewis TA. Crawford RL (1995) Transformation of carbon tetra— chloride via sulfur and oxygen substitution by Pseudomonas sp. strain KC. J Bacteriol 177 : 2204—2208 Nielsen AH (1990) The biodegradation of halogenated organic compounds. .1 Appl Bacterial 69 : 445-470 Sambrook J. Fritsch EL. Maniatis T (1989) Molecular cloning. A laboratory manual. 2nd edn. Cold Spring Harbor Laboratory. Cold Spring Harbor. NY Seeliger S. Cord-Ruwisch R. Schink B ( 1998) A periplasmic and extracellular c-type cytochrome of Geobacter sulfurreducen: acts as a ferric ion reductase and as an electron carrier to other acceptors or to partner bacteria. J Bacteriol 180: 3686-3691 Semprini L. Hopkins GD. McCarty PL. Roberts PV ( 1992) In-situ transformation of carbon tetrachloride and other halogenated compounds resulting from biostimulation under anoxic condi- tions. Environ Sci Technol 26: 2454-2461 Sepdlveda-Torres L del C. Dybas MJ. Criddle CS (1997) Solid phase bioremediation of carbon tetrachloride by Pseudomonas stutzeri strain KC. In: Alleman BC. Leeson A (eds) The Fourth International iii-rim and On-site Bioremediation Symposium. Battle Press. Columbus. pp 33—37 Simon R. Priefer U. Pilhler A (1983) A broad host range mobiliza- tion system for in viva genetic engineering: transposon muta- genesis in gram-negative bacteria. Biotechnology 1 :784-790 429 Sittig M (1985) Handbook of toxic and hazardous chemicals and carcinogens. 2nd edn. Noyes. New York Tatara GM (1996) PhD Thesis. Michigan State University. MI. USA Tatara GM. Dybas Ml. Criddle CS (1993) Effects of medium and trace metals on kinetics of carbon tetrachloride transformation by Pseudomonas sp. strain KC. Appl Environ Microbiol 59: 2126—2131 Tatara GM. Dybas MJ. Criddle CS (1995) Biofactor-mediated transformation of carbon tetrachloride by diverse cell types. In: Hinchee RE. Leeson A. Semprini L (eds) Bioremediation of chlorinated solvents. Battle Press. Columbus. pp 69-76 United Nations Environment Programme (1994) Adjustment to the Montreal protocol on substances that deplete the ozone layer. In: Sands P. Tarasofsky R. Weiss M (eds) Principles of inter- national environmental law. 2A. Documents in international environmental law. Manchester University Press. Manchester. pp 208-223 Wackett LP. Gibson DT (1983) Expression of naphthalene oxida- tion in Escherichia coli results in the biosynthesis of indigo. Science 222: 167-169 Wolk CP. Cal ‘1’. Panoff TM (1991) Use of a transposon with lu- eiferase as a reporter to identify environmental responsive genes in a cyanobacterium. Proc Natl Acad Sci USA 88: 5355-5359 CHAPTER 3 SEQUENCE AND ANALYSIS OF THE GENES INTERRUPTED IN FOUR PSEUDOMONAS S T U T ZERI STRAIN KC MUTANTS WITH IMPAIRED ABILITY TO DEGRADE CARBON TETRACHLORIDE 37 ABSTRACT As indicated in Chapter 2, transposon mutagenesis was used to create isolates of Pseudomonas stutzeri strain KC unable to degrade carbon tetrachloride. The present chapter presents the strategies used to sequence the genes interrupted in four mutants with impaired CC14 degradation capabilities. The DNA sequence was analyzed using various tools, allowing the creation of a possible biosynthesis pathway for pyridine-2,6- bis(thiocarboxylate), a compound that chelates copper to fortuitously degrade carbon tetrachloride. 38 INTRODUCTION The primary objective of this research project was to identify genes involved in the degradation of carbon tetrachloride (CC14) by Pseudomonas stutzeri strain KC. In order to achieve that goal, 3,500 transpositional mutants of stain KC were created via tri- parental matings using a transposon Tn5 containing promoterless luciferase reporter genes from Vibrio fischeri (Wolk et al., 1991). Four mutants named KC657, KC1896, KC2753 and KC3164 showed an impaired CCl4 degradation ability. Three of the four mutants strongly expressed luciferase under iron-limiting conditions and gene expression was attenuated when cells were grown in cultures containing iron. These experiments are described in detail in Chapter 2 of this dissertation (Sepulveda-Torres et al., 1999). The agent responsible for CCl4 degradation was recently identified as pyridine-2,6- bis(thiocarboxylate) (PDTC) chelated to copper (Lee et al., 1999). PDTC is a strong metal chelator that was first described in 1978 as a metabolite produced by a Pseudomonas putida strain grown under iron-limiting conditions (Ockels et al., 1978). A recent report by Lewis et al. (2000) described the characterization of a P. stutzeri strain KC mutant that lost the ability to produce PDTC and degrade CCl4 upon the spontaneous deletion of a 170 kb fragment. This mutant was named CTN]. The CCl4 degradation capacity of strain CTNl was restored when a 25,746 bp piece of DNA (called T31, GenBank accession number AF196567) containing 16 predicted ORFs, was introduced back into the cell using the wide host range cosmid pRK311. 39 The present chapter explains the methodology utilized to determine the sequence and possible functions of the genes mutated in the four strains described in Chapter 2. I also analyzed the sequence of the genes independently identified by Lewis et al. (2000) in order to generate my own conclusion of how the genes sequenced by Lewis are involved in the CCl4 degradation process. I analyzed the mutants generated by both research groups in order to explain how P. stutzeri strain KC synthesizes pyridine-2,6- bis(thiocarboxylate) (PDTC) when grown under iron-limiting conditions. 40 MATERIALS AND METHODS Organisms and culture conditions. Bacteria and plasmids used in this study are listed in Table 3.1. Pseudomonas strains were propagated aerobically at 25 °C and 150 revolutions per minute (rpm) in tryptic soy broth (TSB) (Difco Laboratories, Detroit, MI). Escherichia coli strains were propagated under aerobic conditions at 37 °C and 200 rpm in Luria broth (Sambrook et al., 1989). Antibiotics used were from Sigma (St. Louis, M0) at the following concentrations: ampicillin (Ap) 50 pg ml_l, kanamycin (Km) 70 pg ml_l, rifampicin (Rt) 100 pg ml—l streptomycin (St) 100 pg ml_l, and tetracycline (Te) 15 pg ml_'. The selection medium utilized for conjugation was a modified DRM medium (Lee et al., 1999) containing the following (per liter): KZHPO4, 6g; sodium citrate dihydrate, 6 g; sodium nitrate, 0.5 g; ammonium chloride, 1 g; adjusted to pH 7.9 prior to the addition of agar, 15 g. The medium was autoclaved and cooled to 60 °C prior to the addition of 1 mL of l M MgSO4, 666 pl Ca(NO3)2 and 1 ml of trace elements solution TN2 (Criddle et al., 1990) from sterile solutions. Medium D (Criddle et al., 1990) was used for the determination of CCl4 degradation capacities. 41 Table 3.1 List of strains and plasmids used in this study Strain / Plasmid Comments Source / Reference Pseudomonas stutzeri strain KC CCI4 degrader, aquifer isolate (Criddle et al., 1990) Pseudomonas stutzeri strains KC657, KC1896, KC2753 and KC3164 Mutants created by transpositional;k insertion of pRL1063a, Km , Rf 3, impaired in CCI4 degradation (Sepulveda-Torres et al., 1999) P. stutzeri CCUGb l 1256 Type strain for P. stutzeri species, clinical isolate, genomovar 1 Dr. J. Lalucat, Universitet de les llles Balears, Spain (Stanier et al., 1966) P. stutzeri ATCCb 1759] Clinical isolate, genomovar 2 Dr. J. Lalucat, Universitet de les llles Balears, Spain (Stanier et al., 1966) P. stutzeri DSMb 50227 Clinical isolate, genomovar 3 Dr. J. Lalucat, Universitet de les llles Balears, Spain (Stanier et al., 1966) P. stutzeri l9SMN4 Marine isolate, naphthalene degrader genomovar 4 Dr. J. Lalucat, Universitet de les llles Balears, Spain (Rossello et al., 1991) P. stutzeri DNSP2l Wastewater isolate, genomovar 5 Dr. J. Lalucat, Universitet de les llles Balears, Spain (Rossello et al., 1991) P. balearica DSM 6083 Wastewater isolate, naphthalene degrader, genomovar 6 Dr. J. Lalucat, Universitet de les llles Balears, Spain (Bennasar et al., 1996; Rossello et al., 1991) P. stutzeri DSM 50238 Soil isolate, genomovar 7 Dr. J. Lalucat, Universitet de les llles Balears, Spain (Stanier et al., 1966) P. stutzeri JM300 Soil isolate, genomovar Dr. J. Lalucat, Universitet de les llles Balears, Spain (Carlson & lngraham, 1983) P. putida DSM 3601 Tomato plant isolate, produces 2,6-bis(pyridine thiocarboxylic acid) Dr. R. Crawford, University of Idaho (Ockels et al., 1978) P. fluorescens ATCC 13525 Type strain, used to corroborate CCl4 degradation by non — CCI4 degraders in the presence of strain KC supernatant Michigan State University Department of Microbiology Escherichia coli DHSa Used for plasmid propagation Dr. F. deBruijn, Michigan State University (Hanahan, 1983) E. coli JM109 Used for plasmid propagation Promega (Madison, WI) (Yanish-Perron et al., 1985) E. coli S- l 7~l Ra . . . St RP4 mobilization genes integrated in the chromosome (mob+) Dr. F. deBruijn, Michigan State University (Simon et aL,l983) )iBluescript SK(—) DNA cloning vector Stratagene (La Joya, CA) 42 Table 3.1 (continuation) Strain / Plasmid Comments Source/ Reference pRK311 T R a 'd h t . d Dr. R. Crawford, University c w' e 05 range cosmi of Idaho (Ditta et al., 1985) pT31 25.7 kb DNA fragment of P. stutzeri Dr. R. Crawford, University strain KC cloned into the BamHI site of Idaho (Lewis et al., 2000) of pRK311 pBlue8.3 R This study Ap , 8.3 kb DNA fragment of P. stutzeri strain KC cloned into the EcoRI site of pBluescript SK(—) RKblue This stud p ApR 3, TcR, pBluescript SK(—) y inserted in the BamHl site of pRK311 RKblue8.3 This stud p ApR, TcR, pBluescript SK(—) y containing a 8.3 kb DNA fragment of P. stutzeri strain KC cloned into the BamHl site of pRK3 ll 3 Km, kanamycin; Rf, rifampicin; St, streptomycin; Tc, tetracycline; Ap, ampicillin b ATCC, American Type Culture Collection, Rockville, MD; CCUG, Culture Collection University of Goteborg, Sweden; DSM, Duetsche Sammulung von Microorganismen und Zellkulturen, Braunschweig, Germany R . resrstant Generation and isolation of plasmids containing the genes interrupted by quAB::Tn5. Genomic DNA from the four P. stutzeri strains KC657, KC1896, KC2753 and KC3164 were isolated with the QIAamp® tissue kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. Five micrograms of DNA were restricted with 20 U EcoRI (Gibco BRL, Rockville, MD) for 2 hr in a final volume of 50 pl. The restricted DNA samples were extracted with phenol:chloroform, precipitated with ethanol, washed with 70% ethanol and dissolved in 88 ul of water as previously described (Sambrook et al., 1989). The suspended DNA samples were combined with 10 pl 10X T4 ligase buffer and 2 units of T4 ligase (l U / pl) (Roche Molecular Biochemicals, 43 Indianapolis, IN) and incubated at 16 °C for 20 hr. Ten microliters of T4-ligated DNA were transformed to CaClz-competent E. coli DHSa using the heat-shock method (Sambrook et al., 1989). The cells were transferred to LB-agar medium amended with Km and incubated overnight at 37 °C. Colonies were streaked once, to assure purity, before the plasmids were isolated using the Quantum® prep plasmid isolation kit (Bio- Rad Laboratories, Hercules, CA). Figure 3.1 shows a graphical representation of how the plasmids containing the mutated DNA were generated. 44 Tn5 1063a EcoRI EcoRI Promoter Interrupted gene X EcoRI excision and religation A‘A‘A‘A‘A‘A‘A‘A‘A‘A‘A‘A‘A‘I. Promoter Interrupted gene X DNA sequencing from primers A & B X (MB m Transcriptional fusion Figure 3.1 Cloning and sequencing of genes mutated P. stutzeri strain KC. A) Map of pRL1063a, the plasmid containing the luxAB::Tn5 transposon (Wolk et al., 1991). B) Interruption of gene X by luxAB::Tn5. The P. stutzeri strain KC native promoter now controls the luciferase genes. C) Cloning of the interrupted gene by restricting the DNA with EcoRI, followed by a ligation reaction. D) Sequencing of the luxAB::Tn5 flanking regions using primers A and B. Figure kindly provided by Frans deBruijn. 45 Sequencing of the plasmids containing the genes interrupted by luxAB::Tn5. Double-stranded plasmid DNA was sequenced with a modified dideoxy method of Sanger et a1. (1977). Automated sequencing readings were performed using the ABI model 377 automated DNA sequencer (Applied Biosystems, Foster City, CA) following the protocols recommended by the manufacturer for T aq DNA polymerase cycle sequencing reactions with flourescently labeled BigDyesTM dideoxynucleotide terminators. To determine the sequence of the P. stutzeri strain KC DNA on both sides of the transposon insertion, two oligonucleotides derived from the Tn5-pRL1063a sequence were used as sequencing primers. One primer, corresponding to positions 110- 86 of the Tn5-pRL1063a DNA sequence (5’~TACTAGATTCAATGCTATCAATGAG- 3’), was designed to determine the upstream sequence from the Tn5 in the anti-sense direction. The other primer, corresponding to positions 7758-7781 of the Tn5-pRL1063a DNA sequence (5’-AGGAGGTCACATGGAATATCAGAT-3’) was designed to determine the downstream DNA sequence in the sense direction. These primers were modified from previously described sequencing primers for Tn5-pRL106Ba (Black etal., 1993; Femandez-Pifias et al. , 1994). The sequences of the internal fragments of the DNA inserts were determined, in both orientations, by primer walking. The DNA sequence was deposited to GenBank under accession number AF 149851 and is provided in Appendix B. Cloning of EcoRI fragment containing the wild type genes. Genomic DNA from wild type strain KC was isolated with the GenomicPrep cells and tissue DNA isolation kit (Amersham Pharrnacia Biotech, Piscataway, NJ). Five micrograms of DNA were 46 restricted with 10 units (U) of EcoRI (Life Technologies, Rockville, MD). The restricted fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. The fragments ranging in size from 8 to 9 kb were purified from the gel using the QIAEX 11 gel extraction system (QIAGEN, Valencia, CA) and ligated to pBluescript KS(—) (Stratagene, La Jolla, CA) previously restricted with EcoRI and dephosphorilated with calf intestine phosphatase (CIP) (Life Technlogies). The resulting plasmids were introduced into Escherichia coli JM109 competent cells (Promega, Madison, WI). All plasmids were screened for the presence of the fragment of interest by the polymerase chain reaction (PCR) using primers that amplify fragments containing the transposon insertion sites. The PCR were performed in a volume of 50 pl containing 50 ng DNA, 20 mM Tris pH 8.4, 50 mM KCl, 3 mM MgCl, 0.2 mM of each dNTP, 10 pmole of each primer and 2 U Taq DNA polymerase (Life Technologies). The following PCR program was used: initial denaturation at 95 °C for 3 min, 30 cycles consisting of 94 °C for 1 min, 55 0C for 1 min and 72 °C for 2 min. A single final extension step was performed at 72 °C for 10 min in order to assure chain termination. Two sets of primers were used: primers CC109f (5’ - GTT ACA GCC GCC ACC TAC TGA T — 3’) and CC] 10r (5’- GCT AGG CAG AGA AGA GTC CAC G — 3’) were used to amplify a 1.1 kb fragments spanning from position 2,493 to position 3,604; primers CCl 11f (5’ — GGC TGC TCA GTA TCG GCA GTA T — 3’) and CC112r (5’ — GGG GCG TI’G ACA GAG AAG TAA G — 3’) were used to amplify a 1.4 kb fragment spanning from position 4,892 to position 47 6,276. PCR products were separated by electrophoresis in 1.5% agarose and stained with ethidium bromide. pBluescript SK(—) containing the fragment of interest (pBlue8.3) was restricted with BamHI, treated with CIP and ligated to BamHI-restricted wide host range cosmid pRK311 (Ditta et al., 1985) using T4 DNA ligase (Life Technologies). The resulting TcR ApR plasmid (pRKblue8.3) was introduced to E. coli S-17-1 by transformation using the CaClz — heat shock method (Sambrook et al., 1989), and was transferred to P. stutzeri CCUG 11256 by conjugation. Plasmids pRK311, pT31and pRKblue were also introduced to P. stutzeri CCUG 11256. DNA sequence analyzes. Alignments of the DNA fragments obtained from sequencing were done using Sequencher (Gene Codes, Ann Arbor, MI). Open reading frames (ORFs) were determined by the program CodonUse 3.1 (Conrad Halling, University of Chicago) using the codon use tables of Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas aeruginosa as references, with a codon window of 33 bases and a logarithmic range of 3. Comparison of DNA and protein sequences with the sequences available in the databases was done using the basic local alignment search tool (BLAST) programs available in the intemet search engines of the National Center for Biotechnology Information (Altschul et al., 1997) using the address listed in Table 3.2. Possible promoter regions were localized with the promoter prediction by neural network sofiware administered by the Lawrence Berkeley Laboratory available through the Internet (Reese & Eeckman, 1995), as indicated in Table 3.2. Transcriptional terminators 48 were predicted by a program available in the Wisconsin Package (Genetic Computer Group, Madison, WI) (Brendel & Trifonov, 1984; Butler, 1998; Devereaux et al., 1984). Molecular weights and isoelectic points of proteins were determined by the Lasergene Package (DNAstar, Madison, WI). Transmembrane helices were predicted by the Internet servers of the seven programs included in Table 3.2. Signal peptides were determined by the SPScan program of the Wisconsin Package and two other Internet resources as indicated in Table 3.2. The localization of motifs was performed by comparing the proteins against the five libraries provided in Table 3.2. 49 Table 3.2 Internet-based programs used for DNA and protein analyses Resource type Resource name (Internet address) Reference DNA and BLAST (Altschul et al., 1997) protein (www.ncbi.nlm.nih.gov/BLAST) comparisons Promoter Promoter (Reese & Eeckman, 1995) determination (www.fruitfly.org/seq_tools/promoter.html) Transmembrane TMHMM (Sonnhammer et al., 1998) helices (www.cbs.dtu.dk/services/TMHMM-1.0/) (Tusnady & Simon, 1998) HMMTOP (www.cnzimhulhmmtop) (Hirokawa et al., 1998) SOSUI (azusa.proteome.bio.tuat.ac.jp/sosui/submit.htmI) (Hofmann & Stofell, 1993) TMPred (Nakai & Kaneshisa, 1991) (www.ch.embnet.org/soflware/'I'MPRED_form.htmI) (von Heijne, I992) Psort (psort.nibb.ac.jp/form.html) (Cserzo et al., 1994) TopPred2 (www.8iokemi.su.se/~server/toppred2/toppredServer.cgi) DAS (www.Biokemi.su.se/~server/DAS) Signal peptide SignalP (Nielsen et al., 1997) (www.cbs.dtu.dk/services/SignalP) Psort (Nakai & Kaneshisa, I991) (psort.nibb.ac.jp/form.html) Protein motifs Motif finder (motif.genome.ad.jp/) - searches all the following: PROSITES (Hoffmann el al., 1999) BLOCKS (Henikoff et al., 1999) ProDom (Corpet et al., 1999) PRINTS (Attwood et al., 1999) Pfam (Bateman et al., 1999) 50 Corroboration of CCI4 degradation by P. stutzeri KC657, KC1896, KC2753 and KC3164 when the supernatant from wild type strain KC is provided. In order to asses the CCI4 degradation capacity of the four mutant strains when the CCI4 degrading factor is provided externally, mutant cells were combined with partially purified PDTC using the bioassay developed by Dybas et al. (1995). Corroboration of CCI4 degradation by P. stutzeri CCUG 11256 containing plasmids with DNA from P. stutzeri strain KC. Triplicate 5-ml cultures of P. stutzeri CCUG 11256 harboring pRK311, pT31, pRKblue or pRKblue8.3 were incubated overnight at room temperature and 250 rpm in TSB amended with Ap and Te as needed. The cultures were used to inoculate 20 ml serum vials containing 10 ml of medium D and 500 ng CCI4. The vials were incubated in an inverted position for 24 hr before the concentration of CCI4 remaining in the vials was determined by a gas chromatograph equipped with an electron capture detector (Tatara et al., 1993). Screening of Pseudomonas strains for the presence of the genes interrupted in mutants impaired in CCI4 degradation. Eight Pseudomonas stutzeri strains, representing the eight P. stutzeri genomic groups identified to date (Rossello et al., 1991; Rossello-Mora et al., 1996), were screened for the presence of the genes interrupted in the mutants that lost the ability to degrade CCI4. The strains were analyzed by PCR and by Southern hybridization. The PCR primer pairs used for the study were CC109f — CC110r and CCIIIf — CC112r and the reactions were performed as described in a 51 previous section of this chapter. The probe for Southern hybridization was a DIG-labeled 3.4 kb HindIII fragment corresponding to positions 1653 to 5043 of the 8.3 kb EcoRI fragment containing the wild type genes interrupted in the mutants. The probe was hybridized to 10 pg of genomic DNA as recommended by the manufacturer (Roche Molecular Biochemicals). The strains used for this study were P. stutzeri CCUG 11256, P. stutzeri ATCC 17591, P. stutzeri DSM 50227, P. stutzeri 19SMN4, P. stutzeri DNSP21, P. stutzeri DSM 50238, P. stutzeri JM300, P. balearica DSM 6083 and P. putida DSM 3601. The carbon tetrachloride degradation capacity of the strains was tested in medium D containing 500 ng CCI4 as described previously in this chapter. 52 RESULTS Sequencing of genes interrupted by luxAB::Tn5. Sequence analysis determined that all four mutants had insertions within a 3.1 kb sequence contained within a 8,274 bp EcoRI fragment. Furthermore, two of the four mutants received the transposon insertion in the same position, separated only by 9 bp. Figure 3.2 shows a map of the transposon insertion points in the four mutants. r r , O EcoRI KC657 KC1896 KC2753 (-) ) EcoRI 1 ho 2,621 bp 4,897 bp 5,683 bp 8274 bp KC3164 (6) 5,692 bp Figure 3.2 Mapping of the transposition insertion points for transpositional mutants KC657, KC1896, KC2753 and KC3164. The black flags represent the luxAB::Tn5 transposon. The direction of the flag indicates the direction of the luciferase genes. The exact positions of the transposon insertion sites are provided. Determination of open reading frames. The codon use tables of P. putida, P. fluorescens and P. aeruginosa generated the same open reading frame profile. As seen in Figure 3.3, eight ORF’s were found in the forward direction. Mutant KC657 mapped in ORF 2435—3610 while mutants KC1896, KC2753 and KC3164 mapped in ORF 4460- 6291. 53 1000 2000 3000 4000 5000 6000 7000 8000 Figure 3.3 Open reading frames (ORFs) found in the 8,274 bp EcoRI fragment mutated in KC657, KC1896, KC2753 and KC3164. Panels a, b and c represent the 3 forward reading frames while panels d, e and f represent the 3 reverse reading frames. A small vertical line depicts the methione start codon and a small bold vertical line represents the stop codons. 1, ORF-446; 2, ORF-1009; 3, ORF-2435; 4, ORF-3626; 5, ORF-4099; 6, ORF-4460; 7, ORF-689; 8, ORF-7985. The insertion points in KC657 (0), KC 1896 (0) and KC2753/KC3164 (i) are also provided. Determination of physical characteristics of the open reading frames. The start and stop positions of the ORFs, the putative ribosomal binding site (RBS) (Shine & Delgarno, 1974), as well as the number of amino acids in the encoded proteins, their molecular weights and isoelectric points are indicated in Table 3.3. Table 3.3 Physical characteristics of the open reading frames encoded in the P. stutzeri strain KC 8.3 kb EcoRI fragment interrupted in mutants impaired in CCI4 degradation. Frame Start Endin 7‘ - . b c (1 Base Baseg RBS (posmon) aa kDa pl 2 446 1012 AGGA (437 - 439) 188 20.1 8.65 1 1009 2262 AGAGGA (994 — 999) 417 45.4 10.52 2 2435 3610 AGGA (2423 - 2426) 391 42.8 4.93 2 3626 4036 AGGA (3612 — 3615) 136 15.6 6.50 1 4099 4371 AGGA (4088 — 4091) 90 9.7 5.56 1 4460 6291 GO (4450—4451) 610 65.5 6.71 l 6289 7983 GGAGG (6279 — 6283) 564 60.8 5.42 2 7985 > 8274 GGAG (7970 — 7974) > 96 > 10.1 “ RBS, ribosomal binding site b aa, number of amino acids in the protein encoded by the gene c kDa, protein molecular weight in kiloDaltons d pI, isoelectric point of protein Sequence analysis of the 8.3 kb DNA fragment. The information gathered by comparing the ORFs with DNA and protein databases, motif databases, transmembrane helices prediction and leader peptide determination programs was used to assign possible gene functions. This information is summarized in Table 3.4 and provided in detail in Appendix C. 55 Tabli strain z. I 716-7 1009-— - Puiaii nwm mm 1 I funct I *———-—,,. - , ' -435 - . i - lmerru I KC65 - Memb; protei: im 01\ tnmsfc I‘M 2626 — 4( 1 ‘ Functio 75“\\ 60 \ 0: ‘ [Mel-mp KC189 and KC ‘ Possible Prolem Involve addilio, Onto ‘ Table 3.4 Putative functions of open reading frames (ORFs) found in the P. stutzeri strain KC 8.3 kb fragment interrupted in mutants impaired in CCI4 degradation. ORFs Similarities Motifs Functions 446 - 1012 - No similarities found - No motifs found 1009 - 2262 - No similarities found - Imperfect matches found with - Putative integral membrane protein with unknown ATP-phosphoribosyl transferase, Fe-containing alcohol dehydrogenase, homoserine function dehydrogenase - 12 transmembrane helices predicted by 7 programs 2435 - 3610 —122 a b , - Imperfect matches found with - Interrupted in ' 10 Mer from Mgcobactegmm chemotaxis sensory transducer, KC657 tuberculosis (295120 , 71% p and dihydroxyacid dehydratase, - Membrane bound 57% i6, 389 aaf) amino acid dehydrogenase, protein - may be involved in sulfur transfer - 10.104 MoeB from M. tuberculosis (molybdopterin synthase sulfurylase, Z95150, 67% p, 51% i, 379 aa) - 10- 42 MoeB from Escherichia coli (D90720, 56% p, 39% i, 245 aa) - 10 ThiF from E. coli (activates protein involved in thiamin biosynthesis, P30138, 56% p, 37% i, 235 aa) pyridine nucleotide disulfide oxidoreductase, ATP- phosphoribosyl transferase, purine phosphorilase, purivate kinase, - Transmembrane domains: 0 (SOSUI), 1 (TMHMM), 2 (HMMTOP, TMPred, PSort, DAS, TopPred2) - 11 bp signal peptide found by SignalP 3626 — 4036 - Function unknown - 10_3O hypothetical 16.5 kDa protein va334 from M. tuberculosis (010645, 67% p, 47% i, 134 aa) _ + - 10 I Mec from Streptomyces kasugaensis (restores cysteine methionine nutritional deficiency, M29166, 62% p, 42% i, 96 aa) - Imperfect match found with bacterial ribonuclease P, an enzyme that cleaves extra nucleotides from tRNA precursors 4099 - 4371 —20 . . -Perfect match for TonB-dependent - May be involved in '10 hypothetical protein RVI335 from protein N-terminus recognition sulfur transfer M tuberculosis “73902, 74% P, 51% sequence (79-DSLTVXPA-86). i, 90 aa) - 10.4 MoaD from Archeaglobusfillgidus (adds sulfur to molybdopterin precursor, AEOOO990, 50% p, 32% i, 74 a) 4460 — 6291 —23 . . - Imperfect matches with alanine - Interrupted in - 10 M hygpothjtical [grggmleoZ/n icy; dehydrogenase and KC1896, KC2753 . ' ‘u ”C“ 0"“ , ° P, ° glutamyltranspeptidase. and KC3164 " 278 aa) - Transmembrane domains: 0 - Possible membrane protein. May be involved in addition or removal of H20 or H2. - 10.22 BaiF from E. coli (bile acid dehydroxylase, D90867, 52% p, 30% —i’6 242 aa) - 10 CaiB from E. coli (L-carnitine dehydratase, P31572, 39% p, 30% i, 197 aa) (TMHMM, HMMTOP, SOSUI), 1 (PSort), 5 (TopPred2), 6 (TMPred), 7 (DAS) - 124 amino acid signal peptide predicted by SignaIP 56 Tal W lig sul Deter Strucn ORF- SeqUei resUlta (Adh, aerugi‘ Ranger and [he Table 3.4 (continuation) ORFs Similarities Motifs Functions 6289 - 7983 —55 , , , - Perfect AMP binding domain - Possible AMP— ' ‘0 9th from BUM“ MW“ (23' (194-LLVSSGTESEPK-205). ligase involved in dihydroxybenzoate-AMP ligase, - Transmembrane domains: 0 substrate activation P4087], 43% P, 30% i, 530 33) (TMHMM, l (SOSUI), 2 - 10 52 PchD from Pseudomonas aeruginosa (AMP-ligase in pyochelin biosynthesis, X82644, 44% p, 30% i, 529 aa) - 10”47 SnbA from Streptomyces pristinaespiralis (3-hydroxydipicolinic acid-AMP ligase, X98515, 45% p, 30% i, 545 aa) - 10 9 EntE from E. coli (2,3- dihydroxybenzoate-AMP ligase, P10378, 45% p, 26% i, 526 aa) (PSort), 3, HMMTOP, 4 (TMPred, TopPred2), 5 (DAS). a Match probability b protein name 0 GenBank accession numbers d p, positive matches (amino acids with the same functional side chains) c i, identify matches (identical amino acids) f a, amino acids (number of amino acids overlapping in match alignment) Determination of transcriptional terminators and promoters. A stable stem — loop structure was predicted from positions 2,295 to 2,331, between the termination codon of ORF-1009 and the initiation codon of ORF-2435. As illustrated in Figure 3.4, this sequence forms a stable G-C rich hairpin followed by a string of 5 uridine residues in the resultant mRNA. This structure resembles the Rho-independent terminators of E. coli (Adhya & Gottesman, 1978; Platt, 1986). Similar structures have also been found in P. aeruginosa (Gray et al., 1984) and Burkholderia cepacea (Zylstra et al., 1989). Two transcriptional promoters were predicted between the termination codon of ORF-1009 and the initiation codon of ORF-2435, one corresponding to positions 2343 —— 2388 (87% 57 CCI‘L’J predi Figu re Primar certainty) and the other corresponding to positions 2362 — 2407 (94% certainty). The predicted transcription starts are cyidine-2,368 and guanosine-2,397, respectively. C G G C C T T 35350610667 TGCATAGTTTTT Figure 3.4 Stem —- loop secondary structure observed between positions 2,295 and 2,331. Primary sequence value = 3.99, secondary sequence value = 55. Corroboration of CCI4 degradation by P. stutzeri KC657, KC1896, KC2753 and KC3164 when the supernatant from wild type strain KC is provided. As seen in Table 3.5, the mutants were able to degrade CCI4 when the supernatant of a wild type strain KC culture capable of degrading CCI4 was provided. This result confirms previous observations indicating that the ability to utilize the CCI4 degradation agent is totally independent of its production (Dybas et al., 1995; Tatara et al., 1995) 58 Tabl the St C orro with I degrad Screen mUtanI PrimEr Strain C Produ“ Table 3.5 Degradation of CCI4 by strains KC657, KC1896, KC2753 and KC3164 when the supernatant from a wild type strain KC culture capable of degrading CCI4 is provided. Strain % CCI4 degraded (T = 2.5 h) Non—inoculated culture medium 0 i 1 < 10 kDa m.w. supernatant alone 0 i 14 P. morescens + supernatant 61 i 3 KC657 + supernatant 67 i 1 KC1896 + supernatant 62 i‘ 7 KC2753 + supernatant 61 j: 3 KC3164 + supematant 63 i 2 Corroboration of CCI4 degradation by P. stutzeri CCUG 11256 containing plasmids with DNA from P. stutzeri strain KC. Only the strain harboring pT31 was able to degrade CCI4. Screening of Pseudomonas strains for the presence of the genes interrupted in mutants impaired in CCh degradation. The strains tested did not amplify with the two primer sets used and no signal was observed in the Southern hybridization. The only strain capable of degrading carbon tetrachloride was P. putida DSM 3601, a strain that produces PDTC when grown in iron-limiting conditions. 59 Septil ORF- transr “1111 i: lucifer inserte Lewis degrad; “he“ I Septilv' that the Spannin The rep 5':- (D h) l /i deleCla’r CTN] , ORFS‘ product DISCUSSION The four transpositional mutants of Pseudomonas stutzeri strain KC created by Sepulveda et al. (1999) that showed an impaired ability to degrade CCI4 had mutations in ORF-2435 (KC657) or ORF-4460 (KC1896, KC2753 and KC3164). Furthermore, the transposon insertion in mutants KC2753 and KC3164 mapped to the same location but with inverted orientations. These results explain why KC3164 was unable to translate the luciferase reporter genes at any significant levels because the luciferase genes were inserted in the wrong orientation with respect to the promoter. Lewis et al. (2000) sequenced a 25.7 kb fragment that was able to restore the CCI4 degradation ability to a spontaneous strain KC mutant that lost a 170 kb DNA fragment. When the 8.3 kb EcoRI fragment containing the four mutation points characterized by Sepfilveda was compared with the 25.7 kb sequence reported by Lewis, it was evident that the 8.3 kb fragment corresponds to positions 4,041 to 12,314 of the Lewis sequence, spanning from the end of Lewis’ ORF-C to the beginning of ORF-K. The report by Lewis et al. (2000) included the results of a semi-saturation mutagenesis of the 25.7 kb fragment with transposon mini-Tn5::lacZ1 or mini-Tn5::phoA. No detectable decrease in PDTC production and CCI4 degradation was observed when strain CTNI harbored a T31 fragment mutated in ORFs A, B, C, D, E, M, or the space between ORFs N and 0. On the other hand, when ORFs F, J or P were mutated, PDTC production was impaired and no significant degradation of CCI4 was observed. 60 Mm [1’31]. only com: the n Mutations in ORF-K showed a variable phenotype, depending on the position of the transposon insertion. When smaller DNA fragments were introduced to strain CTN], only partial restoration of the CCI4 degradation capacity was seen when a fragment containing ORFs A to the beginning of O was used. Figure 3.5 illustrates and compares the results independently obtained by Sepulveda and by Lewis. 61 m aisle. AB C D E F G H I J K L M N O P O 446 1009 2435 4460 6289 7985 4099 M36 ‘ ' _ 1868 9 L‘ — 09 G o __ 8.3 kb ‘ ' fragment Figure 3.5 Organization of 16 open reading frames (ORFs) in a 25.7 kb fragment capable of restoring the CCI4 degradation capacity in strain CTN]. Block arrows with letter designations below indicate ORFs identified by Lewis. The numbers underneath the ORF letters indicate the equivalent nomenclature used by Sepulveda. The directions of the arrows indicate the transcription/translation orientations of the genes. White arrows designate ORFs not mutated by Lewis et al. (2000) or ORFs that did not affect CCI4 degradation when mutated. Black arrows designate ORFs that caused a CCI4-negative phenotype. The stripped arrow designates an ORF mutated by Sept'ilveda and by Lewis while the dotted arrow designates an ORF mutated only by Sepulveda. Tn5::lac] insertions are represented by vertical lines with flags. The orientations of the flags indicate the orientation of the lacZ gene and the color indicates the mutation’s effect on the CCI4 phenotype as described above. The flag designated by a letter 03) indicates a Tn5::phoA insertion. Tn5::quAB insertions made by Sept'ilveda are indicated by lines with linear arrows identified by a letter (I). The orientation of the linear arrows represents the direction of the luciferase genes. Partial fragments introduced to strain CTN] by Lewis and their ability to complement the CCI4 degradation mutation are also provided. This figure was modified from (Lewis et al., 2000). 62 [1'31] som that PDT termi predit codon ORF-l Uptake promo AAT C When i scax-ené faClOr The results reported by Lewis et a1. (2000) confirmed that the four mutants obtained by transpositional insertion using Tn5::lux are impaired in the degradation of CCI4 because some of the structural genes needed for the production of PDTC were truncated. The fact that all mutations upstream of Lewis’ ORF-F (Sept'ilveda’s ORF-2435) had no effect on PDTC production confirmed the findings obtained by Sepulveda in the promoter and terminator analyses of the 8.3 kb EcoRI fragment. As seen in Figure 3.6, there is a predicted Rho-independent terminator followed by a promoter between the termination codon of ORF-1009 (Lewis’ ORF-E) and the initiation codon of ORF-2435 (Lewis’ ORF-F). Further analysis of that region also showed the presence of an imperfect ferric uptake regulator protein binding site (Fur box), an A-T rich sequence found in the promoter region of many iron-controlled genes in E. coli (E. coli consensus = 5’—GAT AAT GAT AAT CAT TAT C—3’) (Braun & Hantke, 1991). Fur is believed to bind DNA when iron is abundant in order to repress the synthesis of proteins involved in iron scavenging. Sequences similar to the -10 and -35 sequences recognized by transcription factor 070 were also identified. 63 b SeqL initie bohi 2251 GAGCGGGTTE QAAGGCTGAA GTGACCGGCC ATGCCCCTTC ORF-1009 stop codon 2291 GGACAArggc QIQAAATGCG CGGTQQTEET GCATAogggg pEdicEd Rho-independent terminator 2331 ICATGCTCAC GTCATATGAA EGAACAGCCA ACGGCAATTG -35 sequence 2371 CTATAGTCAT CACCACGAAC GATAATGATT ATCGTTACCA -10 sequence Fur Box 2411 TTGAAATCAA ACAGGATAAG CGATATGCCA CTATCAGCGC .o-o—o—o-u- RBS ORF-2435 start codon Figure 3.6 Organization of the DNA region between ORF-1009 and ORF-2435. Sequences associated to termination of transcription and translation of ORF-1009 and the initiation of transcription and translation of ORF-2435 are indicated. Cysteine residues in bold correspond to possible transcription initiation sites for ORF-2435. ORF-2435 (Lewis’ ORF-F) was mutated in strain KC657 and was also mutated by Lewis et al. (2000) using a Tn5::phoA transposon. It is very similar to Mer (probability of 10422), a putative Mycobacterium tuberculosis protein. It is also very similar to MoeB from M tuberculosis (probability of 10"“) and Escherichia coli (probability of 10“”). Significant similarities were also observed with ThiF from E. coli (probability of 10—35). Mer is a hypothetical protein, named after MoeB, the molybdopterin synthase sulfurylase protein from E. coli. MoeB transfers sulfur to the molybdopterin synthase (MoaD/Man heterodimer) in order to sulfurylate precursor Z of the molybdopterin synthesis pathway (Rajagopalan, 1996). ThiF is also similar to MoeB and it catalyses the adenylation by ATP of the carboxyl-terminal (C-terminus) glycince of ThiS. The adenylation of ThiS is likely to be involved in the activation of ThiS for sulfur transfer from cysteine or from a cysteine-derived sulfur donor in the thiamine biosynthetic 64 path cyst: tuber cyste transt encod trans f. The p] progra domai PTOgra Signal tennjn Lewis 243 (Jr PhOSp int0 01 the p, N0 p‘ 31ml]; prOte pathway (Taylor et al., 1998). MoeB, ThiF and other MoeB-like proteins contain two cysteines separated by two amino acids (C-X-X-C motifs) that are missing in both the M. tuberculosis Mer protein, and the protein encoded by ORF-2435. These paired cysteines have been proposed to bind zinc and form an active center involved in sulfur transfer (Rajagopalan, 1997). The lack of the C-X-X-C motifs in Mer and the protein encoded in ORF-2435 may indicate that these proteins are not directly involved in sulfur transfer. The protein encoded by ORF-2435 is a putative cell membrane bound-protein since five programs (HMMTOP, TMPred, PSort, DAS and TopPred2) predicted 2 transmembrane domains in the vicinity of amino acids 40 to 70 and 200 to 230, and an additional program (TMHMM) predicted only the first transmembrane domain. The program SignalP predicted a possible ten-residue signal sequence at the amino terminus (N- tenninus) that may be removed upon protein translocation through the cell membrane. Lewis et al. (2000) confirmed the membrane topology of the protein encoded by ORF- 2435 by the expression of phoA on an iron-limited medium. PhoA is an alkaline phosphatase from E. coli used to identify genes whose protein products are transported into or through the inner membrane because it is only active when a significant portion of the protein is located in the periplasmic space (Gutierrez et al., 1987). No perfect motifs were found in the protein encoded in ORF-2435 but it has signatures similar to motifs found in different enzymes. Such signatures may provide insights on protein function. Signatures similar to amino acid dehydrogenases, amino acid 65 annr regu: con\' (fisso inter- 1990 bond eta/q annnc pnnei CODCC] adapts Wheri phOSp} The 1] Chemo Orann Neithc OR}:S and R under gCUOn aminotransferases and purine phosphorilases were found in the first membrane-spanning region predicted by all seven programs. Amino acid dehydrogenases are involved in the conversion of an amine group into a carbonyl group, with the release of ammonia and the dissociation of water (Link et al., 1997). Amino acid aminotransferases catalyze the inter-conversion of amino acids by the exchange of the amino group (Scofield et al., 1990). Purine phosphorilases catalyze the phosphorolytic breakdown of the N-glycosidic bond with the formation of the corresponding free base and pentose-l-phosphate (Seeger et al., 1995). Two signatures were found near the C-terminus end of the protein, between amino acids 330 and 370. One of the signatures corresponds to integral membrane proteins that serve as chemotaxis transducers. Such proteins respond to changes in the concentrations of attractant and repellents in the environment and facilitate sensory adaptation through the variation of the level of methylation (Boyd et al., 1983). The other motif corresponds to ATP phosphoribosyltransferase involved in the activation of a phosphorilated ribose involved in the biosynthesis of histidine (Javanovic et al., 1994). The motifs suggest that the protein encoded by ORF-2435 may be involved in chemotaxis signal transduction and/or substrate modification by the transfer of phosphate or amino groups. Neither Sepulveda nor Lewis isolated mutants in ORF-3626 and ORF-4099 (Lewis’ ORFs G and H). The highest similarities were seen with hypothetical proteins va334 and Rv1335, respectively. These proteins are found in the M tuberculosis genome in tandem, which indicates that similar protein pairs may be found elsewhere in bacterial genomes, suggesting the possibility of similar protein functions and the acquisition of 66 SUC aux has 8nd] ()RI fit/sf if u. prote evide invob bler seq uei 1997) 10 a St end3\l such genes by lateral transfer. ORF-3626 shows similarity to an unidentified protein from Streptomyces kasugaensis that complements a methionine-cysteine double auxotroph through an uncharacterized process (Hirasawa et al., 1985). ORF-3626 also has a signature corresponding to an enzyme involved in tRNA maturation by endonucleolitic cleavage (Hansen et al., 1985). ORF-4099 shows low similarity (]0_ 4) to MoaD-like proteins from Archeaglobus fulgidus and even lower similarities to MoaD-like proteins from Helicobacter pilori and M. tuberculosis. No similarities were observed with the E. coli MoaD. These MoaD-like proteins were named for their similarity to the E. coli MoaD and there is no empirical evidence of their function. MoaD is part of the molybdopterin synthase heterodimer involved in the synthesis of molydopterin from precursor Z and is the substrate for MoeB. MoeB forms a thiocarboxylate at the MoaD C-terminus glycine-glycine (G-G) sequence that is believed to serve as the sulfur donor for molybdopterin (Rajagopalan, 1997). The G-G sequence is also shared with ubiquitin, which in eukaryotes is attached to a series of transfer proteins via thioester linkage at its terminal glycine. ORF-4099 ends with two glycines in tandem. Motif analyses of ORF-4099 revealed that it encodes for a protein with a perfect signature for TonB-dependent receptor signature (X10 to ”5 — DSLTVXPA, X73 in the case of ORF-4099), even though it appears to be a cytoplasmic protein. TonB is an inner membrane-anchored protein that extends through the periplasmic space in order to couple cytoplasmic membrane energy to active transport of iron-bearing siderophores across the 67 Ton radu coup phys eka dunn ngna (DRE- Sepul highe: (’pl'Ob; and 3 With outer membrane. The interactions between TonB and the outer membrane receptors are believed to cause a transformational change in the loaded receptor, leading to the release of the siderophore into the periplasmic space (Braun, 1995; Postle, 1990). The TonB box has been emphasized as an important mediator of some physical interactions between TonB and TonB-dependent receptors. It is likely that the conformation of the TonB box rather than the specific amino acid sequence dictates productive interactions of energy coupling with TonB (Kadner, 1990; Larsen et al., 1997). Since MoeB and MoaD interact physically during the biosynthesis of the molybdopterin cofactor, it would be worth exploring the possibility of physical interactions between ORF-2435 and ORF—4099 during the production of PDTC. Such interactions may be mediated by the TonB signature and/or the adjacent G-G carboxyl-terminus motif present in ORF-4099. ORF-4460 (Lewis’ ORF I) was mutated in strains KC1896, KC2753 and KC3164 by Sepulveda but Lewis et al. (2000) were unable to identify any mutants in this region. The highest similarity was found with a hypothetical protein Rv3272 from M. tuberculosis (probability of 10—23). ORF-4460 is also similar to bile acid dehydroxylase (probability of 10-22) and L-carnitine dehydratase (probability of 10_6), two enzymes from E. coli that introduce double bonds in their substrates. Higher similarities were observed with L- carnitine dehydratase - like proteins from different organisms. No transmembrane helices were predicted by TMHMM, HMMTOP or SOSUI while TMPred, Psort, DAS and TopPred2 predicted 6, 1,7 and 5 transmembrane domains, respectively. A 124 amino acid signal peptide was predicted by SignalP. Imperfect motif matches were observed with alanine dehydrogenase, phosphoribosylamine-glycine ligase and 68 glut. info: hydr this pied;- predi ORF- sidert other obser Subti/i invoh Simila Siderol Perfec heliceg Predic domai motif meml‘. glutamyltranspeptidase. A conclusive function cannot be derived from the motif information. On the other hand, a function related to the addition or removal of water or hydrogen can be suggested based on the functions of the two E. coli proteins similar to this ORF. The topology of this protein cannot be completely elucidated from the prediction programs due to the differences in the number of transmembrane domains predicted. ORF-6289 (Lewis’ ORF-J) is similar to several AMP-ligases involved in the activation of siderophore precursors by adenylation. The activated precursors are then combined with other intermediates to produce the mature siderophore. The highest similarity was observed with 2,3-dehydroxybenzoate-AMP ligase (probability of 1045) from Bacillus subtiIis (Rowland et al., 1996) and E. coli (probability of 10‘”) (Rusnak et al., 1989), involved in the biosynthesis pathway of the siderophore enterobactin. A significant similarity was also observed with an AMP-ligase involved in the biosynthesis of the siderophore pyochelin in P. aeruginosa (probability of 10-52) (Serino et al., 1997). A perfect AMP binding domain was observed in residues 194 to 205. No transmembrane helices were predicted by TMHMM, but several membrane spanning segments were predicted by SOSUI (1 domain), PSort (2 domains), HMMTOP (3 domains), TMPred (4 domains), TopPred2 (4 domains) and DAS (5 domains). The BLAST similarities and the motif searches indicate that the protein encoded by ORF-6289 may be an integral membrane protein involved in reagent activation by adenylation. 69 ()RJ Kit rece] memb C-lei'n indire( that h IDIErm Irladtj (3000 TOnB. ORF-7985, truncated in Sepulveda’s 8.3 kb EcoRI fragment, corresponds to Lewis’ ORF- K (687 amino acids, molecular weight 75.3 kD). It is similar to the ferric yersiniabactin receptor (FyuA) from E. coli (GenBank accession number 238065, 26% identities and 43% positives over a 645 amino acid stretch with a probability of 10—34) (Rakin et al., 1994) and Yersenia pestis (GenBank accession number Z35104, 25% identities and 42% similarities over a 645 amino acid stretch with a probability of 1043) (Rakin et al., 1995). FyuA is an outer membrane protein involved in the uptake of ferric yersiniabactin in a process that requires TonB and energy from the proton motive force (Klebba et al., 1993; Moeck & Coulton, 1998; Rakin et al., 1994). One popular concept of TonB function is that it spans the periplasm and physically interacts with the siderophore-loaded receptor, inducing conformational changes in the receptor that lead to the release of the siderophore to periplasmic binding proteins. TonB has also been hypothesized to induce the intake of loaded siderophores by transiently juxtaposing the inner and outer membranes when the N-terminus of TonB is anchored to the inner membrane while the C-terminus spans the outer membrane bilayer. A third hypothesis suggests that TonB indirectly influences siderophore uptake by energizing a mobile periplasmic messenger that interacts with outer membrane receptors (Klebba et al., 1993). The possible interactions between ORF-K and TonB may be the topic for future investigations. In addition to the predicted TonB box at amino acids 84 to 92 reported by Lewis et al. (2000), an additional sequence similar to signatures found towards the C-terminus of TonB-dependent siderophore receptors was found in amino acids 543 to 585 of ORF-K. A 25 to 50 amino acid signal peptide was predicted for this protein, depending on the 70 preve Senen (Heml protei aSSQm facili: prOVIL ORF- “Dial program used. Lewis observed a variable effect when this ORF was mutated. A mutation near the beginning of the ORF impaired PDTC production and CC14 degradation while a mutation on the last third of the gene was neutral. This result indicates that the first 2 thirds of the gene are enough to produce a functional protein. The BLAST similarities and the motif searches suggest that the protein encoded by ORF- K is a putative TonB-dependent outer membrane siderophore receptor. Lewis’ ORF-L encodes for a 743 amino acid long protein (molecular weight 83.3 kD) of unknown function, whose mutation does not affect the CCI4 degradation ability. This protein does not have any perfect motifs but has several sequences similar to motifs with known functions including a chaperonin cpn60 - like sequence and a sequence similar to bacterial type II secretion system protein B . Chaperonins are cytoplasmic ATPases that prevent misfolding and promote refolding and assembly of unfolded polypeptides generated under conditions of stress in a variety of organisms from bacteria to mammals (Hemmingsen et al., 1988). The proteins belonging to bacterial type II secretion system protein B are cytoplasmic ATPases that facilitate protein secretion, DNA uptake and assembly of type-IV fimbriae in E. coli and other bacteria (Whitchurch & Mattick, 1994). The protein encoded by ORF-L lacks an ATP binding site. If this protein is indeed a facilitator of protein folding or translocation, the energy needed for the process should be provided from an external source. ORF-M encodes for a 764 amino acid long protein (molecular weight 83 kD) that does not affect PDTC production and CCla degradation upon mutation. It is similar to putative 71 pror pho: cola invo amin PVric Side . CDCOC and 2: El (11” pro r; 0'3 Whicl aminotransferases from Streptomyces coelicolor (GenBank accession number CAB39702, 50% positives and 35% identities over a 466 amino acid overlap with a probability of 10—70) (Redenbach et al., 1996) and E. coli (GenBank accession number P42588, 54% positives and 39% identities over a 407 amino acid overlap with a probability of 10‘“) (Blattner et al., 1997). Lower similarities were observed with proven aminotransferases. ORF-M has a perfect aminotransferases class III pyridoxal phosphate attachment site in amino acids 524 to 561. Pyridoxal phosphate is an essential cofactor for reactions which act on the C-2 (alpha carbon) atom of amino acids and involve cleavage of any of the amino acid bonds. It is the coenzyme in a large number of amino-acid conversions such as transaminase, decarboxylase and dehydratase reactions. Pyridoxal phosphate is covalently attached to the enzyme by the amino group of a lysine side chain (Michal, 1999). Both BLAST and motif searches suggest that the protein encoded by ORF-M is a putative aminotransferase. ORF-N encodes for a 392 amino acid long protein (molecular weight 40.6 kD) that was not mutated by Lewis. It is similar to a putative transmembrane efflux protein from Streptomyces coelicolor (GenBank accession number CAB66188, 40% positives and 26% identities over a 466 amino acid overlap with a probability of 10- 9) (Redenbach et al., 1996) and Bacillus subtilis (GenBank accession number CABI6085, 43% positives and 25% identities over a 308 amino acid overlap with a probability of 109) (Ogasawara et al., 1994). This ORF has 8 to 12 predicted transmembrane helices, depending on the program used, and there are three predicted sites for the cleavage of a leader sequence, which range from 30 to 120 amino acids. Even though this ORF does not have perfect 72 So (J) 4.. \l dehy 6096 10‘th posni motifs, sequences similar to motifs found in integral membrane transporters were found. Some examples include hemolysin and alkaline protease secretion proteins (Hess et al., 1986), sugar transport proteins (Blattner et al., 1997), sodium - galactoside symporters (Yazyu et al., 1984), glycerol-3-phosphate transporter (Eiglmeier et al., 1987), and formate — nitrite transporters (Sawers & A, 1989). The aforementioned information suggests that the protein encoded in ORF-N is a perrnease involved in solute transport. ORF-O encodes for a 513 amino acid long protein with a predicted molecular weight of 54.7 kD. This protein was not mutated by Lewis and it is similar to an acyl-CoA dehydrogenase from Deinococcus radiolarians (GenBank accession number AAF10499, 60% positives and 39% identities over a 376 amino acid overlap with a probability of 1040) (White et al., 1999) and B. subtilis (Genbank accession number CAA74221, 56% positives and 39% identities over a 375 amino acid overlap with a probability of 10450) (Tosato et al., 1997). Two perfect motifs found in acyl-CoA dehydrogenases were observed in residues 256 to 268 and 464 to 483. Acyl-CoA dehydrogenase catalyses the first step in the degradation of fatty acids by the process of B-oxidation. It removes two hydrogens, leading to the formation of a double bond that will eventually be cleaved to yield acetyl-CoA and an acyl-CoA shortened by two carbons. Acetyl-CoA is used in the tricarboxylic acid cycle for the production of reducing power that leads to the generation of cellular energy (Michal, 1999). The protein encoded in ORF-O is apparently linked to the B—oxidation pathway. 73 kl: is (Gt acu eta in I] “Eu: ubiqi nun] meth; The I; dicart Pmnu a Sho metab acle Weigh diPlCt. 22255 firsr i, acid a ORF-P encodes a 351 amino acid long protein with a predicted molecular weight of 38.2 kD. Mutations in this ORF impaired production of PDTC and the degradation of CC14. It is similar to hydroxyneurosporene methyltransferase from Rhodobacter spheroides (GenBank accession number P54906, 43% positives and 31% identities over a 180 amino acid overlap with a probability of 10—9) involved in the biosynthesis of carotenoids (Lang et al., 1995). Several sequences similar to motifs found in methyltransferases were seen in the protein encoded by ORF-P. Some of the motifs include RNA methyltransferase (Gustafsson et al., 199]), C—5 cytosine-specific methylase (Som et al., 1987), and ubiquinone — menaquinone methyltransferase (Daniels et al., 1992). The sequence similarity and motif information suggest that the protein encoded in ORF-P is a putative methyltransferase. The last intermediate in the production of PDTC should be dipicolinic acid (pyridine-2,6- dicarboxilic acid, CAS registration number 499-83-2). Dipicolinic acid is a compound produced by B. subtilis and a few other bacteria genera, and its production is confined to a short period during bacterial sporulation. It is not essential for the structure or metabolism of growing cells, but its absence results in heat-sensitive spores. Dipicolinic acid combines with calcium in the endospore core, representing about 10% of the dry weight of the endospore (Paulus, 1993). Dipicolinic acid is produced by the heterodimer dipicolinate synthase (DpaA/DpaB, also called SpoV/SpoF, GenBank accession number 222554), in a single step, from 2,3-dihydrodipicolinate. 2,3-dihydrodipicolinate is the first intermediate in the biosynthesis branch leading to the production of diaminopimelic acid and L-lysine from L-aspartate (Chen et al., 1993; Daniel & Errington, 1993) (Figure 74 '2) fr;- sy: Silt 3.7). None of the predicted proteins found in the 8.3 kb EcoRI fragment or the 25.7 kb fragment identified by Lewis show similarity to dipicolinate synthase. If P. stutzeri KC synthesizes dipicolinic acid from 2,3-dihydrodipicolinate, a different set of enzymes should be involved in the process. 75 FiEu LagI MOCl; C=O | E CH3 COOH i C-H / CH I pyruvate O O I 2 -—->>-——§> cnz §§/\§5 57 I / N \ H—C-NH2 HO on I H-C-NHZ C 0011 | 2,3-dihydrodipicolinic acid COOH L-aspartate 2 H20 L-aspartate- dipicolinic 4’semIaIdChYde acid synthase / B. subtilis coon // I O O HzN-C-H \\ // l /C/\\ c\ . H bacterial ‘— ‘l—_ ((1312) 3 HO 0 cell wall dipicolinic acid H-C-NH2 I COOH L,L-2,6-diaminopimelic acid ““2 \ l I COOH CH2 1 I H-C-NH2 (CH2) 3 | I I (CH2)3 H-C-NH2 I I _ _ COOH HIC NHZ L-lysine COOH D,L-2,6-diaminopimelic acid Figure 3.7 Pathway leading to bacterial cell wall precursors and L-lysine from L-aspartate. Modified from (Michal, 1999) and (Paulus, 1993). 76 The branch leading to dipicolinic acid in B. subtilis is also provided. Even though there is no empirical data to explain how the essential ORFs found by Sept'ilveda and by Lewis are involved in the PDTC biosynthesis pathway, a speculative pathway can be proposed based on the similarity profiles. Lewis et al. (2000) proposed that ORF-2435, ORF-3626 and ORF-4099 (Lewis’ ORFs F-H) serve to effect sulfur transfer to an oxygen-substituted (acyl or hydroxyl) carbon, and that ORF-6289 (Lewis’ ORF-J) activates an acyl-group by adenylation. They proposed that the thiocarboxylate groups of PDTC are formed by condensation of a sulfur carrier (ORF-4099) and the adenylated precursor. I propose a more explicit pathway that takes into consideration motif information. An overview is provided in Figure 3.8. A detailed explanation of a speculative biosynthesis pathway is proposed in Figure 3.9 and in the subsequent paragraphs. 77 Figllre explanat 3.5. 2435 4099 4460 6289 7985 L M N O P 3626 sulfur donor activation dipicolinic acid synthesis and activation receptor exportation (non-essential) regulation Elflflfltt Figure 3.8 Overview of the proposed biosynthesis pathway for PDTC. A detailed explanation of the information provided in this figure is provided in the caption of Figure 3.5. 78 Figure 3.9 Speculative pathway for the synthesis of PDTC in Pseudomonas stutzeri strain KC. Upon the detection of environmental stimuli by ORF -243 5, ORF-P methylates the C—terminus of ORF-2435 (indicated by a star), triggering the PDTC biosynthetic pathway. ORF’s 3626 and 4099 activate the sulfur donor while ORF’s 4460 and 6289 synthesize and activate dipicolinic acid from 2,3-dihydrodipicolinate. PDTC is exported out of the cell, possibly by ORF-N with help of ORF-L. For simplicity, ORF numbers are used to represent the polypeptides encoded by the corresponding ORF. 79 C CDCC. pr( PFC oustide outer membrane protein-K periplasm cell membrane I I protein-M cytosol l protein2435 l protein-N K I / _P .\ I /. protein- \ / C \N C To be exported: \ HS/ \SH . . . . . . . . . Via protein-N ? Possrble activation pyridine-2,6-bis(thiocarboxyllc acrd) of C-terminus . . W th h l f t -L ? glycine in l e p 0 pro ein protein4099 / o\ l /o \C \ C/ . / N \ O protein6289-AMP AM P - protein6289 protein4099-gly-g-S— adenylated dipicolinic acid 2ADP + 2ATP $> protein2435-S-H or protein3626-S-H ? / o\ l /o x-s $ x \C \\N C/ no/ \on dipicolinic acid O . “ . oxidized protein2435--S-C-gly-protein4099 ‘2 co factor reduced cofactor or protein4460 O Membrane bound? / II o O protein3626--S-C-gly-protein4099 ? \\C \ C// N HO/ \OH T 2,3-d'h d d' ' 1' ' 'd protein3626/ protein4099 + protein2435 ? I Y I0 IPICO "NC 30! Synthesis and activation of dipicolinic acid Activation of sulfur donor 80 Tht in P Sept pred term the l termi The 1 prodt (Lewi sulfur 068d ( The p- hydrog dthVdr the St dehFdr The mutagenesis experiment performed by Lewis indicates that the first protein involved in PDTC synthesis is encoded by ORF-2435 (Lewis’ ORF-F) which was mutated by Sepulveda and by Lewis. This gene encodes for an integral membrane protein with two predicted transmembrane helices. It has a sequence similar to motifs found near the C- tenninus of chemotaxis sensors that facilitate sensory adaptation through the variation of the level of methylation of glutamate and glutamine residues located towards the C- terminus of the protein. The level of methylation is regulated by a methyltransferase. The fiinction of this protein may be to sense the levels of the stimuli that trigger PDTC production from 2,3-dihydrodipicolinate. The similarity profiles obtained for the proteins encoded by ORF-3626 and ORF-4099 (Lewis’ ORFs G and H) suggest that these proteins may work together in the process of sulfur transfer. The interactions between protein-3626, protein-4099 and protein-2435 need clarification and may be the subject of future research. The protein encoded by ORF-4460 (Lewis’ ORF-I) is similar to proteins that remove hydrogen or water from their substrates, yielding a double bond. The conversion of 2,3- dihydrodipicolinate to dipicolinic acid requires the removal of two hydrogen atoms and the subsequent formation of a double bond. ORF-4460 may encode for the dehydrogenase involved in that process. The protein encoded by ORF-6289 (Lewis’ ORF-J) has a perfect AMP-binding motif and is highly similar to enzymes that activate siderophore precursors by adenylation. This 81 prc acit thic tran proc protein may be responsible for the adenylation of the two carboxyl groups of dipicolinic acid, activating this compound for the transformation of the carboxyl groups into thiocarboxyl groups. Once the adenylated intermediate is formed, sulfur can be transferred from the protein-3626/protein-4099 complex to the adenylated intermediate, producing PDTC. The proteins encoded downstream of ORF-6289 appear to be involved in PDTC transport, reception and synthesis regulation, rather than in the biosynthetic pathway per se. ORF-K encodes for a TonB-dependent siderophore receptor. This outer membrane bound protein may be responsible for bringing metal-PDTC into the cell. Protein-L is not similar to known proteins but contains sequences similar to motifs found in molecular chaperons and proteins that facilitate translocation through the cell membrane. Protein-M is similar to aminotransferases and has a perfect motif for pyridoxal phosphate, an essential cofactor in many aminotransferase reactions. ORF-N encodes for a putative permease that facilitates solute transport through the cell membrane. The role of an aminotransferase is not clear at this point, but the permease may be responsible of transporting PDTC out of (and maybe into) the cell with the help of cytoplasmic facilitators like Protein-L. ORF-O and ORF-P are transcribed in the opposite orientation than the rest of the genes involved in PDTC production. Protein-O is similar to acyl-CoA dehydrogenases and it has a perfect acyl-CoA binding site. Such an enzyme will be involved in the process of B-oxidation of fatty acids. The function of this ORF in PDTC production/processing is 82 not pose Prot knor synt initiz bios; respc plasr (_Figt not clear. On the other hand, the physiological roles of PDTC are yet to be elucidated. A possible role for protein-O might me assigned when more information is available. Protein-P is similar to methyltransferases and has sequences similar to motifs found in known methyltransferases. Protein-P may be involved in the regulation of PDTC synthesis. This putative methylase may modify Protein-2435 (Lewis’ ORF-F) in order to initiate or attenuate PDTC synthesis. Protein-2435 is the first enzyme in the PDTC biosynthesis pathway and has sequences similar to chemotaxis transducers that trigger responses to environmental stimuli, depending on their level of methylation. When plasmids M22 and JS68 containing a partial fragment of T3] lacking ORF’s O and P (Figure 7.6) are introduced into strain CTN], only 10% or no CC14 degradation capacity are observed, respectively, indicating that proteins 0 and P are important for PDTC production. Their possible roles as regulators come to mind once again. One of the main differences in the mutants found by Sept’llveda and by Lewis is that Sept'llveda was unable to find mutants corresponding to the proteins found in the upper half of the pathway. This result can be explained when the differences between their systems and experimental conditions are taken into consideration. Sepulveda started with a modified P. stutzeri KC strain capable of growing in the presence of rifampicin while retaining the wild type CCI4 degradation capacity. The bacterium was mutated with Tn5::lux and exposed to l4CC14 for 5 days before l4C-labeled non-volatile products trapped in the culture medium were analyzed. If other receptors and permeases can compensate for the lack of a functional protein-K or protein-N, for example, Sept'ilveda’s assay could not be used to identify such a mutant, even if the affinity of the other proteins 83 for PD' over a l CCI4 dc compen compen mutatin, and utili Sepi'llve Injzzlur reasons. for P. st interestil that 1051 the T115: COnsu-UC receptor some of prOdUcn AS "1d“ able to \ genes in for PDTC is lower, because the assay only shows the non-volatile product accumulated over a long period of time. Lewis reported that mutations in ORFs L and M do not affect CCI4 degradation, which suggests that other proteins present in P. stutzeri strain KC can compensate for the lack of functional proteins encoded by ORFs L and M. The compensatory action of other proteins present in the strain KC genome could be tested by mutating ORFs K, L, M or N in wild type strain KC and monitoring PDTC production and utilization by these cells. Sepr’rlveda tried to introduce pRK311, pT31, pRKblue and pRKblue8.3 into the four Tn5 :1qu transpositional mutants but the mutants did not accept any DNA, for unknown reasons. When these plasmids were introduced into P. stutzeri CCUG 11256 (type strain for P. stutzeri), only the cells harboring pT31 were able to degrade CCI4. It would be interesting to see if strain CCUG 11256 and strain CTN] (the spontaneous KC mutant that lost a 170 kb fragment) are able to degrade CC14 when the 8.3 kb EcoRI mutated in the Tn5::lux mutants and Lewis’ ORF’s O and P are introduced into the cell. Such a construct will leave out the upper half of the PDTC biosynthesis pathway encoding for a receptor and a permease. If these experiments are successful, they would confirm that some of the genes present in the upper half of the pathway are not essential for PDTC production and CCI4 degradation. As indicated in the results section, P. putida DSM 3601, a PDTC producing bacteria, was able to degrade CCI4 but its DNA did not hybridize with a 3.4 kb probe containing the genes interrupted in KC657, KC1896, KC2753 and KC3164. No amplification of strain 84 resul diffe: syntr The inves how reseal P. Stu DSM 3601 DNA was observed when primers that amplify the genes interrupted in strain KC were used, even when the annealing temperature was decreased by 5 °C. These results suggest that P. stutzeri strain KC and P. putida DSM 3601 produce DPTC using different mechanisms. The elucidation of the similarities and differences of the PDTC synthesis pathways of these organisms may be the subject of future research. The genetic information presented in this chapter has opened new doors for the investigation of the physiological role of PDTC, the mechanisms of PDTC regulation and how this molecule can be efficiently used for the decontamination of CCI4. More research is needed in order to fully understand the fascinating capabilities of PDTC and P. stutzeri strain KC. 85 Adhy Altscl Attw Bate! Bear 313‘ REFERENCES Adhya, S. & Gottesman, M. (1978). Control of transcription. Annu Rev Biochem 47, 967-996. Altschul, S. F., Madden, T. L., Schfiffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST; a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402. Attwood, T. K., Flower, D. R., Lewis, A. P., Mabey, J. E., Morgan, S. R., Scordis, P., Selley, J. & Wright, W. (1999). PRINTS prepares for the new millenium. Nucleic Acid Res 27, 220-225. Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Finn, R. D. & Sonnhammer, E. L. (1999). Pfam3.l: 1313 multiple alignments match the majority of proteins. Nucleid Acids Res 27, 260-262. Bennasar, A., Rossello-Mora, R. A., Lalucat, J. & Moore, E. R. (1996). 16S rRNA gene sequence analysis relative to genomovars of Pseudomonas stutzeri and proposal of Pseudomonas balearica sp. nov. Int J Syst Bacteriol 46, 200-205. Black, T. A., Cai, Y. & Wolk, C. P. (1993). Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development development in Anabaena. Mol Microbiol 9, 77-84. Blattner, F. R., Plunkett, G. I. I. I., Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, F. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B. & 86 F Bani: Brau“ Brau Bre; Bur Shao, Y. (1997). The complete genome sequence of Escherichia coli K-12. Science 277, 1453-1474. Boyd, A., Kendall, K. & Simon, M. I. (1983). Structure of the serine chemoreceptor in Escherichia coli. Nature 301, 623-626. Braun, V. & Hantke, K. (1991). Genetics of bacterial iron transpor. In Handbook of Microbial Iron Chelates (ed. G. Winkelmann), pp. 107-138. CRC Press, Boca Raton, FL. Braun, V. (1995). Energy-coupled transport and signal transduction through the Gram- negative outer membrane via TonB-Ebe-Ebe-dependent receptor proteins. FEMS Microbiol Rev 16, 295-307. Brendel, V. & Trifonov, E. N. (1984). A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res 12, 441 1-4427. Butler, B. A. (1998). Sequence analysis using GCG. Meth Biochem Anal 39, 79-97. Carlson, C. & lngraham, J. (1983). Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa and Paracoccus denitrificans. Appl Environ Microbiol 45, 1247-1253. Chen, N. Y., Jiang, S. Q., Klein, D. A. & Paulus, H. (1993). Organization and nucleotide sequence of the Bacillus subtilis diaminopimelate operon, a cluster of genes encoding the first three enzymes of diaminopimelate synthesis and dipicolinate synthase. J Biol Chem 268, 9448-9465. Corpet, F., J, Gouzy, J. & Kahn, D. (1999). Recent improvements of the ProDom database of protein domain families. Nuclei Acid Res 27, 263-267. 87 Cridd Cserzr Daniel Daniel: Devere: Ditta, Dybas, Criddle, C. S., DeWitt, J. T., Grbic—Galic, D. & McCarthy, P. L. (1990). Transfromation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions. Appl Environ Microbiol 56, 3240-3246. Cserzo, M., Bernassau, J., Simon, I. & Maigret, B. (1994). New alignment strategy for transmembrane proteins. J Mol Biol 243, 388-396. Daniel, R. A. & Errington, J. (1993). Cloning, DNA sequence, functional analysis and transcriptional regulation of the genes encoding dipicolinic acid acid synthetase required for sponilation in Bacillus subtilis. J Mol Biol 232, 468-483. Daniels, D. L., Plunkett, G. 1., Burland, V. D. & Blattner, F. R. (1992). Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. Science 257, 771-778. Devereaux, J., Haeberli, P. & Smithies, O. (1984). A comparative set of sequence analysis programs for the VAX. Nucleic Acid Res 12, 387-395. Ditta, G., Schmidhauser, T., Yokobson, E., Lu, P., Liang, X. W., Finlay, D. H., Guiney, D. & Helinski, D. R. (1985). Plasmids related to the broad host vector pRK290 useful for gene cloning and for monitorig gene expression. Plasmid 13, 149-153. Dybas, M. J., Tatara, G. M. & Criddle, C. S. (1995) Localization and characterization of the carbon tetrachloride transformation activity of Pseudomonas sp. strain KC. Appl Environ Microbiol 61, 758-762. Eiglmeier, K., Boos, W. & Cole, S. (1987). Nucleotide sequence and transcriptional startpoint of the glpT gene of Escherichia coli: extensive sequence homology of 88 the glycerol-3-phosphate transport protein with components of the hexose-6- phosphate transport system. Mol Microbiol 1, 251-258. Fernandez-Pinas, F. , Leganés, F. & Wolk, C. P. (1994). A third genetic locus required for the formation of heterocysts in Anabaena sp. strain PCC 7120. J Bacteriol 176, 5277-5283. Gray, G. L., Smith, D. H., Baldridge, J. S., Harkins, R. N., Vasil, M. L., Chen, E. Y. & Heyneker, H. L. (1984). Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa. Proc Nat Acad Sci USA 81, 2645-2649. Gustafsson, C., Lindstroem, P. H. R., Hagervall, T. G., Esberg, K. B. & Bjoerk, G. R. (1991). The trmA promoter has regulatory features and sequence elements in common with the rRN A P] promoter family of Escherichia coli. J Bacteriol 173, 1757-1764. Gutierrez, C., Barondess, J., Manoil, C. & Beckwith, J. (1987). The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. J Mol Miol 195, 289-297. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580. Hansen, F. G., Hansen, E. B. & Atlung, T. (1985). Physical mapping and nucleotide sequence of the rnpA gene that encodes the protein component of ribonuclease P in Escherichia coli. Gene 38, 85-93. Hemmingsen, S. M., Woolford, C., Van der Vies, S. M., Tilly, K., Dennis, D. T., Georgopoulos, C. P., Hendrix, R. W. & Ellis, R. J. (1988). Homologous plant 89 and bacterial proteins chaperone oligomeric protein assembly. Nature 333, 330- 334. Henikoff, J. G., Henkoff, S. & Pietrokovski, S. (1999). New features in the BLOCKS database servers. Nucleic Acids Res 27, 226-228. Hess, J., Wels, W., Vogel, M. & Goebel, W. (1986). Nucleotide sequence of a plasmid- encoded hemolysin determinant and its comparison with corresponding chromosomal hemolysin sequence. FEMS Microbiol Lett 34, 1-1 1. Hirasawa, K., Ichihara, M. & Okanishi, M. (1985). Nucleotide Sequence of Mec+ gene region of Streptomyces kasugaensis. J Antibiot (Tokyo) 38, 1795-1798. Hirokawa, T., Boon-Chieng, S. & Mataku, S. (1998). Classification and secondary structure prediction system for membrane proteins. Bioinformatics 14, 378-3 79. Hofmann, K. & Stofell, W. (1993). Tmbase - a database of membrane spanning protein segments. Biol Chem (Hoppe-Seyler) 347, 166. Hoffmann, K., Bucher, P., Falquet, L. & Bairoch, A. (1999). The PROSITE database, its status in 1999. Nucleic Acids Res 27, 215-219. Javanovic, G., Kostic, T., Jankovic, M. & Savic, D. J. (1994). Nucleotide sequence of the Escherichia coli K-12 histidine operon revisited. J Molec Biol 239, 433-435. Kadner, R. (1990). Vitamin 812 transport in Escherichia coli: energy coupling between membranes. Mol Microbiol 4, 2027-2033. Klebba, P. E., Rutz, J. M., Liu, J. & Murphy, C. K. (1993). Mechanism of TonB- catlyzed iron transport through the enteric bacterial cell envelope. J Bioenerg Biomembr 25, 603-6] 1. 90 Lang, H. P., Cogdell, R. J., Takaichi, S. & Hunter, C. N. (1995). Complete DNA sequence, specific Tn5 insertion map and gene assignment of the carotenoid biosynthesis pathway of Rhodobacter sphaeroides. J Bacterial 177, 2064-2073. Larsen, R. A., Foster-Hartnett, D., McIntosh, M. A. & Postle, K. (1997). Regions of E coli TonB and FepA proteins essential for in vivo physical interactions. J Bacteriol 179, 3213-3221 . Lee, C. H., Lewis, T. A., Paszczynski, A. & Crawford, R. L. (1999). Identification of an extracellular catalyst of carbon tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2,6-bis(thiocarboxylate). Biochem Biophys Res Commun 261, 562-566. Lewis, T. A., Cortese, M. S., Sebat, J. L., Green, T. L. & Crawford, R. L. (2000). A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCI4 - dechlorinating agent pyridine-2,6-bis(thiocarboxilic acid). Environ Microbiol In Press. Link, A. J., Robison, K. & Church, G. M. (1997). Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrphoresis 18, 1259-1313. Michal, G. (1999). Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology. John Wiley & Sons, Inc., New York. Moeck, G. S. & Coulton, J. W. (1998). TonB-dependent iron acquisition: mechanism of siderophore-mediated active transport. Mal Microbiol 28, 675-681. 91 Nakai, K. & Kaneshisa, M. (1991). Expert systems for predicting protein localization sites in Gram-negative bacteria. Proteins: Structure, Function and Genetics 11, 95-110. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1-6. Ockels, W., Rt'imer, A. & Budzikiewicz, H. (1978). An F e(II) complex pryridine-2,6-di- (monothiocarboxylic acid) - a novel bacterial metabolic product. Tetrahedron Lett 36, 3341-3342. Ogasawara, N., Nakai, S. & Yoshikawa, H. (1994). Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Res 1, 1-14. Paulus, H. (1993). Biosynthesis of the aspartate family of amino acids. In Bacillus subtilis and other Gram-positive Bacteria: Biochemistry, Physiology and Molecular Genetics (ed. A. Sonenshein, J. A. Hoch and R. Losick), pp. 237-267. American Society for Microbiology, Washington, D C. Platt, T. (1986). Transcription terminator and the regulation of gene expression. Annu Rev Biochem 55, 339-372. Postle, K. (1990). TonB and the Gram-negative dilemma. Mol Microbiol 4, 2019-2025. Rajagopalan, K. V. (1996). Biosynthesis of the molybdenum cofactor. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (ed. F. C. Neidhardt), pp. 674-679. ASM Press, Washington DC. 92 Rajagopalan, K. V. (1997). Biosynthesis and processing of the molybdopterin cofactors. Biochem Soc Trans 25, 757-761. Rakin, A., Saken, E. & Harmsen, D. (1994). The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function. Mal Microbiol 19, 253-263. Rakin, A., Urbitsch, P. & Heesemann, J. (1995). Evidence for two evolutionary lineages of highly pathogenic Yersenia species. J Bacterial 177, 2292-2298. Redenbach, M., Kieser, H. M., Denapaite, D., Eichner, A., Cullum, J., Kinashi, H. & Hopwood, D. A. (1996). A set of ordered cosmids an a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3 (2) chromosome. Mol Microbiol 21, 77-96. Reese, M. G. & Eeckman, F. H. (1995). Novel neural network algorithms for improved eukaryotic promoter site recognition. In The 7th International Genome Sequencing and Analysis Conference (ed. L. Hunter and T. E. Klein), Hilton Head Island, South Carolina. Rossello, R. A., Garcia-Valdés, E., Lalucat, J. & Ursing, J. (1991). Genotypic and Phenotyic Diversity of Pseudomonas stutzeri. Syst Appl Microbial 14, 150-157. Rossellb-Mora, R. A., Lalucat, J., Timmis, K. N. & Moore, E. R. B. (1996). Strain JM300 represents a new genomovar within Pseudomonas stutzeri. Syst Appl Microbiol 19, 596-599. Rowland, B. M., Grossman, T. H., Osburne, M. S. & Taber, H. W. (1996). Sequence and genetifc organization of a Bacillus subtilis operon encoding 2,3- dehydroxybenzoate biosynthetic enzymes. Gene 178, 119-123. 93 Rusnak, F., Faraci, W. S. & Walsh, C. T. (1989). Subcloning, expression, and purification of the enterobactin biosynthetic enzyme 2,3-dihydroxybenzoate-AMP ligase: demonstration of enzyme-bound (2,3-dihydroxybenzoyl)adenylate product. Biochemistry 28, 6827-6835. Sambrook, J., Fritsch, E. L. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain - terminating inhibitors. Proc Nat Acad Sci USA 74, 5463-5467. Sawers, G. & A, B. (1989). Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression. J Bacteriol 171, 2485-2498. Scofield, M. A., Lewis, W. S. & Schuster, S. M. (1990). Nucleotide sequence of Escherichia coli asnB and deduced arnio acid sequence of asparagine synthetase B. J Biol Chem 265, 12895-12902. Seeger, C., Poulsen, C. & Dandanell, G. (1995). Identification and characterization of genes (xapA, xapB and xapR) involved in xanthosine catabolism in Escherichia coli. J Bacteriol 177, 5506-5516. Sepr'ilveda-Torres, L. del C., Rajendran, N., Dybas, M. J. & Criddle, C. S. (1999). Generation and initial characterization of Pseudomonas stutzeri KC mutants with impaired ability to degrade carbon tetrachloride. Arch Microbiol 171, 424-429. Serino, L., Reimmann, C., Visca, P., Beyeler, M., Della Chiesa, V. & Haas, D. (1997). Biosynthesis pf pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa. J Bacteriol 179, 248-257. 94 Shine, J. & Delgarno, L. (1974). The 3'-terminal sequence of Escherichia coli l6S ribosomal RNA: complementary to nonsense triplets and ribosome binding sites. Proc Nat Acad Sci USA 71, 1342-1346. Simon, R., Priefer, U. & Piihler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1, 784-791. Som, S., Bhagwat, A. S. & Friedman, S. (1987). Nucleotide sequence and expression of the gene encoding the EcoRII modification enzyme. Nucleic Acids Res 15, 313- 332. Sonnhammer, E. L. L., Von Heijne, G. & Krogh, A. (1998). A hidden Markov model for predicting transmembrane helices in protein sequences. In Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ed. J. Glasgow), pp. 175-182. AAAI Press. Stanier, R., Palleroni, N. & Doudoroff, M. (1966). The aerobic pseudomonas: a taxonomic study. J Gen Microbiol 43, 159-271. Tatara, G. M., Dybas, M. J. & Criddle, C. S. (1993). Effects of medium and trace elements on kinetics of carbon tetrachloride transforamtion by Pseudomonas sp. strain KC. Appl Environ Microbiol 59, 2126-2131. Tatara, G. M., Dybas, M. J. & Criddle, C. S. (1995). Biofactor mediated transformation of carbon tetrachloride by diverse cell types. In Bioremediation of Chlorinated Solvents, vol 3(4) (ed. R. E. Hinchee, A. Leeson and L. Semprini), pp. 69-76. Battelle Press, Columbus, Ohio. 95 Taylor, S. V., Kelleher, N. L., Kindsland, C., Chiu, H.-J., Costello, C. A., Backstrom, A. D., McLafferty, F. W. & Begley, T. P. (1998). Thiamin biosynthesis in Escherichia coli. J Biol Chem 273, 16555-16560. Tosato, V., Albertini, A. M., Zotti, M., Sonda, S. & Bruschi, C. V. (1997). Sequence completion, identification and definition of the fengycin operon in Bacillus subtilis 168. Microbiology 143, 3443-3450. Tusnady, G. E. & Simon, I. (1998). Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J Mal Biol 283, 489-506. von Heijne, G. (1992). Membrane protein structure prediction, hydrophobicity analysis and the positive-inside rule. J Mol Biol 225, 487-494. Whitchurch, C. B. & Mattick, J. S. (1994). Escherichia coli contains a set of genes homologous to those involved in protein secretion, DNA uptake and assembly of type-IV fimbriae in other bacteria. Gene 150, 9-15. White, 0., Eisen, J. A., Heidelberg, J. F., Hickey, E. K., Peterson, J. D., Dodson, R. J., Haft, D. H., Gwinn, M. L., Nelson, W. C., Richardson, D. L., Moffat, K. S., Qin, H., Jiang, L., Pamphile, W., Crosby, M., Shen, M., Vamathevan, J. J., Lam, P., McDonald, L., Utterback, T., Zalewski, C., Makarova, K. S., Aravind, L., Daly, M. J., Minton, K. W., Fleischmann, R. D., Ketchum, K. A., Nelson, K. E., Salzberg, S., Smith, H. O., Venter, J. C. & Fraser, C. M. (1999). Genome sequence of athe radioresistant bacterium Deinococcus radiadurans R]. Science 286, 1571-1577. 96 Wolk, C. P., Cai, Y. & Panoff, J. M. (1991). Use of a tansposon with luciferase as a reporter to identify environmental responsive genes in a cyanobacterium. Proc Nat Acad Sci USA 88, 5355-5359. Yanish-Perron, C., Vierira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUCl9 vectors. Gene 33, 103-1 19. Yazyu, H., Shiota-Niiya, S., Shimamoto, T., Kanazawa, H. & Futai, M. (1984). Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coli. J Biol Chem 259, 4320- 4326. Zylstra, G. J., Olsen, R. H. & Paballou, D. P. (1989). Genetic organization and sequence of the Pseudomonas cepacea genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase. J Bacteriol 171, 5915-5921. 97 CHAPTER 4 RECOMMENDATIONS FOR FUTURE RESEARCH 98 The main goal at the outset of this project was to characterize genes involved in the degradation of CCI4 by Pseudomonas stutzeri strain KC, when the identity of the agent responsible for CCI4 dechlorination was still unknown. I was the pioneer in the discovery of genes involved in the production of the compound that was later identified as pyridine-2,6-bis(thiocarboxylic acid) (PDTC) (Figure 1.1). The discovery of a spontaneous Pseudomonas stutzeri strain KC mutant unable to degrade CCI4 by the Crawford group at the University of Idaho, provided both an independent corroboration of my work and additional supplementary information. Although our joint efforts constitute a significant advancement in the understanding of the CCI4 degradation capacity of P. stutzeri strain KC, many questions remain to be answered by future investigations. The hypothetical PDTC biosynthesis pathway provided in Chapter 3 needs to be empirically corroborated. The information gathered using the DNA and protein similarity searches, as well as the protein motif searches, indicate that open reading frames (ORFS) 3626 and 4099 may be responsible for donating the two sulfurs found in PDTC. A possible interaction with ORF-2435 cannot be discarded. The isolation and characterization of the proteins encoded by these genes will allow the design of experiments to test the interactions between the polypeptides as well as their sulfur- transfer capabilities. The aforementioned pathway assumes that the carbon and nitrogen atoms in the PDTC ring come from 2,3-dihydrodipicolinic acid, the first intermediate in the branch leading to 99 the biosynthesis of L-lysine from L-aspartate (Figure 3.7). If this is the case, radiolabeled PDTC should be obtained when l4C-labeled L-aspartate or pyruvate is supplied in the culture medium because these two compounds provide the atoms for 2,3- dihydrodipicolinic acid. In the proposed pathway, ORF-4460 introduces a double bond into 2,3-dihydrodipicolinic acid to produce dipicolinic acid. The isolation of the protein would allow the in vitro characterization of the reaction in the presence of various oxidized cofactors. Computer programs could be used to model the active site of the protein and determine if 2,3- dihydrodipicolinic acid and oxidized cofactors would fit inside the active site. If this protein is involved in this particular step, the accumulation of 2,3-dihydrodipicolinic acid should be observed in strains KC1896, KC2753 and KC3164 because all of them were mutated in this ORF. ORF-6289 encodes for a putative AMP-ligase probably involved in the activation of the two carboxyl-groups of PDTC by adenylation. The mutants impaired in ORF-6289 generated by the Crawford group should be unable to carry out this particular reaction and must accumulate dipicolinic acid in the cytoplasm. Once this protein is isolated, the reaction could be carried out in vitro, allowing the formation of adenylated dipicolinic acid and the isolation of the enzyme-bound adenylated dipicolinic acid. The adenylation of dipicolinic acid by other AMP-ligases could also be studied in similar assays. 100 Crude cell lysates of wild type strain KC and of mutants impaired in different steps of the PDTC biosynthesis pathway may be used to study the accumulation of biosynthetic intermediates. Such experiments may provide further information about gene function. Lewis’ ORF-K encodes for an outer membrane siderophore receptor that may be the receptor for PDTC. Since siderophore receptors may interact with structurally similar siderophores, it would be worth investigating the possibility of the interactions of ORF-K with PDTC analogues and the interactions of PDTC with the siderophore receptors that showed similarity to ORF-K at the DNA and protein levels. Similar experiments could be designed to determine if metal-loaded PDTC is taken into the cell by ORF-K (or proteins similar to ORF-K); or if PDTC remains in the culture medium while the metal is transported into the cytoplasm. It is my belief that Lewis’ ORF-P is involved in the regulation of PDTC synthesis by changing the state of methylation of ORF-2435, the first protein involved in PDTC biosynthesis. ORF-2435 is an integral membrane protein that has a sequence similar to chemotaxis transducers that are controlled by the level of methylation of some residues near their C-termini. The isolation of the proteins encoded by ORF-P and ORF-2435 may provide the framework for the study of interactions among them. Nevertheless, this task may be difficult because of the membrane topology of ORF-2435. I was unable to identify mutants in the genes corresponding to Lewis’ ORFs L and M and N. On the other hand, mutants in these ORFs did not affect PDTC degradation when the 10] Crawford group mutated them. I think these proteins are not essential for PDTC production because other proteins can carry out the same functions. Lewis’ ORFs O and P appear to be essential, presumably due to their possible regulatory role. The substitutability hypothesis can be tested by the introduction of a plasmid containing the 8.3 kb EcoRI fragment harboring the genes directly involved in PDTC biosynthesis, ORF-O and ORF-P into the type strain of P. stutzeri and P. stutzeri CTN]. If this plasmid can confer the CCI4 degradation capacity to the aforementioned strains, the substitutability hypothesis would be sustained. The identification of the genes needed for the synthesis of PDTC also opens the door to investigations regarding the molecular mechanisms of transcriptional control of PDTC production. As indicated in Figure 3.6, a sequence very similar to the consensus sequence of the E. coli ferric uptake regulator (Fur, Fur box) was found in the promoter region of the PDTC operon. Fur is believed to bind to the Fur box when ferrous iron is abundant, in order to repress the transcription of genes needed for the biosynthesis and uptake of iron-scavenging compounds. Since PDTC is only produced when P. stutzeri strain KC is grown under iron-limiting conditions, the discovery of a Fur box in the promoter region of the PDTC operon is congruent with the mechanism of regulation of iron-scavenging processes. Furthermore, I demonstrated the iron-dependent promoter activation by following the expression of the promoterless reporter luciferase genes when strain KC was grown with different iron concentrations (Figure 2.1). Experiments designed to assess the effects of mutations in the Fur box on PDTC production will provide evidence of the involvement of Fur in the control of PDTC biosynthesis. The 102 PDTC promoter could be fused to a promoterless reporter gene whose expression is monitored under iron abundance or limitation, in hosts that contain a functional fur gene and in fitr-minus hosts. The discovery of a Fur box in the promoter region of the PDTC biosynthesis genes also has implications for the utilization of PDTC and P. stutzeri KC in CCI4 remediation projects. If iron-limitation cannot be attained, it is imperative to place the PDTC biosynthesis genes under the control of a promoter that would be activated under the particular circumstances prevailing in the contaminated samples. The transfer of the PDTC biosynthesis genes into hosts already adapted to the environmental conditions that predominate in the contaminated site should also be considered. Sequence information suggests that the PDTC operon starts in ORF-2435 and may be transcribed until a hairpin structure identified in positions 20,593 to 20,616 between Lewis’ ORF-N and ORF-O. ORFs O and P appear to be part of a transcript independent from the larger PDTC operon. There is a possible promoter from position 24,477 to position 24,432 and a hairpin structure from position 20,336 to position 20,316 (between ORFs O and N). This information can be empirically verified by the analysis of mRNA isolated under the conditions leading to PDTC biosynthesis, using the PDTC genes as probes for Northern blots or reverse transcription. The exact transcript beginning could be determined by the primer extension technique or by S] nuclease analysis. The position of the 3’-end of the transcript could be estimated by reverse transcription, 103 varying the primer closer to the 3’-end until no cDNA could be generated with the specific primer set. The control of PDTC synthesis by feedback inhibition should be studied in detail. If PDTC inhibits its own synthesis, P. stutzeri strain KC cultures grown under iron limitation should not transcribe the PDTC genes if external PDTC is provided. This hypothesis can be tested with Northern blots or reverse transcription. If feedback inhibition is observed, interactions between PDTC and the enzymes involved in its biosynthesis should be empirically corroborated. PDTC was originally discovered as a metabolite produced by Pseudomonas putida DSM 360] grown under iron-limiting conditions. Even though P. putida DSM 360] shares this metabolic capability with strain KC, the bacteria apparently use different mechanisms for PDTC production, as indicated by the DNA-based studies described in Chapter 3. The DNA from P. putida DSM 3601 and 8 P. stutzeri strains incapable of degrading CCI4 did not amplify with polymerase chain reaction (PCR) primers designed for the amplification of ORF-2435 or ORF-4460, even when the annealing temperature was lowered by 5°C. The same negative result was observed on a Southern blot when a 3.5 kb fragment spanning from ORF-2435 to ORF-4460 was used to hybridize with 10 pg of genomic DNA from P. putida DSM 360] and 8 P. stutzeri strains. These results should be verified by the utilization of different PCR primer sets and by low stringency Southern blots. The characterization of the genes involved in PDTC biosynthesis in P. putida DSM 3601 would allow a direct comparison of the pathway used by these bacteria for the 104 production and regulation of PDTC. The information obtained from these comparisons may increase our knowledge about the native role of PDTC. The physiological experiments described in Chapter I, along with the genetic studies depicted in Chapters 2 and 3 have greatly enhanced our understanding of the CCI4 degradation capacity of P. stutzeri strain KC. The experiments suggested in Chapter 4 would further extend our comprehension of this process at the molecular level. The combined efforts of past and future experimentation will allow us to fully elucidate the fascinating CCI4 degradation capacity of P. stutzeri strain KC. 105 APPENDIX A PHYLOGENY AND TAXONOMY OF PSEUDOMONAS S T U T ZERI STRAIN KC “Pseudomonas strain KC represents a new genomovar within Pseudomonas stutzeri” to be submitted to The International Journal of Systematic and E valutianaty Microbiology 106 Pseudomonas strain KC represents a new genomovar within Pseudomonas stutzeri Lycely del C. Sepulveda-Torresl, J izhong Zhouz, Caterina Guasp3, Jorge Lalucat3, David Knaebel“, Jody L. Plank“, and Craig s. Criddle51 I Department of Microbiology and National Science Foundation Center for Microbial Ecology, Michigan State University, East Lansing, MI 48823, USA 2 Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA 3 Microbiologia, Departament de Biologia, Universitat de les llles Balears and Institut Mediterrani d’Estudis Avancats, Palma de Mallorca, Spain 4 Biology Department, 5805 Clarkson University, Potsdam, NY 13699 5 Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305-4020, USA ‘ o e a Current address: Envrronmental Scrence Center, Syracuse Research Corporation, North Syracuse, NY 13215, USA 107 1 Current address: Department of Biochemistry, Duke University, Durham, NC 27708, USA IAuthor for correspondence: Craig S. Criddle. Tel: + 650 723 9032. Fax: + 650 725 9474 e-mail: criddle@ce.stanford.edu Keywords: Pseudomonas KC, phylogeny and taxonomy, genomovar, carbon tetrachloride biodegradation, pyridine-2,6-bis(thiocarboxylate) Subject category: Evolution, Phylogeny and Biodiversity Running title: Phylogeny and taxonomy of Pseudomonas strain KC Non-standard abbreviations: DMSO, dimethyl sulfoxide; FAME, fatty acid methyl ester; gv, genomovar; ITS], 16S — 23S intemally-transcribed spacer region; PDTC, pyridine- 2,6-bis(thiocarboxylate); TSB, tryptic soy broth; U, units; UPGMA , unweighted pair- group mean analysis 108 SUMMARY Pseudomonas sp. strain KC is a denitrifying aquifer isolate that produces and secretes pyridine-2,6-bis(thiocarboxylate) (PDTC), a compound that chelates copper to fortuitously transform carbon tetrachloride without producing chloroform. Although KC has been successfully used for full-scale bioremediation of carbon tetrachloride, its taxonomy has proven difficult to resolve, as it retains pr0perties of both P. stutzeri and P. putida. In the present work, a polyphasic approach, comprising phenotypic characteristics (carbon substrate utilization patterns, antibiotic resistance profiles and composition of cellular fatty acids) and genotypic information (DNA-DNA hybridization, DNA fingerprinting, 16S rDNA sequencing, ITS sequencing and gyrB PCR) is used to establish that strain KC is a member of the species Pseudomonas stutzeri. Moreover, we conclude that strain KC represents a new genomovar (genomovar 9) within the species P. stutzeri. 109 INTRODUCTION Bacterial strain KC (ATCC deposit no 55595, DSM deposit no 7136) is a denitrifying bacterium originally isolated from an aquifer in Seal Beach, California (Criddle et al., 1990). Under iron-limiting conditions, strain KC induces genes for the production and secretion of pyridine-2,6-bis(thiocarboxylate) (PDTC), a molecule that chelates copper and can rapidly dechlorinate CCI4 yielding C02 (~ 50%), and nonvolatile compounds (~ 50%), under anoxic conditions (Criddle et al., 1990; Dybas et al., 1995; Lee et al., 1999; Lewis & Crawford, 1993; Sept'ilveda-Torres et al., 1999). This activity is important for bioremediation applications in aquifer sediments because it is rapid, with half-lives of only a few minutes (Tatara et al., 1995) and occurs without accumulation of chloroform. Strain KC attaches to aquifer sediment, but it can also exist in a free-swimming, highly motile form that is chemotactic towards nitrate, and it can sustain dechlorination activity during migration (Witt et al., 1999a; Witt et al., 1999b). Emerson (1999) reported that strain KC reproducibly forms colonies of complex morphology on agar motility plates containing nitrate or nitrite. Five other species of pseudomonads tested under identical conditions were unable to form such complex colonies. Recent developments further underscore the unique environmental significance of this strain. Lewis et al. (2000) have reported that a laboratory culture of strain KC had spontaneously lost a 170 kb fragment containing genes necessary for PDTC biosynthesis on a 25 kb fragment of the lost DNA. This fragment was not detected in three other P. stutzeri strains. Strain KC has also assumed great significance for biotechnology because of its use in one of the first full- scale field aquifer bioaugmentation applications. Large volumes of strain KC were 110 grown on-site and injected into a CCI4-contaminated aquifer in Schoolcrafi, Michigan (Hyndman et al., 2000). The resulting biocurtain for CCI4 degradation has now been maintained for over two years, with efficient removal of CCI4. Strain KC was originally classified as a Pseudomonas stutzeri — like organism for its ability to reduce nitrate and use maltose, citrate, malonate and glyerol as carbon sources and a preliminary fatty acid profile (Criddle et al., 1990) and some past results have referred to it as a P. stutzeri. Nevertheless, no exhaustive studies were performed to elucidate the strain KC taxonomy. The present investigation was performed to conclusively establish the systematic classification of strain KC based on physiological and genotypic studies. The results obtained from DNA-DNA hybridization, DNA fingerprinting, l6S rRNA sequence, ITS] sequence analysis and gyrB PCR studies were combined with substrate utilization, antibiotic resistance and fatty acid methyl ester (FAME) analyses to establish that strain KC should be classified as a type strain for a novel P. stutzeri genomovar. lll METHODS Strains and growth conditions: The strains used in this study, their source of isolation and relevant references are provided in Table A]. The bacteria were grown aerobically in tryptic soy broth (TSB), nutrient broth or LB medium at 30 °C (Sambrook et al., 1989). 112 Table A.l Bacterial strains used in this study Strain Other designations and origins of isolation References Pseudomonas sp. KC P. stutzeri ATCC 17591 gv 2T P. stutzeri DSM 50227 gv 31' P. stutzeri l9SMN4 gv 4T , ‘l' P. stutzeri DNSP21 gv 5 . T P. balearica DSM 6083 gv 6 P. stutzeri DSM 50238 gv 7T P. stutzeri JM300 gv 8? P. putida ATCC 12633T P. putida DSM 3601 , " T 1' P. stutzeri CCUG 11256 gvl t 0 ATCC 55595,DSM 7136, aquifer isolate, carbon tetrachloride degrader ATCC 17588, Stanier strain 22], clinical isolate Stanier strain 224, clinical isolate ATCC l 1607, clinical isolate DSM 6084, marine isolate, naphthalene degrader DSM 6082, wastewater isolate waste water isolate, naphtalene degrader ATCC 17832, Stanier strain 419, soil isolate DSM 10701, soil isolate DSM 50202, lactate enrichment tomato plant isolate, Produces 2,6-bis(pyridine thiocarboxylate) (Criddle et al., 1990) (Stanier et al., 1966) (Stanier et al., 1966) (Van Niel & Allen, 1952) (Rossello et al., 1991) (Rossello et al., 1991) (Bennasar et al., 1996; Rossello et al., 1991) (Stanier et al., 1966) (Carlson & lngraham, I983) (Skennan et al., 1980; Stanier et al., 1966) (Ockels et al., 1978) ATCC, American Type Culture Collection, Rockville, MD, USA; CCM, Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia; CCUG, Culture Collection University of Goteborg, Sweden; CECT, Coleccion Espafiola de Cultivos Tipo, Valencia, Spain; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulteren, Braunschweig, Germany; LMG, Laboratorium Microbiologie Rijksuniversiteit, Gent, Germany I type strain of genomovar T type strain 113 Determination of carbon source utilization: Three individual colonies of each strain were picked from fresh nutrient agar plates and used to inoculate 50 ml erlenmeryer flasks containing 10 ml of TSB. The cultures were grown at 30 °C and 150 rpm. for 20 h. Cultures were centrifuged at 2,500 X g for 10 min, washed twice in PBS and normalized to an optical density of 0-195 — 0-205 in a spectrophotomer at 600 nm. 150 pl aliquots were added to BIOLOG® GN2 plates (Biolog) and the plates were incubated at 30 0C for 48 h. Substrate consumption was followed by reading the plates in a microtiterplate reader at an optical density of 590 nm after 4, 24 and 48 h of incubation. Antibiotic susceptibility test: Triplicate 20 h cultures in TSB were used to inoculate 150 mm x 15 min nutrient agar plates, bi-directionally, using a sterile cotton swab. Filter disks (0-7 cm diameter) containing antibiotics were placed on the surface of the plates prior to incubation at 30 °C for 24 h. The following antibiotics were used for the susceptibility test: 10 pg ampicillin ml—l, 10 units (U) penicillin G ml—l, 20 pg amoxillin ml.l and 10 pg ml—l, streptomycin 10 pg ml—l, clavulanic acid ml—', 30 pg erythromycin ml—l, 30 pg tetracycline ml_l, 1°25 pg trimethropin ml_I and 23°75 pg sulfamethoxazole ml 4, 1 pg oxacillin ml _1, 30 pg cephalothin ml 4, 30 pg vancomycin ml 4, 2 pg clindamycin ml_], and 300 U polymyxin B ml—l. Fatty acid analysis: Bacterial isolates were grown overnight on tryptic soy agar at 28 °C and harvested with a sterile loop. Saponification, methylation and extraction were performed by Microbial ID using a previously described procedure (Sasser, 1990a). 114 Methylated fatty acids were separated by a gas chromatograph equipped with a flame ionization detector, in a 25 m X 0.2 mm phenyl methyl silicone fused silica capillary column using hydrogen as the carrier gas and nitrogen as the makeup gas. Numerical comparisons of the fatty acid profiles were carried out as previously described (Sasser, 1990b) DNA fingerprinting: DNA to be used for fingerprinting was isolated using the GenomicPrep cells and tissue DNA isolation kit (Amersham Pharmacia Biotech) following the manufacturer’s recommendations. PCR for REP, BOX and ERIC fingerprinting were performed in 25 pl reactions containing 50 ng DNA, 2 pmole each primer, 1°25 pmole each dNTP, 2 U Taq DNA polymerase, 20% v/v Gitschier buffer (Kogan et al., 1987), 0°8% BSA, 10% v/v dimethyl sulfoxide (DMSO), and 4% v/v Tween 20. Primers REPlR-I and REP2-I (V ersalovic et al., 1991) were used for REP fingerprinting, primer BOXAIR (Versalovic et al., 1994) while primers ERICIR and ERIC2 (V ersalovic et al., 1991) were used for ERIC fingerprinting. PCR cycles for REP, ERIC and BOX PCR were as follows: initial denaturation at 95 °C for 7 min, 35 cycles consisting of 94 °C for 1 min, 44 °C for 1 min for REP (52 °C for ERIC or 53 °C for BOX) and 65 °C for 8 min. A single final extension step was performed at 65 0C for 15 min in order to assure chain termination. PCR products were separated by electrophoresis in 2% agarose (w/v) and stained with ethidium bromide. The GelCompar image analysis system (Applied Maths) was used to calculate similarities between all fingerprinting profiles. Dendrograms of relationships were deduced by the unweighted pair-group mean analysis (UPGMA) cluster algorithm (V auterin & Vauterin, 1992). 115 DNA-DN A hybridizations: Nucleic acids from bacterial strains were isolated following the method of Marmur (1961). DNA-DNA hybridizations were performed, using a modification of the hydroxyapatite method described previously (Ziemke et al., 1998). Reference DNA’s were double-labeled with DIG-1 l-dUTP and biotin-16-dUTP using the nick-translation kit as recommended by the manufacturer (Boehringer Mannheim). 16S rRNA cloning, sequencing and analysis: The 168 rRNA gene was PCR amplified with modified universal eubacterial primers fDl (5’-CCA TCG ATG TCG ACA GAG TTT GAT CCT GGC TCA G—3’) and rPl (5’-GAC TAG TGG ATC CAC GGT TAC CTT GTT ACG ACT T-3’) (Zhou et al., 1995) as previously described (Weisburg et al., 1991). The sequence of the 16S rRNA gene of strain KC was submitted to GenBank under the accession numbers AF 67960 and AF 063219. Multiple sequence alignment was done with the PILEUP program in the Genetics Computer Group software package (Devereaux et al., 1984). The alignment was edited for the appropriate analysis by using the SUBALIGN and GDE programs from the Ribosomal Database Project (Maidak et al., 1999). The phylogenetic analyses were performed in the DNA distance program ARB using Neighbor-Joining with Felsenstein correction (Stunk et al., 2000). Amplification of the 16S rRNA gene with primers specific for P. stutzeri strains: A 1160 bp fragment of the 16S rRNA gene of strain KC and several P. stutzeri strains was amplified with the P. stutzeri- specific PCR primers fpslSO (5’-GTG GGG GAC AAC GTT TC-3’) and rp5127l (5’-CTA CGA TCG GTT TTA TGG-3’) as previously 116 described (Bennasar eta1., 1998a). The PCR products were purified using the WizardTM PCR preps DNA purification kit as recommended by the manufacturer (Promega). 10 pl of purified PCR products were restricted with 10 U of the endonuclease BamHI, for 1 h at 37 °C in a total volume of 30 pl. The restricted fragments were separated by electrophoresis in a 1.5 % (w/v) agarose gel and stained with ethidium bromide. Amplification and sequencing of the 168-238 internally transcribed spacer region (ITSl): ITS] was amplified by PCR with oligonucleotide primers 16F945 and 23R458 (Lane et al., 1985) designed to anneal to conserved positions in the 3’ and 5’ regions of the bacterial l6S rRNA and 23S rRNA genes, respectively. Primers 16F945 (5’-GGG CCC GCA CAA GCG GTG G-3’) and 23R458 (5’-CTT TCC CTC ACG GTA C-3’) targeted positions 927-945 of the Escherichia coli 16S rRNA gene (Brosius et al., 1978) and positions 458-473 of the E. coli 23S rRNA gene (Brosius et al., 1980), respectively. PCR amplification cycles were performed as per Guasp et al. (2000). The sequence of the ITS] region was determined by Taq cycle sequencing using fluorescent dye-labeled dideoxynucleotides. The primers used for sequencing were rrnl6S (5’-GAA GTC GTA ACA AGG-3’) and rrn23S (5’-CAA GGC ATC CAC C-3’) (Jensen et al., 1993) designed to anneal to conserved positions in the 3’ and 5’ regions of the bacterial 16S rRNA and 23S rRNA genes, respectively. Primers rrnl6S (5’-GAA GTC GTA ACA AGG-3’) and rrn23S (5’-CAA GGC ATC CAC CTG-3’) targeted positions 1491-1505 of the Escherichia coli 16S rRNA gene (Brosius et al., 1978) and positions 21-35 of the E. coli 23S rRNA gene (Brosius et al., 1980), respectively. The ITS] sequence of strain KC was submitted to GenBank under accession number (to be submitted). ITSl 117 sequences were aligned using the computer program CLUSTAL W (Thompson et al., 1994), with a final manual adjustment (Rabaut, 1996). Evolutionary distances were calculated from pairwise sequence similarities (Jukes & Cantor, 1969) and estimations of relationships were generated using the Fitch program within the Phylogeny Inference Package (PHYLIP) (Felsenstein, 1989). gyrB-based PCR amplification: PCR amplification using primers specific for the Pseudomonas putida gyrB gene was performed as previously described (Yarnamoto & Harayama, 1995). 118 RESULTS Substrate utilization, antibiotic resistance and fatty acid analysis. Fourty of the 95 carbon sources tested were used differently by the bacterial strains, as seen in Table A2. This result is consistent with previous reports that indicate the high degree of physiological heterogeneity within P. stutzeri strains (Palleroni et al., 1970; Rossello et al., 1994a; Stanier et al., 1966). Strain KC is similar to P. stutzeri strains in its ability to grow on dextrin, glycogen, maltose and a-ketobutyric acid and its incapacity of utilizing D-arabinose, D-sorbitol and phenyl ethylamine, three carbon sources used only by the P. putida strains. Strain KC was the only organism, of the eleven tested, capable of growing on m-inositol. Even though significant differences in carbon source utilization were observed, the behavior of strain KC and P. stutzeri strains in the antibiotic resistance test and the fatty acid analysis was more homogeneous. As seen in Table A3, the antibiotic susceptibility results obtained for strain KC coincided with the consensus for the majority of P. stutzeri strains and diverged from the P. putida pattern for any of the 12 antibiotic tested, except trimethroprin/sulfamethoxazole, an antibiotic combination that showed variability among the strains tested. 119 * Pseudomonas stutzeri strains tested: CCUG 1126T (gv l), ATCC 17591 (gv 2), DSM 50227 (gv 3), 19SMN4 (gv 4), DNSP21 (gv 5), DSM 50238 (gv 7), JM300 (gv 8): Pseudomonas balearica DSM 6083T (gv 6); Pseudomonas putida strain tested: ATCC 12633T (9), DSM 3601 (10) I +, positive for substrate utilization; —, negative for substrate utilization; w, weak positive 120 Table A.2 Substrate utilization by strain KC and various Pseudomonas stutzeri, P. balearica and P. putida strains * * BIOLOG Pseudomonas stutzeri strains P. balearica P. putida.” carbon source KC 1 2 3 4 5 7 8 6 9 10 dextrin t + + + + + + + t + + + + + + glycogen + + + + + L-arabinose _ _ _ _ _ _ D- arabitol D-fructose m-inositol maltose D-mannitol I | I | ++| +++++| €+ .4. + +++| + ++| D-mannose D-sorbitol _ trehalose formic acid a-hydroxybutiric acid y-hydroxybutiric acid p-hydroxyphenylacetic acid itaconic acid a-keto butyric acid a-keto valeric acid propionic acid D-saccharic acid sabacic acid ' succinamic acid glucuronarnide alaninamide L-alanyl-glycine L-histidine _ hydroxy-L-proline L-leucine L-omithine L-phenylalanine L-pyroglutamic acid D-serine L-serine L-threonine D,L-camitine y-amino butyric acid phenyl ethylamine _ putrescine w 2-amino ethanol _ 2,3butanediol _ _ _ _ _ __ _ _ I I I II II II II II +| | |+i+| +| ass-2| 2:2] + ++£| ssl ++| + ++| i ++££+£I £+| +++| ++| || ++£++| +++| +++| |£+| €+| +++| £+| +| ++++| I€I I++I | s+s| | I ++| ++| ++| i+++| +| I+I |++++++| I+€|I |+sss+s| |+€€++| I |+++S£| |+£€| ++++| I|++| +£+S£S++++£++++£| +I + +| +| I I I +| +| +| ++++| Isl +ill | I 2| ++| +| €£++| 2s| sssl ++| +| I | | + I + + +€| 2I I S ++| +€| SI +++++++++| i l | 121 Table A.3 Antibiotic susceptibility test for strain KC and various Pseudomonas stutzeri, P. balearica and P. putida strains Antibiotic Pseudomonas stutzeri strain; P. balearicaI 1" putida KC 1 2 3 4 5 7 8 6 ATCCT 12633T ampicillin MS i MS MS MS MS 5 1 MS MS MS R I penicillin G R R R R R MS R R R R amoxicillin / S S S S S S S S MS R clavulanic acid streptomycin MS MS MS MS MS S MS MS MS R erythromycin S MS S S S S MS MS MS R tetracycline S S S S S S S S MS MS trimethroprin / MS R R R MS S MS 8 R R sulfamethoxazole I Pseudomonas stutzeri strains tested: CCUG1126 (gv , type strain), ATCC17591 (gv 2), DSM50227 (gv 3), 19SMN4 (gv 4) DNSP21 (gv 5), DSM50238 (gv 7), JM300 (gv 8); Pseudomonas balearica DSM6083 (gv 6, type strain) I ATCC, American Type Culture Collection, Rockville, MD, USA I R, resistant, diameter of inhibition < 1 cm; MS, moderate sensitiveness, diameter of inhibition 1°0 - l°7 cm; S, sensitive, diameter of inhibition > 1°7 cm T type strain The fatty acid distribution of strain KC is very similar in composition and abundance to the profiles observed for P. stutzeri and P. balearica strains (Table A4). This result is congruent with previous observations (Rossello et al., 1994a; Stead, 1992; Veys et al., 1989) indicating that a differentiation of P. stutzeri strains on the basis of fatty acid patterns is not possible. Even though the fatty acid profile of P. putida strains were similar to the profiles of strain KC and the P. stutzeri strains, a significant difference in the abundance of two fatty acids was observed. The abundance of fatty acids 16:0 and 18:1 m7c were 14 — 18% and 27 — 40%, respectively, for all P. stutzeri strains (including 122 strain KC) and P. balearica. The abundance of these two fatty acids was 25% and 15%, respectively, in the case of the two P. putida strains tested. Table A.4 Cellular fatty acid composition of strain KC and several Pseudomonas stutzeri, P. balearica and P. putida strains % of total fatty acids Fatty acid Pseudomonas stutzeri strain; KC gvl gv2 gv3 gv4 gvS gv7 gv8 gv6 9 10 10:0 ND 1 0-25 0°18 0°28 0°18 027 ND ND 029 ND 025 10:0 30H 3°36 3°70 2°42 2°50 3°45 3°65 2°86 3°93 3°46 3°52 3°89 11:0 iso 30H ND ND 0-09 ND 010 ND ND ND ND ND 020 12:0 8°12 11-5 7-43 10-25 7-44 11° 1 3 10°26 11°59 10-29 3-24 2°67 12:0 2011 ND 0°16 0°10 ND 0°11 ND ND ND ND 6°16 7-03 12:0 3011 3°90 4°13 2°42 2°89 2°99 3°52 3°30 3°31 3°53 4°64 4°82 12:1 3011 ND ND ND ND ND ND ND ND ND ND 0°21 13:0 iso ND ND 0°11 ND 0°08 ND ND ND ND ND ND 14:0 0-79 1-25 0°61 1°18 0-90 1-30 1°06 1-71 0°87 0-44 0-34 15:0 ND ND ND ND ND ND ND ND ND ND 0°25 15isoZOH/ 33°56 32°56 28°33 38°33 32°28 37°33 35°49 34°97 30°44 38°10 31°62 16:1w7c 16:0 14°73 14°69 16°84 16°29 17°84 13°95 17°81 15°62 16°61 25°20 26°56 17:0 iso ND ND 0°49 0°41 0°43 ND ND ND 1°28 ND 0°81 17:0 cyclo ND ND ND 0°35 0°20 ND ND 0°97 1°00 3°11 4°29 18:1 007C 35°28 31°57 39°56 27°23 33°18 28°85 29°21 27°13 30°79 15°59 16°48 18:0 ND ND 0°55 ND 0°36 ND ND 0°78 ND ND 0°37 19:0 cyclo ND ND 0°15 ND 0°14 ND ND ND 0°51 ND ND (08c . Pseudomonas stutzeri strains tested: CCUG 1126 (gv 1, type strain), ATCC 17591 (gv 2), DSM 50227 (gv 3), 19SMN4 (gv 4) DNSP21 (gv 5), DSM 50238 (gv 7), JM300 (gv 8); Pseudomonas balearica DSM 6083 (gv 6, type strain); Pseudomonas putida ATCC 12633 (9, type strain), Pseudomonas putida DSM 3601 (10) I ND, not detected 123 DNA-DNA similarity studies. Strain KC did not show similarity values higher than 70% with any of the type strains of the 7 genomovars of P. stutzeri described so far (Table A.5). DNA-DNA similarity values are usually higher than 70% for members of the same gv, between 40 and 60% for members of different gv and under 20% when a P. stutzeri strain is compared with other Pseudomonas species (Rossello et al., 1991). DNA hybridizations between strain KC and most P. stutzeri genomovars were in the 40 to 60% range, as expected for inter-genomovar hybridizations, and low similarity indices were observed when KC was hybridized to P. balearica, P. putida and P. aeruginosa type strains. Table A.5 DNA — DNA similarity results for strain KC and several Pseudomonas strains Strain % DNA — DNA similarity KC 100 p. stutzeri ATCC‘ 17589 : Essa m a x25 use 28 areas a a :Ndmzo eons: m u aomoN tease. m .o ”Rm: 8.? toes... m a ”ma: 8.? tease. m .o ”an: 00 H4 toast. N .n Somt 00 ._.< tomcat. .& .m ”Ewing 05 .80 com: 825 magnum 9.36:8 BE. .mfimbm .2332 uoeosohzmmk was on one do menopause 2% one due .xom Bassoon .5 morass <28: and so Ban seasoned Swish 3. any... 126 2033 N >0 >0 >0 >0 >0 >0 >0 >0 >0 >0 >0 >0 >0 >0 >0 >0 a . ._: E .: z :3. : |Ill_ Q__,;,__; I: I... o :33: Z. a E: _:: c E . y. z. 0.; 0 : ii. .0. .. ._ _ 5.77 1:; .__ .w. ._ in T M iii: ;:_. .: .1: l m. _ _W _ , . 0:: .~ 2...: : _.: 5 gig U 13...: 3 .: . .Igai of . 5:; 31:. u 0:: . 2 .. if m «T L::: *::m,_ u 1. :5: 3;: U :. .2. .. 2 g a : E. : m :. 0:. . T : 9::L... » if: .:_:_ _ _ _ _ _ _ __________:*__Jtfi QM Nd M." md o.N©~.o Wm 2: on o0 ov cm 055 mam x9. 52: £53m E 127 16S rDNA, gyrB PCR and ITSl region sequencing. l6S rDNA sequence comparisons with sequences available in the Ribosomal Database Project demonstrate that the 16S rDNA gene of strain KC clustered with P. stutzeri in the Neighbor-Joining analysis, with a bootstrap confidence of 86% (Figure A.2). When the sequence was compared to 16S rDNA genes from strains assigned to different P. stutzeri genomovars, strain KC clustered within the P. stutzeri phylogenetic branch, being gv 3 the closest group with 9903 8%, followed by gv 4 with a 99°24% sequence identity. The 16S rDNA of strain KC can be amplified using the P. stutzeri specific primers fp5158 and rp31271 (Bennasar et al., 1998a). The 16S rDNA of some P. putida strains can also be amplified with these primers but the 1,160 bp PCR product cannot be restricted by BamHI to yield a 645 bp fragment and 465 bp fragment (Bennasar et al., 1998a). As seen on Figure A.3, only P. stutzeri strains and strain KC had PCR products that could be digested with BamHI. No PCR products were obtained from strain KC with the gyrB primers specific to P. putida, indicating that strain KC is not likely to be a P. putida strain. 128 33332 2: 53» 3583 3:285 05 co 83:53 3:090:00 92503 05 3863 mowficoocom 2F 3%on 838mm RESBE 2: E 05356 328308 088 8 @2888 mm 0M E83 .«o 26w <29 mg 05 can? 3:850 8b 308330 nutmwoom N.< unsure 129 #00: 002 “.533 35.6mm 38:13 353% if it. 08:8 252236 'IL «.3 Hcom: 00.5. fictbofimc: bagosmtw If #80: 00.5w aflmmnm :nzxxwgogesscw L $3 P9.5— 00 H< uncsmfmu “dramasmi ommcm 00 H< 305%:ch 33:53me $3 2d $09 {one MM $39055 munoficmamé *3 0 >0 8va 8.2633 8=o5on=mmt 8m< ~305ka anesehzmmk Ty: m mums?” wanesomamnm m-w.: :5 «0 35:5 .Szesehzmut ~ 0 $5an 3550383» #32 $302 mom Qmunmgkgxefiohzmam xiv _ >w Fawn: 00H< .2333. uuxofisgmnk _ >w _RR 82 ENE: azosogam 8. £25 v >w vZEmm: tuna: wagfiohnmnm N >m an: 00H< .2935. grcfiofiami N >m wow: 00 .5. =omoN .2333. aurosogmnk .33 ll :88; ozoauwo—Ea .585“. autosenzui 130 Figure A.3 BamHI restriction digests of the 168 rRNA gene amplified by PCR using the Pseudomonas stutzeri- specific primers fpsl 58 and rp51271. KC, Pseudomonas sp. strain KC; M, 100 bp DNA ladder; U, undigested sample; D, BamHI restricted sample; 1160, length of undigested fragment (bp); 465 and 695, length of restricted fragments (bp); 1, P. stutzeri CCUG 11256 (type, gv 1); 2, P. stuzeri ATCC 17591 (gv 2); 3, P. stutzeri DSM 30227 (gv 3); 4, P. stutzeri 19SMN4 (gv 4); 5, P. stutzeri DNSP21 (gv 5); 6, P. stutzeri DSM50238 (gv 7); 7, P. balearica DSM 6083 (type, gv 6); 8, P. putida ATCC 12633 (type); 9, P. aeruginosa ATCC 10145 (type); 10, E. coli K-12 ATCC 10798 131 ITS] sequence analysis results agree with 16S rDNA sequence comparisons. Once again, strain KC clustered with the P. stutzeri phylogenetic group, as seen in Figure A.4. ITSl sequences are identical within all the strains of a P. stutzeri genomovar, and deletions or insertions in this portion of DNA can be used as a taxonomic tool to differentiate strains at the gv level (Guasp et al., 2000). Strain KC is closely linked to the gv 3 — gv 4 cluster. When the ITSl sequences of strain KC and strain 19SMN4 (gv 4) are compared, differences are only observed towards the end of the sequence. The ITS] region of strain KC has an 11 bp and a 2 bp insertion separated by 4 bases. Nine mismatches are also observed in the vicinity of these insertions. The sequence differences observed in the ITSI region indicate that strain KC could be a new genomovar of P. stutzeri. 132 Figure A.4 Dendrogram depicting phylogenetic relationships among strain KC, several P. stutzeri strains and type strains of other Pseudomonas species, as estimated by comparing the ITSl sequence. Scale bar 0°] nucleotide substitutions per nucleotide position. The EMBL accession numbers of the sequences used for this analysis are provided in parenthesis: P. putida (need #), P. cichorii (AJ279242), P. syringae (D863 56), P. agarici (AJ279243), P. corrugata (need #), P. chlororaphis (AJ279240), P. fragi (AJ279241), P. tolaasii (M279244), P. fluorescens (need #), P. pseudoalcaligenes (AJ27945), P. mendocina (L28159), P. stutzeri DSM 50238 (AJSl909), P. alcaligenes (need #), P. aeruginosa (L28148), P. stutzeri JM300 (AJ390581), P. stutzeri ATCC 17591 (A125190l), P. stutzeri DSM 50227 (AJ251903), Pseudomonas sp. strain KC (to be submitted), P. stutzeri 19SMN4 (AJ241906), P. stutzeri CCUG 11256 (AJ251910), P. stutzeri DNSP21 (AJ2 l 908). 133 P. putida P. cichorii P. syringae P. agarid P. corrugata P. chlororaphis P. fragi P. talaasii P. fluorescens P. pseudoalcaligenes I L— P. merulocina P. stutzeri DSM50238 gv 7 i l P. balearica DSM 6083 gv 6 Ralcaligena P. aeruginosa PstutzeriJM300gv8 P. stutzeri ATCC17591 gv 2 P. stutzeri DSM50227 gv 3 KC P. stutzeri 19SMN4 gv 4 I. P. stutzeri CCUG]1256 gv 1 L P. stutzeri DNSP21 gv 5 0-1 134 DISCUSSION Based on the overall evidence from phylogenetic and genetic studies, we conclude that strain KC should be classified as a member of a new genomovar within the species P. stutzeri. Pseudomonas stutzeri is a motile, non-fluorescent, denitrifying, Gram-negative, rod-shaped bacterium that is widely distributed in nature. It was first described in 1895 (Burri & Stutzer, 1895) as Bacillus denitrificans II and re-classified fifty-seven years later as P. stutzeri (Van Niel & Allen, 1952). Members of the newly described species were distinguishable from other non-fluorescent pseudomonas by the dry, wrinkled colonies of fresh isolates and the ability to use maltose and starch as sole carbon sources, as well as the ability to produce large amounts of molecular nitrogen from nitrate. Many strains of this species are of special interest for their ability to degrade environmental pollutants (Baggi et al., 1987; Rosello-Mora et al., 1994b). It has not been possible to assign each of the P. stutzeri genomovars to new species due to their highly diverse phenotypes which do not allow the identification of phenotypic traits unique to each genomovar (Rossello et al., 1991; Rossello et al., 1994a). To date, only one of the P. stutzeri genomovars has been re-classified as a new species based on distinct phenotypic characteristics (Bennasar et al., 1996). Strain KC has the unique ability to produce PDTC enabling carbon tetrachloride degradation, and it can grow on m-inositol, unlike any P. stutzeri strain studied in this work. On the other hand, other phenotypic characteristics like resistance to antibiotics and the composition of cellular fatty acids, correlate with the patterns observed in other P. stutzeri strains. 135 DNA similarity, as measured by DNA-DNA re-association studies (Johnson, 1973; Johnson & Palleroni, 1989) has been the standard genotypic method for assigning a strain to a given genomovar (Rossello et al., 1991). When strain KC is compared to P. stutzeri strains, the similarity indices are below the 70% threshold; the value used classically to differentiate between species and members of the same genomovar. On the other hand, the similarity indices remained between the 40 to 60% observed when members of different genomovars are compared (Rossello et al., 1991). It should be noted that unlike many species that show homogeneity at the genotypic levels, P. stutzeri have proven to be highly diverse. Comparison of genomic maps for different P. stutzeri strains revealed a high degree of genomic plasticity as chromosomal re-arrangements can occur without apparent consequences in strain fitness (Ginard et al., 1997). This genomic plasticity may have played an important role in P. stutzeri’s ability to colonize and persist in diverse environments. Of interest in this regard is the recent report of Lewis et al. (2000) indicating that strain KC can spontaneously lose large chromosomal fragments (~ 170 kb) without loss of viability. This trait is consistent with the characteristics of the P. stutzeri species. When smaller pieces of DNA with phylogenetic relevance like 16S rDNA, ITSl and gyrB are analyzed to deduce phylogenetic relationships, strain KC clusters in the P. stutzeri phylogenetic branch. Strain KC exhibited over a 99% similarity index with the 16S rDNa gene of members of gv 3 and 4. 16S rRNA gene sequence comparisons support the natural relationship among the genomovars and have further sustained the genomovar concept because similarities of 16S rRNA genes is 99-9 to 100% for 136 members of the same genomovar and 98-0 to 99-7% for members of different genomovars (Bennasar et al., 1996). Strain KC shows more than 98% 16S rDNA sequence similarity with the type strains of P. stutzeri genomovars and similarities of 96%, with P. putida, P. aeruginosa and P. balearica. These results are consistent with previous observations reporting 16S rDNA similarity indices of less than 97% for strains of different species (Stackebrant & Goebel, 1994). PCR amplification of gyrB also excluded the possibility that strain KC belongs to the species P. putida because the gyrB gene of strain KC did not amplify with P. putida-specific primers. The intragenic, 16S-23S internally-transcribed spacer region (lTSl) has also been used as a tool to confirm genomovar assignments (Guasp et al., 2000). The sequence of ITSl is assumed to be less susceptible to selective pressures, due to its non-coding fimction, and should have accumulated a higher percentage of mutations than the rRNA genes (Tyrrell et al., 1997). Comparison of ITS] sequences indicate that the considerable variation in length and sequence make these regions good candidates for discriminating among closely related taxa (Giirtler & Stanisich, 1996). Strain KC clustered with P. stutzeri strains (Figure A.4) showing more than 80% identity at the sequence level with P. stutzeri strains and less than 70% sequence identity with other closely related species such as P. putida, P. aeruginosa and P. mendocina. The results reported in this publication demonstrate that strain KC is a member of the P. stutzeri species. Its phenotype fits the description of the overall phenotype of the species, except for the ability to grow on m-inositol and the capability to degrade carbon 137 tetrachloride. We therefore propose that strain KC be classified the sole representative of a new genomovar, genomovar 9, following the enumeration of Rossello et al. (1991, 1996). The isolation and characterization of new strains belonging to the same gv as strain KC may help to clarify if the unique phenotypic characteristics of strain KC are sufficient to propose its reclassification as a new species within the genus Pseudomonas; or if these attributes simply reflect the unusual physiological traits within the diverse P. sutzeri species. 138 ACKNOWLEDGEMENTS The authors gratefully acknowledge Mrs. Carmen M. Medina-Ferret for technical assistance in DNA fingerprinting analysis. This work was supported, in part, by grants from the National Science Foundation Center for Microbial Ecology (BIR-9120006) and by the NIEHS Superfund Basic Research Program of the Institute for Environmental Toxicology (ESO4911) at Michigan State University. 139 REFERENCES Baggi, G., Barbieri, P., Galli, E. & Tollari, S. (1987). Isolation of a Pseudomonas stutzeri strain that degrades o-xylanes. Appl Environ Microbial 53, 2129-2132. Bennasar, A., Rossello-Mora, R. A., Lalucat, J. & Moore, E. R. (1996). 16S rRNA gene sequence analysis relative to genomovars of Pseudomonas stutzeri and proposal of Pseudomonas balearica sp. nov. Int J Syst Bacterial 46, 200-205. Bennasar, A., Guasp, C., Tesar, M. & Lalucat, J. (1998a). Genetic relationships among Pseudomonas stutzeri strains based on molecular typing methods. J Appl Microbial 85, 643-656. Bennasar, A., Guasp, C. & Lalucat, J. (19981)). Molecular methods for the detection and identification of Pseudomonas stutzeri in pure culture and environmental samples. Micrab Ecol 35, 22-33. Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978). Complete nucloetide sequence of a 16S ribosomal RNA gene form Escherichia coli. Proc Natl Acad Sci USA 75, 4801-4805. Brosius, J., Dull, T. J. & Noller, H. F. (1980). Complete nucleotide sequence of a 23S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci USA 77, 201- 204. Burri, R. & Stutzer, A. (1895). Ueber Nitrat zerstorende Bakterien und den durch dieselben bedingten Stickstoffverlust. Zbl Bakt 11, 257-265, 350-364, 392-398, 422-432. 140 Carlson, C. & lngraham, J. (1983). Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa and Paracoccus denitrificans. Appl Environ Microbial 45, 1247-1253. Criddle, C. S., DeWitt, J. T., Grbic-Galic, D. & McCarthy, P. L. (1990). Transfromation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions. Appl Environ Microbial 56, 3240-3246. Devereaux, J., Haeberli, P. & Smithies, O. (1984). A comparative set of sequence analysis programs for the VAX. Nucleic Acid Res 12, 387-395. Dybas, M. J., Tatara, G. M. & Criddle, C. S. (1995). Localization and chracterization of the carbon tetrachloride transformation activity of Pseudomonas sp. strain KC. Appl Envrion Microbial 61, 758—762. Emerson, D. (1999). Complex pattern formation by Pseudomonas strain KC in response to nitrate and nitrite. Microbiology 145, 633-641. Felsenstein, J. (1989). PHYLIP-phylogeny inference package (version 3.2). Cladistics 5, 164-166. Ginard, M., Lalucat, J., Tiimmler, B. & Romling, U. (1997). Genome organization of Pseudomonas stutzeri and resulting taxonomic and evolutionary considerations. Int J Syst Bacteriol 47, 132-143. Guasp, C., Moore, E. R. B., Lalucat, J. & Bennasar, A. (2000). Utility of intemally- transcribed 16S-23S rDNA spacer regions for the definition of Pseudomonas stutzeri genomovars and other Pseudomonas species. Int J Sys Bacterial 50, (part4). 141 Giirtler, V. & Stanisich, V. A. (1996). New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. Microbiology 142, 3-16. Hyndman, D. W., Dybas, M. J., Forney, L., Heine, R., Mayotte, T., Phanikumar, M. S., Tatara, G., Tiedje, J., Voice, T., Wallace, R., Wiggert, D., Zhao, X. & Criddle, C. S. (2000). Hydraulic Characterization and design of a full scale biocurtain. Ground Water In Press. Jensen, M. A., Webster, J. A. & Straus, N. (1993). Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl Environ Microbial 59, 945-952. Johnson, J. L. (1973). Use of nucleic acid homologies in the taxonomy of anaerobic bacteria. Int J Syst Bacterial 23, 308-315. Johnson, J. L. & Palleroni, N. J. (1989). Deoxiribonucleic acid similarities among Pseudomonas species. Int J Sys Bacterial 39, 230-235. Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian protein metabolism (ed. H. N. Munro), pp. 21-132. Academic Press, New York. Kogan, S., Doherty, M. & Gitschier, J. (1987). An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl J Med 317, 985-990. Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. & Pace, N. R. (1985). Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA 82, 6955-6959. Lee, C.-H., Lewis, T. A., Paszczynski, A. & Crawford, R. L. (1999). Identification of an extracellular catalyst of carbon tetrachloride dehalogenation from 142 Pseudomonas stutzeri strain KC as pyridine-2,6-bis(thiocarboxylate). Biochem Biophys Res Commun 261, 562-566. Lewis, T. A. & Crawford, R. L. (1993). Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC. Appl Environ Microbiol 59, 1635-1641. Lewis, T. A., Cortese, M. S., Sebat, J. L., Green, T. L. & Crawford, R. L. (2000). A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCI4 - dechlorinating agent pyridine-2,6-bis(thiocarboxilic acid). Environ Microbial In Press. Maidak, B. L., Cole, J. R., Parker, C. T., Garrity, G. M., Larsen, N., Li, B., Lilburn, T. G., McCaughey, M. J., Olsen, G. J., Overbeek, R., Pramanik, S., Schmidt, T. M., Tiedje, J. M. & Woese, C. R. (1999). A new version of RDP (Ribosomal Database Project). Nucleic Acids Res 27, 171 -173. Marmur, J. (1961). A procedure for the isolation of DNA from microorganisms. J Mol Biol 3, 208-218. Ockels, W., Romer, A. & Budzeikiewicz, H. (1978). An Fe(II) complex of pyridine- 2,6-di-(monothiocarboxilic acid) - a novel bacterial metabolic product. Tetrahedron Lett 36, 3341-3342. Palleroni, N., Doudoroff, M., Stanier, R., Solanes, R. & Mandel, M. (1970). Taxonomy of the aerobic pseudomonas: the properties of the Pseudomonas stutzeri group. J Gen Microbial 60, 215-31. Rabaut, A. (1996). Se-Al: sequence alignment editor. Version 1.0 alpha 1. Department afZaalogy. University of Oxford South Parks Roads. Oxford 0X1 4JD. UK. 143 Rossello, R. A., Garcia-Valdés, E., Lalucat, J. & Ursing, J. (1991). Genotypic and Phenotyic Diversity of Pseudomonas stutzeri. Syst Appl Microbial 14, 150-157. Rossello, R. A., Lalucat, J., Dott, W. & Kfimpfer, P. (1994a). Biochemical and chemotaxomic characterization of Pseudomonas stutzeri genomovars. J Appl Bacterial 76, 226-233. Rosella-Mora, R. A., Lalucat, J. & Garcia-Valdés, E. (1994b). Comparative biochemical and genetic analysis of naphthalene degradation among Pseudomonas stutzeri strains. Appl Environ Microbial 60, 966-972. Rossello-Mora, R. A., Lalucat, J., Timmis, K. N. & Moore, E. R. B. (1996). Strain J M300 represents a new genomovar within Pseudomonas stutzeri. Syst Appl Microbial 19, 596-599. Sambrook, J., F ritsch, E. L. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sasser, M. (1990a). Technical note # 101: Identification of bacteria by gas chromatography of cellular fatty acids. MIDI, Inc, Newark, DE . Sasser, M. (1990b). Technical note # 102: "Tracking" a strain using the Sherlock Microbial Identification System. MIDI, Inc., Newark, DE . Sepfilveda-Torres, L. del C., Rajendran, N., Dybas, M. J. & Criddle, C. S. (1999). Generation and initial characterization of Pseudomonas stutzeri KC mutants with Impaired ability to degrade carbon tetrachloride. Archiv Microbial 171, 424-429. Skerman, V., McGowan, V. & Sneath, P. (1980). Approved list of bacterial names. Int J Syst Bacterial 30, 225-420. 144 Stackebrant, E. & Goebel, B. (1994). Taxonimic note: a place for DNA-DNA reassociation and 16S sequence analysis in the present species definition in bacteriology. Int J Syst Bacterial 44, 846-849. Stanier, R., Palleroni, N. & Doudoroff, M. (1966). The aerobic pseudomonas: a taxonomic study. J Gen Microbial 43, 159-271. Stead, D. (1992). Grouping of plant-pathogenic and some other Pseudomonas spp. by using cellular fatty acids profiles. Int J Sys Bacterial 42, 281-295. Stunk, 0., Gross, 0., Reichel, B., May, M., Hermann, S., Struckmann, N., Nonhoff, B., Lenke, M., Vilbig, A., Ludwig, T., Bode, A., Schleifer, K. H. & Luwig, W. (2000). ARB: a sofware environment for sequence data. Nucleic Acids Res In Press. Tatara, G. M., Dybas, M. J. & Criddle, C. S. (1995). Effect of medium and trace metals on kinetics of carbon tetrachloride transformation by Pseudomonas stutzeri KC. Appl Environ Microbiol 59, 2126-2131. Thompson, J. D., Higgins, D. G. & Gibson, T. G. (1994). Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22, 4673-4680. Tyrrell, G. J., Bethune, R. N., Willey, B. & Low, D. E. (1997). Species identification of enterococci via intergenic ribosomal PCR. J Clin Microbial 35, 1054-1060. Van Niel, C. & Allen, M. (1952). A note on Pseudomonas stutzeri. J Bacterial 64, 413- 422. 145 Vauterin, L. & Vauterin, P. (1992). Computer aided comparison of electrophoresis patterns for grouping and identification of microorganisms. European Microbiology 1, 37-41. Versalovic, J., Koeuth, T. & Lupski, J. (1991). Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nuclei Acids Res 19, 6823-6831. Versalovic, J., Schneider, M., de Bruijn, F. J. & Lupski, J. R. (1994). Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Molec Cell Biol 5, 25-40. Veys, A., Callewaert, W., Waelkens, E. & Van den Abbeele, K. (1989). Application of gas-liquid chromatography to the routine identification of nonfermenting gram- negative bacteria in clinical specimens. J Clin Microbial 27, 1538-1542. Weisburg, W. W., Barns, S. M., Palletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacterial 173, 697-703. Witt, M. E., Dybas, M. J., Worden, R. M. & Criddle, C. S. (1999a). Motiltity- enhanced bioremediation of carbon tetrachloride-contaminated aquifer sediments. Environ Sci Technol 33, 2958-2964. Witt, M. E., Dybas, M. J., C, W. D. & Criddle, C. S. (1999b). Use of bioaugmentation for continuous removal of carbon tetrachloride in model aquifer columns. J Environ Eng Sci 16, 475-485. Yamamoto, S. & Harayama, S. (1995). PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and 146 taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbial 61, 1104-1109. Zhou, J., Fries, M. R., Chee-Sanford, J. C. & Tiedje, J. M. (1995). Phylogenetic analyses of a new group of denitrifiers capable of anaerobic growth on toluene and description of Azaarcus tolulyticus sp. nov. Int J Syst Bacterial 45, 500-506. Ziemke, F., Hofle, M. G., Lalucat, J. & Rossello-Mora, R. (1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacterial 48, 179-86. 147 APPENDIX B SEQUENCE OF A 8,274 BASE PAIR ECORI FRAGMENT MUTATED IN FOUR PSEUDOMONAS S T U T ZERI STRAIN KC TRANSPOSITIONAL MUTANTS WITH IMPAIRED ABILITY TO DEGRADE CARBON TETRACHLORIDE 148 RBS CDS Translation RBS CDS miscelaneous Translation Miscellaneous -35 seq —lO seq Fur box RBS CDS Miscellaneous Translation 437 446 - - 439 1012 MSYRPGTARPRGTGRLPTKPNPVETLPFPSLLARPHALQSCWPT QLTEPLRDGRPCPVSPAPAGRRQGWADTARVKMAQVQAVRADVS CEILPRQPVFSPIGRTLNMDGMPGLVAVALAKPKLEVSGVGFEC PAAAVVAQQPIESVGLPDRLSARSGDLVQEVEQRDDVCFPFASV AGLLARKEENLS 994 — 999 1009 - 2262 12 transmembrane helices indicated by underlined residues MTKSRVVGLQLLFGWMNLVLAVPSIYLMLGMPLVMRQHGWSGAE IGLFQLAALPAIFKFLLAVPVQRVRLGRGHFVHWLLLLCALLLA LYWLIGRHNLIGDRIMLFALTFAISIAATWADIPLNALAVQWLP RSEQLRAGSIRSAALFVGAIVGGGVMIMVQARVGWQAPFWLLGV GLLIGALPFLLLRRHAALPEQAEPRETTDPPPGVMADWASFFHQ PGARQWTLLLLTSFPFLGATWLYLKPLLLDMGMQLERVAFIVGI VGGTAGALFSLLGGQLVQMLGIARAIAWYLLAALGALALLTFSV WAQLGAAWLIASALCVAASMGAISALMFGLTMFFTRNRRNASDX ALQTTMFTVARLAVPIAAGVLLDRVGYTGMLLAMTLALLLSFAL ACRVREKVESSAQSILEHERV Rho-independent terminator 2295 - 2331 2346 - 2351 2372 - 2376 2391 - 2409 2423 - 2426 2435 - 3610 2 transmembrane helices indicated by single underlines, sequence similar to chemotaxis transducer signature indicated by double underlines MPLSALVAPAGELSRAEINRYSRHLLIPDVGMIGQRRLKNAKVL VIGAGGLGSPTLLYLAAAGVGTIGIIDFDRVDDSNLQRQVIHGV DTVGELKVDSAKKAIARLNPFVQVETYTDRLERDMAIELFSRYD LIMDGTDNFATRYLVNDACVLANKPYVWGSIFRFEGQASVFWEN APNDLGLNYRDLYPEPPPPEMAPSCSEGGVFGILCASIASIMAT EAVKLITGIGEPLLGRLVVYDALDMRYRELPVRRLPNRQPITDL AEDYQVFCGLGLPKGDTADAVPGISVTELKKRMDQDEVPVLIDV REPTEWDIVRIPGAILVTKSPTAAQTLRERYGADANLVIVCKSG RRSADVTAELLNLQMRNVRNLEGGVLAWVKDVDSSLPSY 149 RBS CDS Translation RBS CDS Miscelaneous Translation RBS CDS Translation RBS CDS Miscelaneous 3612 - 3615 3626 - 4036 MALLIKRQALGQVLAQARRDHPLETCGIVASSLEAQLATRVIPM RNQAASQTFFRLDSQEQFQVFRSLDDRNEFQRVIYHSHTASEAY PSREDIEYAGYPEAHHLIVSTWENAREPARCFRILRGKVIEESI SIVE 4088 — 4091 4099 - 4371 perfect TonB dependent recognition sequence indicated by a single underline, glycine- glycine C-terminus domain indicated by a double underline MSISVIVPTLLRPLTNGEKTVFTQGNSVAEAIENLEHQFPGLKA RLVSAEHVHRFVNIYVNEDDIRFSDGLNTPLKAGDSLTVLPAVA 9.9 4450 - 4451 4460 - 6291 MPTLLNEFSLLHSSTSFPPNWNELQLSLTEQARLLGICPLAISP PVDMEGAAFQLQHPAISPIQAHFASPAGWLPNRHLSELLLQAGS GLMSVHGRASGRAQPLGVDYLSTLTAVMTLHGTLAAAVGQLRGG AFDQVQLSPLGCGLLSIGQYLAGATAPEDREAFLPGGSDPHLRP PFRSADGITFELETLDSTPWRSFWTAVGIESELAGTAWKGFLLR YARAVSPLPAACLTALARLRYAKIQQLAAQAGVAVVPVRTDAQR REDPDYRQSLATPWQFESFPPSPERHRDTAFPSLLPLQGMRVIE SCRRIQGPLAGHLLASLGAEVIRLEPPGGDPLRAMPPCAEGCSV RFDALNHLKSVHEVDIKSAHGRQLVYELARDADVFLHNWAPGKA HEMQLDAEHLRRVQPHLVYAYAGGWGRAPVNAPGTDFTVQAWSG VSAAIARQSGIRGGSLFTVLDVLGGAIAALGVTAALLNRAVTGT GTYVESSLLGAADLLMHSSGKASRGILSGVYPTLSGLIAIDCQH PDQFQSLAMLLDIPATADTCQQTLAERLRKRPASEWETVLNERG IGACVVIEDLKQLAADTRISECLTRKSYFSVNAPWRFL 6279 — 6283 6289 - 7983 AMP-binding domain indicated by underlined residues 150 Translation RBS CDS Translatation 1 GAATTCCAGG 51 TCCCAACGGT 101 TCGATCAGCG 151 TGGTTCAAGT 201 GCTGCAGCCG 251 TCCATTGTCA 301 GTCCGTTAGC 351 CAAAAAATGC 401 CCAGTCATTG 451 TTACAGGCCA 501 AGCCAAACCC 551 CACGCGCTGC 601 TGGCAGGCCG 651 GGGCCGATAC 701 GACGTGTCCT 751 ACGGACACTG 801 CGAAGCCGAA 851 GCCGTCGTGG 901 GAGTGCGCGC 951 TCTGTTTTCC 1001 AACCTCTCAT 1051 TGGATGAATC 1101 GCCACTTGTA 1151 TCCAGCTTGC MNNAGIIDLVPAEERQRWVQDGTYPNQPVFTLFAAKAEAHPDKK AVLSPQGDVTYGELLDAALRMAHSLRDSGIVAGDVVAYQLTNHW LCCAIDLAVAALGAIVAPFPPGRGKLDIQSLVRRCDARAVIVPQ AYEGIDLCEVIESLRPTLLSMRRLIVQGKPREGWITLDELMSTE PLDLASLPRVCPNSPVRLLVSSGTESEPKLVAYSHNALVGGRGR FLQRIASDGEDFRGMYLVPLGSSFGSTATFGVLCWLGGSLVVLP KFDVDEAIKAIAAFRPGFILGVPTMLQRIAAQPALESIDKSSLR GLIVGGSVIDEATVRKCRDAFGCGFISLYGSADGVNCHNTLDDP IEVVLTSVGKPNPAVCAIRLVDDEGREVRQGEVGEITARGPLTP MQYVNAPELDERYRDPQGWVKTGDLGYINDKGYLVLAGRKKDVI IRGGANISPTQIBGLVMAHPDVVTVACIPVPDDDLGQRVCLSVT LREGAAKFSLKAITDFLRELGLEVNKLPEYLRFYRALPLTPAGK IDKKALTEEARELGTSGICPAGPGQSTPERSLREYA 7970 - 7974 7985 > 8274 MRGQPMMMATALICAFVPGPQLAFAAPGSAASPDSTTLPEITVT AEKIERPLERVPASVAVIDGWDAEQSGITSLKQLEGRIPGLSFQ PFGQAGMNm CTGGTGACAT GATGACCATG GCACAGTCGG TCGACAAAAT AGTCTTGCCA GCCGGTAGCA CCCCCGAATG TAATGGTTCT GCGACCAAAC GGCACTGCAC TGTCGAAACA AATCGTGTTG TGTCCGGTCT CGCCCGGGTG GTGAGATTCT AACATGGACG GCTGGAAGTG CGCAGCAGCC AGCGGCGATC GTTCGCTTCC GACCAAGTCG TGGTGCTGGC ATGCGCCAGC CGCGCTGCCG CCTGCCCGTC ACCCGACCTT ACGATAGTGC CCTGGAAGCA TCGCTCAGGA GCTCTTGTGT TCGCGCATAT CATTACTATA ACCCAACTAA GCCCGAGAGG CTGCCGTTCC GCCGACGCAG CACCCGCTCC AAGATGGCTC TCCTCGACAG GCATGCCCGG TCCGGTGTAG CATCGAAAGC TGGTACAGGA GTCGCTGGCC CGAGTGGTAG GGTACCCAGC ATGGCTGGAG GCGATATTCA 151 GTAGCAGGAG CATGCGGTCC AGCCTGCCAA TTGGCCAATC CGCTGCGGAT GCGCTCACCT CAATCTGTGT ATGCATCATT AAATACGGAA AACTGGACGA CTTCTCTCCT CTGACCGAGC TGCTGGACGG AGGTGCAGGC CCCGTGTTTT CTTGGTGGCG GGTTCGAGTG GTCGGACTTC GGTCGAGCAA TGCTCGCCCG GTCTGCAACT ATCTACTTGA CGGCGCAGAG AATTCCTGTT CGGCCTTTCA GCTCGTTTCC GACCGAGGCC TCGTCGGGCA GCGTGGTTCT CTCACCTCCA TTCTCGCATA TTACCGATGG AATCAATGTC CTACCCACGA TGCTCGACCG CGCTGCGCGA CGGCAGGGAT GGTCAGGGCC CGCCGATAGG GTCGCACTGG CCCGGCCGCC CCGACCGTCT CGGGATGACG CAAAGAGGAA GTTGTTCGGC TGCTCGGCAT ATCGGGTTGT GGCTGTGCCG 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 GTGCAGCGTG GCTCTGTGCG TGATCGGCGA GCCGCCACGT GCCGCGTAGT TCGTAGGCGC GTGGGCTGGC CGCCCTGCCC CCGAGCCGCG GCAAGCTTCT GACCAGTTTC TGTTGGACAT GTCGGCGGCA GCAAATGTTG TGGGCGCGCT GCATGGCTGA CTCGGCGCTG ACGCGTCGGA GCGGTGCCGA CATGCTCCTG GTCGGGTGCG GAGCGGGTTT CTGAAATGCG AGAACAGCCA ATCGTTACCA TGGTGGCGCC CGCCACCTAC GAACGCCAAG TGCTCTATCT GATCGGGTTG TACCGTGGGC TAAATCCCTT ATGGCGATCG CAACTTCGCA AACCCTATGT TTCTGGGAAA TCCGGAGCCT TGTTCGGCAT GTCAAGCTGA GTACGACGCT CAAATCGACA GGTCTGGGGT CGTAACGGAA TAGACGTGCG ATCTTGGTGA CGGGGCAGAT CCGACGTGAC TGCGCCTCGG CTACTACTGG TCGCATAATG GGGCCGACAT GAACAGTTGC CATTGTTGGC AGGCCCCCTT TTCCTGCTGT CGAGACTACA TCCACCAGCC CCCTTCCTCG GGGCATGCAG CCGCAGGCGC GGCATAGCAC GGCACTTTTG TTGCCAGCGC ATGTTCGGGT CTATGCCCTG TCGCCGCCGG GCAATGACCC GGAAAAGGTG GAAGGCTGAA CGGTCCTTTT ACGGCAATTG TTGAAATCAA GGCTGGGGAA TGATACCCGA GTGTTGGTCA AGCTGCAGCA ACGACTCCAA GAGCTCAAGG TGTCCAGGTC AGCTGTTTTC ACCCGTTACT GTGGGGCTCG ACGCCCCGAA CCGCCGCCCG TCTTTGCGCA TCACGGGCAT TTGGATATGC ACCGATCACC TGCCCAAAGG CTCAAGAAGC CGAGCCCACC CCAAATCGCC GCCAACCTGG CGCCGAGTTG GCGCGGACAT CGCTGTACTG CTGTTCGCGC TCCGCTAAAT GCGCCGGCAG GGCGGCGTCA CTGGCTGCTA TGCGTAGACA GATCCTCCAC AGGGGCGCGG GCGCGACGTG CTAGAGCGCG ACTGTTCAGC GGGCCATTGC ACGTTCAGCG CCTCTGCGTG TGACCATGTT CAAACCACCA GGTGTTGCTC TGGCGCTGCT GAATCTTCGG GTGACCGGCC GCATAGTTTT CTATAGTCAT ACAGGATAAG CTGAGCCGCG TGTGGGCATG TCGGCGCCGG GGTGTGGGCA CCTTCAGCGC TGGACAGTGC GAAACCTATA GCGCTACGAC TGGTCAACGA ATATTCCGTT CGACCTGGGC AGATGGCCCC TCCATCGCAT CGGCGAGCCA GCTATCGGGA GACCTGGCCG TGACACGGCG GGATGGATCA GAGTGGGACA CACCGCAGCG TGATCGTCTG CTAAACCTGG 52 TTCGTGCACT GCTAATCGGA TGACCTTCGC GCGCTAGCGG CATCCGTTCC TGATCATGGT GGGGTCGGAC CGCCGCACTG CGGGCGTGAT CAATGGACAT GCTGTACCTC TGGCCTTCAT CTGCTCGGCG CTGGTACCTG TCTGGGCCCA GCAGCCAGCA CTTCACCCGA TGTTCACCGT GACCGGGTGG GCTTTCCTTC CACAGTCGAT ATGCCCCTTC TCATGCTCAC CACCACGAAC CGATATGCCA CCGAGATCAA ATCGGGCAGC CGGTCTTGGC CCATCGGGAT CAGGTCATCC AAAGAAAGCC CCGATCGCCT CTGATCATGG CGCCTGCGTG TCGAAGGGCA CTGAACTACC CTCGTGCTCC CGATCATGGC CTACTGGGTC GCTTCCTGTG AGGACTATCA GACGCCGTGC GGACGAGGTG TCGTCCGTAT CAGACACTGC CAAGTCCGGG GCATGCGCAA GGTTGCTGTT CGGCATAATC CATCAGCATT TGCAGTGGTT GCAGCGCTGT GCAGGCGCGC TGCTGATTGG CCCGAGCAGG GGCGGACTGG TGCTGCTGCT AAACCTTTAT CGTTGGCATC GACAGCTAGT CTGGCGGCGC ACTGGGGGCG TGGGCGCCAT AATCGGCGCA TGCGCGACTG GCTACACCGG GCGCTCGCCT ACTCGAGCAC GGACAATGGC GTCATATGAA GATAATGATT CTATCAGCGC CCGTTACAGC GTCGGTTGAA TCTCCGACTC AATCGACTTT ACGGGGTGGA ATTGCGCGAC GGAACGGGAC ACGGTACCGA CTGGCCAACA GGCGTCCGTG GCGACCTGTA GAGGGCGGTG CACCGAGGCG GGCTGGTGGT CGCCGCCTGC GGTGTTCTGC CAGGGATCAG CCTGTGCTGA TCCGGGCGCA GCGAGCGATA CGGCGCTCTG TGTTCGCAAC 3551 3601 3651 3701 3751 3801 3851 3901 3951 4001 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451 5501 5551 5601 5651 5701 5751 5801 5851 CTCGAAGGTG TAGCTACTGA CGCTGGGGCA TGTGGAATCG CCCAATGCGC AGGAGCAATT CGGGTCATCT GGACATCGAG CATGGGAGAA AAAGTCATCG TTCAATCAGC GTCGATTTCA AAAAGACAGT CTTGAACACC TGTGCATCGT CAGATGGGCT CCTGCCGTCG GACCCCTGGC GTTTTGAATA CACTTCGTTT AGGCCAGATT ATGGAAGGAG GGCCCACTTC AGCTGCTGCT AGCGGTAGGG CGTCATGACG GCGGTGCATT AGTATCGGGC GTTCCTGCCG CTGACGGCAT AGCTTTTGGA GAAAGGTTTT CCTGTCTCAC GCAGCGCAAG CCGCGAGGAC AGTCTTTCCC CTGCTGCCGC GGGACCGCTG GGCTGGAGCC GAAGGCTGTT CGAAGTCGAT CCCGCGATGC GAAATGCAAC TTACGCCTAT CCGACTTCAC CAATCCGGCA CGGCGCGATC GCGTTTTGGC TAGGAGGCCT GGTTCTGGCT TGGCGTCTTC AACCAGGCGG CCAGGTGTTC ACCACTCTCA TATGCGGGCT CGCCCGAGAG AAGAAAGTAT AATGCCTCAG GTGATCGTTC TTTTACCCAA AGTTCCCTGG TTCGTCAATA CAACACGCCA CCGGTGGCTG GATTCAAATC TGCCGACACT CCGCCGAATT ATTGGGCATT CCGCATTCCA GCCTCACCAG GCAGGCGGGC CCCAACCGCT CTGCACGGAA TGATCAGGTT AGTATCTGGC GGCGGCTCCG CACATTCGAG CCGCCGTCGG CTGCTTCGCT GGCGCTCGCC CGGGTGTTGC CCCGATTACC GCCGTCCCCC TACAGGGGAT GCCGGGCATC GCCGGGTGGC CGGTGCGCTT ATCAAATCCG GGATGTCTTT TGGATGCTGA GCGGGAGGCT CGTCCAGGCC TCCGCGGCGG GCGGCACTGG CTGGGTGAAG AGAAGATGGC CAAGCACGTC ACTGGAAGCC CATCACAAAC CGATCTCTGG TACCGCGAGT ATCCGGAAGC CCCGCCCGTT CTCCATTGTG CCACAGCTAG CCACATTGCT GGCAACTCGG CCTTAAGGCC TCTACGTCAA CTCAAGGCCG ACTCGCACCT CAGGGGCAAG CTTAAATGAA GGAATGAACT TGCCCGCTCG GCTGCAGCAT CCGGCTGGCT AGCGGTCTTA GGGCGTGGAT CGCTGGCCGC CAGCTTTCTC AGGCGCCACG ATCCGCATTT CTGGAAACGC CATTGAATCG ACGCGAGGGC CGCCTGCGTT GGTCGTGCCC GGCAGTCACT GAAAGGCATC GCGCGTCATC TGCTGGCATC GATCCGTTGC TGACGCGCTG CCCATGGGCG CTGCACAACT ACATCTGCGC GGGGCCGGGC TGGTCGGGTG CTCGCTGTTC GTGTGACGGC 153 GACGTGGACT ATTGCTTATC GCGATCACCC CAGTTAGCGA CTTCTTTCGG ATGATCGCAA GAAGCCTATC GCATCACCTG GTTTCCGGAT GAATAGCGAC AGGCAAAAGG GCGCCCGCTG TGGCAGAGGC CGGCTGGTCA CGAAGACGAC GTGACAGTTT CCGGACACCG TGCAACGCTT TTTTCCCTGC GCAACTTAGC CAATCTCGCC CCGGCTATTT GCCAAATCGA TGTCGGTGCA TATCTTTCGA AGCCGTGGGG CACTGGGATG GCACCAGAAG GAGGCCGCCA TCGACAGCAC GAATTGGCCG CGTGTCGCCT ACGCAAAGAT GTCCGCACCG GGCTACGCCA GAGACACCGC GAATCCTGTC GCTGGGCGCC GAGCCATGCC AACCACCTCA GCAGTTGGTC GGGCGCCCGG AGGGTTCAGC TCCCGTCAAT TGTCCGCCGC ACCGTGTTGG CGCGTTGCTC CTTCTCTGCC AAGCGTCAGG ACTTGAAACC CAAGAGTAAT CTCGACTCGC CGAGTTCCAA CGAGCAGGGA ATTGTGTCCA ACTTCGTGGA TTTCCAATAT AGTTCTACAT ACCAATGGGG CATCGAGAAC GTGCGGAACA ATCCGCTTCT GACCGTGCTG CTGAAAGAAT TTGTCTCTCG TGCATTCATC CTGACGGAAC GCCTGTGGAT CTCCTATTCA CACCTCTCGG CGGCCGTGCT CACTTACCGC CAGCTGCGTG CGGGCTGCTC ATCGTGAGGC TTTCGTTCCG ACCGTGGCGA GTACGGCCTG CTACCTGCCG CCAACAATTG ATGCGCAACG TGGCAGTTCG ATTTCCGTCA GACGCATTCA GAAGTCATTC GCCCTGCGCC AATCCGTTCA TACGAGCTCG CAAGGCCCAT CACATCTCGT GCCCCGGGTA CATTGCACGT ATGTGCTGGG AATCGAGCAG 5901 5951 6001 6051 6101 6151 6201 6251 6301 6351 6401 6451 6501 6551 6601 6651 6701 6751 6801 6851 6901 6951 7001 7051 7101 7151 7201 7251 7301 7351 7401 7451 7501 7551 7601 7651 7701 7751 7801 7851 7901 7951 8001 8051 8101 8151 8201 TCACGGGCAC CTGCTGATGC GTATCCCACG AGTTCCAGTC TGCCAGCAGA GGAAACGGTG ACCTCAAGCA AAGTCTTACT GGCATCATCG CGGTACCTAC AAGCGCATCC TACGGCGAGC TTCGGGGATC GGTTGTGCTG GCCCCCTTCC CCGCTGCGAC ATCTGTGCGA CGCCTGATTG GCTGATGAGC CGAACTCGCC AAGCTGGTGG CCTGCAGCGC TTCCGCTGGG TGGCTGGGTG CATCAAGGCG CCATGCTGCA TCCAGCCTGC CGTGCGCAAA GTTCCGCCGA GTTGTGCTGA TCTGGTGGAC TCACCGCCCG CTGGACGAGC GGGCTACATC ACGTCATCAT CTGGTCATGG CGATGATGAT GTGCAGCGAA GGACTGGAGG GCCTCTGACA CCCGCGAGCT ACTCCCGAGC TGATGATGGC GCGTTTGCTG GGAAATCACC CCGCCAGCGT ACTAGCCTCA GGGTACTTAT ACAGCAGCGG CTATCGGGAC GCTGGCCATG CGCTGGCGGA CTGAACGAAC GCTCGCCGCC TCTCTGTCAA ACCTGGTTCC CCGAACCAGC CGACAAGAAG TCCTCGATGC GTGGCCGGCG CGCAATCGAC CTCCGGGACG GCGCGAGCGG GGTTATCGAG TTCAGGGCAA ACCGAGCCGC GGTGCGTCTG CGTACTCGCA ATCGCGTCCG TTCGTCCTTC GTTCGCTGGT ATTGCGGCAT ACGCATCGCC GTGGTTTGAT TGCCGTGATG CGGCGTGAAC CCAGCGTCGG GACGAAGGCC CGGGCCATTG GTTACCGCGA AACGACAAGG CCGTGGGGGC CGCATCCCGA CTCGGGCAGC GTTTTCCCTG TGAACAAGCT CCGGCGGGAA GGGCACCTCG GCAGCTTACG TACAGCTTTG CGCCAGGCTC GTCACAGCCG GGCGGTGATC AACAACTGGA GTCGAGAGCT CAAGGCGTCG TGATCGCCAT TTGCTGGACA GCGCTTACGC GGGGCATCGG GACACCCGCA CGCCCCCTGG TGCTGAGGAA CCGTATTCAC GCCGTGCTGT AGCCCTGCGG ACGTGGTGGC CTGGCAGTGG CGGCAAGCTG TGATCGTCCC TCACTGCGCC GCCTCGCGAA TGGATCTCGC CTGGTGTCTT CAATGCGTTG ATGGCGAAGA GGCTCCACTG CGTATTGCCC TTCGGCCGGG GCTCAACCGG CGTCGGCGGC CGTTTGGCTG TGCCATAACA CAAGCCCAAT GGGAGGTCCG ACTCCAATGC CCCGCAAGGC GTTATCTGGT GCCAATATCA TGTCGTGACC GGGTGTGCCT AAAGCGATCA CCCCGAGTAC AGATCGATAA GGCATTTGTC GGAGTACGCA ATCTGTGCCT CGCGGCTTCG AGAAAATCGA GATGGCTGGG AGGACGCATT 154 CATTGCTGGG AGGGGCATCT CGACTGCCAA TTCCTGCCAC AAGCGACCCG CGCCTGTGTA TCTCTGAATG AGGTTCCTAT CGCCAACGTT GCTGTTTGCC CGCCGCAAGG ATGGCTCACA TTACCAGCTC CAGCGCTCGG GATATCCAAT GCAAGCGTAC CCACCCTGCT GGATGGATTA CAGCCTACCC CAGGCACCGA GTTGGTGGTC TTTTCGCGGC CCACCTTCGG AAGTTCGACG CTTCATTCTC CGTTGGAGAG TCGGTCATCG CGGCTTCATC CCCTGGACGA CCGGCGGTCT GCAAGGCGAG AGTACGTCAA TGGGTGAAGA CCTAGCCGGT GCCCGACCCA GTTGCGTGCA TTCCGTCACC CCGACTTCCT CTACGCTTCT AAAAGCGCTA CCGCTGGGCC TGATATGCGC TTGTACCAGG CCTGACTCCA GCGCCCGCTG ACGCCGAGCA CCTGGTCTGT CGCCGCCGAT TGTCCGGCGT CACCCAGATC TGCGGATACC CTTCGGAATG GTCATCGAAG CCTCACTCGC GAACAACGCT GGGTGCAGGA GCCAAAGCCG TGACGTGACC GCCTGCGTGA ACCAACCACT TGCCATCGTC CGCTGGTTCG GAAGGCATCG ATCCATGCGC CGCTCGATGA AGGGTGTGCC GTCGGAGCCC GCGGGCGCTT ATGTACCTCG TGTGTTGTGC TGGATGAAGC GGCGTACCCA CATCGACAAA ACGAGGCCAC AGCCTCTACG CCCCATCGAA GCGCGATTCG GTTGGCGAAA CGCGCCGGAG CCGGAGATCT CGCAAGAAGG GATTGAAGGC TTCCTGTTCC TTGCGCGAGG GCGCGAACTG ACCGCGCTCT ACCGAGGAAG GGGGCAGTCG GGTCAACCGA GCCACAGTTG CGACGCTACC GAAAGGGTGC GTCAGGCATC CATTCCAGCC 8251 GTTCGGGCAA GCAGGTATGA ATTC 155 APPENDIX C MOTIF INFORMATION ABOUT PROTEINS ASSOCIATED TO THE CARBON TETRACHLORIDE DEGRADATION CAPACITY OF PSEUDOMONAS ST U T ZERI STRAIN KC 156 Table C.1 Transmembrane domains for proteins identified by Sepulveda and by Lewis, as predicted by TMHMM, HMMTOP, SOSUI and TMPred. ORF “ TMHMM b HMMTOP ° 5080] d TMPred ° # from to # from to # from to # from to tmhf aa8 aa tmh aa 33 tmh aa aa tmh aa aa 446 0 0 0 0 1009 12 11 33 12 11 35 12 8 30 ll 11 29 45 63 45 65 43 65 42 60 76 94 75 94 71 93 75 93 106 128 104 128 106 128 106 122 139 161 139 159 140 162 145 163 170 188 169 187 167 188 169 188 226 244 228 248 227 249 225 241 259 277 258 278 256 278 260 276 290 308 289 309 285 307 291 307 317 339 318 342 318 340 316 336 352 374 352 372 353 375 377 398 378 396 382 400 378 399 2435 l 43 65 2 46 70 0 2 43 61 206 230 206 224 3626 0 0 0 0 4099 0 0 0 4460 0 0 0 6 109 127 139 155 221 239 428 447 456 474 509 527 6289 0 3 88 107 1 87 108 4 89 107 234 258 243 264 271 290 274 292 328 346 K. 0 0 0 4 22 41 124 141 470 488 525 543 L 0 0 0 5 226 242 264 281 303 320 444 460 640 659 Rd 0 2 49 73 2 56 76 7 23 41 82 103 84 105 57 75 85 101 194 211 219 239 552 569 591 607 157 Table C.l (continuation) ORF TMHMM HMMTOP SOSUI TMPred # from to # from to # from to # from to tmh aa aa tmh aa aa tmh aa aa tmh aa a N 12 13 35 12 11 11 16 38 10 ll 28 50 72 50 51 73 52 72 79 97 79 84 106 81 98 101 119 103 144 166 170 186 144 166 144 168 190 208 227 170 188 169 208 230 237 255 208 230 207 241 263 274 290 239 261 237 271 293 304 321 272 290 273 303 325 338 354 302 324 300 333 355 368 386 337 359 329 365 386 365 387 364 O O 0 O 2 361 377 454 470 P 0 0 0 5 21 37 191 210 247 267 275 295 325 343 a ORF, open reading frame b TMHMM, transmembrane helix on a hidden Markov model (Sonnhammer et al., 1998) http://www.cbs.dtu.dk/services/TMHMM-1 .0/ c HMMTOP, prediction of transmembrane helices and topology of proteins (Tusnady & Simon, 1998) http://www.enzim.hu/hmmtop/server/submit.html d SOSUI, classification and secondary structure prediction system for membrane proteins (Hirokawa et al., 1998) http:/azusa.proteome.bio.tuat.ac.jp/sosui/submit.html e TMPred, transmembrane prediction (Hofmann & Stofell, 1993) http://www.ch.embnet.org/software/TMPRED_form.htrnl f thm, transmembrane helices g aa, amino acid 158 Table C.2 Transmembrane domains for proteins identified by Sepulveda and by Lewis, as predicted by PSort, DAS and TopPred2. oar " Psort b DAS ‘ TopPred2 d # from to # from to # from to tmh ‘ a f aa tmh aa aa tmh aa aa 446 0 2 1 12 1 18 0 173 178 1009 11 47 63 ll 10 31 12 15 35 78 94 50 63 45 65 103 1 19 76 93 74 94 145 161 103 115 102 122 172 182 146 159 143 163 228 244 171 187 168 188 260 276 228 238 225 245 294 310 259 277 258 278 322 338 287 309 290 310 358 374 314 339 323 343 382 398 381 397 354 374 378 398 2435 2 56 72 2 44 68 2 50 70 204 220 206 218 203 223 3626 0 0 0 4099 0 4460 1 456 478 7 35 41 5 56 76 109 124 108 128 143 150 139 159 230 235 458 478 321 325 506 526 456 477 514 521 6289 2 89 105 5 91 105 4 88 108 247 263 248 263 244 264 281 287 271 291 310 314 452 472 462 465 K 0 5 25 36 3 20 40 128 139 126 146 176 178 524 544 478 482 531 538 L 0 2 444 451 0 537 544 M 0 4 29 33 4 55 75 63 71 84 104 88 103 551 571 560 565 586 606 159 Table C.2 (continuation) ORF PSort DAS TopPred2 # from to # from to # from to tmh aa aa tmh aa aa tmh aa aa N 8 81 97 l 1 12 37 12 14 34 98 l 14 58 65 50 70 148 164 80 1 12 79 99 168 184 151 163 102 122 207 223 171 186 145 165 241 257 209 234 168 188 274 290 241 262 215 235 369 385 274 288 238 258 306 308 271 291 339 345 298 318 367 387 332 352 368 388 O 0 1 362 369 O P 0 5 170 175 0 196 202 254 262 281 284 299 307 a ORF, open reading frame PSort, prediction of protein sorting signals ad localization sites in amino acid sequences (Nakai & Kaneshisa, 1991) http://psort.nibb.ac.jp/form.html c DAS, dense alignment surface method for the prediction of membrane a-helices in prokaryotic membrane proteins (Cserzo et al., 1994; von Heijne, 1992) http://www.Biokemi.su.se/~server/DAS d TopPred2, topology prediction of membrane proteins (von Heijne, 1992) http://www.Biokemi.su.se/~server/toppredServer.cgi c tmh, transmembrane helices f . . aa, amino acrd 160 Table C.3 Leader peptides for proteins identified by Sepulveda and by Lewis, as predicted by SignalP, PSort, and SPScan. ORF “ SignalP b PSORT ° SPScan d 446 No signal peptide No signal peptide No 1009 446 No signal peptide Cleave 1 l8 -— 119 TW-AD 2435 Cleave 10 - 11 No signal peptide No APA-GE 3626 Cleave 11 — 12 No signal peptide No ALG-QV 4099 No signal peptide Uncleavable No N-terminal sequence 4460 Cleave 124 - 125 No signal peptide No LAA-AV 6289 No signal peptide No signal peptide No signal peptide K Cleave 48 -— 49 No signal peptide Cleave 42 — 43 SAA-SP FA-AP L Cleave 96 — 97 No signal peptide No ATA-RQ M No signal peptide No signal peptide No signal peptide N Cleave 1 l8 - 119 Cleave 30-31 Cleave 7O — 71 SLA-NP FY-Al GA-LV Cleave 118 — 119 LA-NP O No signal peptide No signal peptide No signal peptide P No signal peptide No signal peptide No signal peptide a ORF, open reading frame their cleavage site (Nielsen et al., 1997) http://www.cbs.dtu.dk/services/SignalP sequences (N akai & Kaneshisa, 1991) http://psort.nibb.ac.jp/form.html d SPScan, sport protein scan (Butler, 1998) 161 SignalP, identification of prokaryotic and eukaryotic signal peptides and prediction of PSort, prediction of protein sorting signals and the localization sites in amino acid Table C.4 Information about some motifs found by BLOCKS a in ORF-2435. b ID Description Sequence in proteinc / Comments BL00538D Bacterial chemotaxis 333 - 369 sensory transducer QtlreeygAdan1vivcksGRrsAthaEllNLngn integral membrane proteins, respond to changes in concentration of attractants and repellents in the environment by changing levels of methylation in C-terminus BL00886D Dihydroxy-acid and 6- 36 - 82 phosphogluconate rrlkNAkVLVlgaGGlgspTLyLAAAgvgtlglinDerdSNLqr dehydratase introduces a C=O bond in a former C-OH bond BLOOO74D Amino acid 40 - 74 dehydrogenases NAKVLViGaGglGSpTlLYLAAAGvgthllDFDR introduces a C=O bond in a former C-NHZ bond BL00573A pyridine nucleotide- 45 - 62 disulfide VlGAGgleptlLYLAaA oxidoreductase sequence located ~ 110 amino acids upstream of C-X-X-C redox active disulfide bond BL01316A ATP-phosphoribosyl- 344 - 368 transferase nLVIVcKSGRrSaDVTaeLlNleR involved in the first step of histidine biosynthesis BLOOl 10E Pyruvate kinase 254 - 309 PLpNRQplTDLAEdyQVFcGLglkaDtadaVPgltheLkKRMd QdevpV involved of final stage of glycolysis, activated by AMP and sugar phosphates BL00557D F MN-dependent 26 - 67 a-hydroxyacid llpDmaigqrrlknAkvaigAgGLGsPTLlyLAAAGvgtl dehydrogenase introduces a C=O bond in a former C-OH bond BL01305 MoaA/nitB family 228 - 237 GIGEPLLgRl involved in the synthesis of molybdopterin precursor Z from guanosine BLOO443D Glutamine 32 - 47 aminotransferase MleRRLknaKVLVlG involved in asparagine biosynthesis a BLOCKS, motif finder (Henikoff et al., 1999) http://motif.genome.ad.jp b ID, BLOCKS identification number for the specific motif c upper case residues indicate perfect matches 162 Table C.5 Information about a motif found by BLOCKS in ORF-3626. a ID Description Sequence in protein / Comments BL00648A Bacterial ribonuclease 62 - 83 P protein component FquRSLdDRnEFQRVIsthT Endonucleolitic cleavage of RNA, removing the 5’-extra nucleotide from tRNA precursor a See legend provided in Table C.4. Table C.6 Information about a motif found by PROSITEa Pattern in ORF-4099. [D b Description Sequence in protein / Comments P800430 TonB-dependent perfect match X78 79 — DSLTVXPA -86 receptor proteins pattern seen in many TonB-dependent outer membrane signature 1 receptors a http://motif.genome.ad.jp (Hoffmann et al., 1999) b ID, PROSITE identification number for the particular motif Table C.7 Information about some motifs found by BLOCKS a and PRINTS b in ORF-4460. ID Description Sequence in protein / Comments BLOO836G Alanine dehydrogenase 465 - 496 and pyridine nucleotide GAiAALGVTaALLnrangTthVessllGAa transhydrogenase alanine dehydrogenase-involved in the assimilation of L- alanine as energy source through tricarboxylic acid cycle, pyridine nucleotide transhydrogenase - couples transhydrogenation of NaDH and NADP to respiration BL00462E y—glutamyltranspeptidase 335 - 347 ngdPLraMcha transfer of glutamyl group from polypeptide to an amino acid FADPNR3 AD-dependent pyridine 59 - 484 (PRINTS) nucleotide reductase VLdVLGGalAALthAALLNRAVTGT signature signature found in many reductases and dioxigenases a See legend provided in Table C.4. b (Attwood et al., 1999) 163 Table C.8 Information about some motifs found by BLOCKS and PROSITE pattern in ORF-6289. “ ID Description Sequence in protein / Comments BL00455 AMP-binding domain 196 - 211 (BLOCKS) vSSGTESEPKLVAYSH PS00455 AMP-binding domain 194 - 205 (PROSITE) LLVSSGTESEPK perfect signature for AMP-binding a See legends provided in Table C4 and Table C.6. Table C.9 Information about a motif found by Pfamal in ORF-K. ID Description Sequence in protein / Comments TonB_boxC TonB-dependent 585 - 687 receptor C-terminal signature found in a variety of TonB-dependent receptors region a http://motif.genome.ad.jp (Bateman et al., 1999) Table C.10 Information about some motifs found by BLOCKS in ORF-L. a ID Description Sequence in protein / Comments BL00296F Chaperonins cpn60 271 - 308 proteins GWqIYGRAYViAMTSGmIDeARnganLKRAAapG11V prevents misfolding and promotes the refolding and proper assembly of unfolded polypeptides generated under stress conditions BL00662F Bacterial type 11 65 - 110 secretion protein tiAQAcRyLrsterlpalstcthmatArQAlsadetLSpL system protein B involved in translocation of the type IV pilin, DNA uptake and protein export a See legend provided in Table C4. 164 Table C.11 Information about some motifs found by PROSITE pattern and BLOCKS in ORF-M. 3 ID Description Sequence in protein / Comments P800600 Aminotransferases class-Ill 524 - 561 (PROSITE) pyridoxal-phosphate WILDEIQTGLGRTGKMFACEWEDVSPDIlVLSKSL attachment site SGG perfect match with PROSITE pattern, pyridoxal attachment in one of the lysine residues BL00600B Aminotransferases class-Ill 404 - 429 pyridoxal-phosphate GLERVFLSNSGTAEVEAALKLALAAAS attachment site pyridoxal attachment site = lysine residue BL00600F Aminotransferases class-111 549 - 561 pyridoxal-phosphate PDIIVLSKSLSGG attachment site pyridoxal attachment site = lysine residue BL00600C Aminotransferases class-111 434 - 449 pyridoxal-phosphate LLyCTNGYHGKTLGAL attachment site pyridoxal attachment site = lysine residue a See legends provided in Table O4 and Table C.6. Table C.12 Information about some motifs found by BLOCKS in ORF-N. a ID Description Sequence in protein / Comments BL00543D HlyD family secretion proteins 13 -35 GLSaLlllamGMPmmltYAigIL inner membrane bound proteins, secrete hemolysin and alkaline protease BL00216B Sugar transport proteins 57 - 106 tFGLaALLSPwAGALvQRMGTRAGlICmFlLvGLsFS LMAVLpGFGGLVT 104 - 153 LVTALLLCGTAqSLANPATnQAlAhstVARKAGV VGLkQSGVQASALLA BL01022E PTR2 family proton/oligopeptide symporter 82 - 1 17 iCMFleGLsFSLmAVlPGngLVTALLLcGTAqSl BL00872A Sodium: galactoside symporter 53 - 101 LtastfgLAAALLsPWAGaLVqRaGLicmFLLVGlsFS1 MAvagF BL01005C Fonnate and nitrate transporter 20 - 43 iAmGMpMmlealGIlnglVADL BL00942B glpT family of transporters 239 - 281 vSvStIGamvSCFgamgilSrletpiADKlkdetiLlngF i glycerol-phosphate uptake a See legends provided in Table C4. 165 Table C.13 Information about some motifs found in ORF-O by PROSITE pattern and BLOCKS. a ID Description Sequence in protein / Comments PS00072 Acyl-COA dehydrogenases 256 - 268 (PROSITE) signature 1 AMSEPEAGSDANG involved in B-oxidation of fatty acids PS00073 Acyl-CoA dehydrogenases 464 - 483 (PROSITE) signature 2 QIFGGMGYCTELPIERYYRD BL00072E Acyl-COA dehydrogenase 461 - 503 SAVQIFGGMGYcTElPIERYYRDARVFRIYdGTSEI HRImlAR BL00072D Acyl-COA dehydrogenase 374 - 424 VGArAVGMAsKlLEmSVDfAKQRsQFGAPIGsFQm VQKMLADchElYgAR BL0007ZB Acyl-COA dehydrogenase 280 - 292 WILNGSKhFISdA BLOOO72C Acyl-COA dehydrogenase 319 - 359 GLeLGPIQEMMGththGLFFtDCRIapquLGEPG RGm BL00072A Acyl-COA dehydrogenase 184 - 194 LGLwAMHquE a See legends provided in Table C4 and Table C.6. Table C.14 Information about some motifs found in ORF-P by BLOCKS. a ID Description Sequence in protein / Comments BL0123OB NA methyltransferase trmA 187 - 199 family proteins FLDLgCGmevaI involved in tRNA maturation BL00094A C-S cytosine-specific DNA 185 - 205 methylase rRFLDLgCGPvaAlALARAl BL01183B ubiE/COQS methyltransferase 245 - 289 deigngDLIwcavathPDlathRklRaaLaPGGvFVsIh ae involved in biquinone biosynthesis BL00533B Porphobilinogen deaminase lOl - 151 cofactor binding site aKrFfakqssDychAwafrlrSLergTrLPEquVrAleEap vPTchw BL00379 CDP-alcohol 111 - 147 phospatidyltransferase DYngaWAfRLRslrDFGterequVrAleeAPVPt involved in biosynthesis of acidic phospholipids a See legends provided in Table C4 and Table C6. 166 REFERENCES Attwood, T. K., Flower, D. R., Lewis, A. P., Mabey, J. E., Morgan, S. R., Scordis, P., Selley, J. & Wright, W. (1999). PRINTS prepares for the new millenium. Nucleic Acid Res 27, 220-225. Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Finn, R. D. & Sonnhammer, E. L. (1999). Pfam3.l: 1313 multiple alignments match the majority of proteins. Nucleid Acids Res 27, 260-262. Butler, B. A. (1998). Sequence analysis using GCG. Meth Biochem Anal 39, 79-97. Cserzo, M., Bernassau, J., Simon, 1. & Maigret, B. (1994). New alignment strategy for transmembrane proteins. J Mal Biol 243, 388-396. Henikoff, J. G., Henkoff, S. & Pietrokovski, S. (1999). New features in the BLOCKS database servers. Nucleic Acids Res 27, 226-228. Hirokawa, T., Boon-Chieng, S. & Mataku, S. (1998). Classification and secondary structure prediction system for membrane proteins. Bioinformatics 14, 378-3 79. Hoffmann, K., Bucher, P., Falquet, L. & Bairoch, A. (1999). The PROSITE database, its status in 1999. Nucleic Acids Res 27, 215-219. Hofmann, K. & Stofell, W. (1993). Tmbase - a database of membrane Spanning protein segments. Biol Chem (Happe-Seyler) 347, 166. Nakai, K. & Kaneshisa, M. (1991). Expert systems for predicting protein localization sites in Gram-negative bacteria. Proteins: Structure, Function and Genetics 11, 95-110. 167 Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1-6. Sonnhammer, E. L. L., Von Heijne, G. & Krogh, A. (1998). A hidden Markov model for predicting transmembrane helices in protein sequences. In Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ed. J. Glasgow), pp. 175-182. AAAI Press. Tusnady, G. E. & Simon, 1. (1998). Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J Mol Biol 283, 489-506. von Heijne, G. (1992). Membrane protein structure prediction, hydrophobicity analysis and the positive-inside rule. J Mal Biol 225, 487-494. 168 E lll ' E llllllllllllll 0