o ’9?!" \JI‘I: mung-romp :8"! ‘9?! mmn.l 0‘“): . .vv.».-L.¢In‘-C 4 . 5 a I Mm ”mama“ . ... 1.. 7 s E“? 2 ‘ s3? 1 a 21:99. I 33".» .. VA . :J 35:? i. ‘3. .v!!! n‘ o >51: < .V‘QII v gamma”. .I . 3 '11- l 0.. 1! v! .uul..1. .A .15.; ‘ I . (‘4 g G . I! I10 3" . la!!! ..0..lva i! . I a: ; f . .II I I: #5.."- 0.5 O f 00" D. 1.. ('00! . luv-3 ‘fi‘lllauiu .963": (III a!..l!lop... CO 9!! ' .~ V ¢|a‘.l.. Hm MlllHlllHlllllllllllllllillllIllslhlillllblllllIllllllHll 3 1293 008803 This is to certify that the dissertation entitled Dissimiltory Reduction of Nitrite and Nitric Oxide by Denitrifying Bacteria presented by Weizhang Ye has been accepted towards fulfillment of the requirements for PILD Microbiology Mk professor degreein MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or beiore date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity institution cmma-QJ DISSIMILATORY REDUCTION OF NITRITE AND NITRIC OXIDE BY DEN ITRIFYING BACTERIA BY Weizhang Ye A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health ABSTRACT DISSIMILATORY REDUCTION OF NITRITE AND NITRIC OXIDE BY DENITRIFYING BACTERIA BY Weizhang Ye Denitrifying bacteria usually prefer oxygen as the electron acceptor and use other nitrogen oxides only when oxygen is limited. Understanding of denitrification is of importance for agriculture and environment. The key step of denitrification is the reduction of nitrite since it converts a mineral form of nitrogen to gaseous forms. This thesis is focused on understanding the pathway, mechanism, and regulation of nitrite and nitric oxide reduction. Cytochrome cd1 nitrite reductase purified from Pseudomonas stutzeri J M300 produced NO as the major product and N20 as a minor product. With the addition of Fe(II), both production of NO and N20 was enhanced. Similar enhanced effects of Fe(II) was also observed for the copper-containing nitrite reductase from Achromobacter cycloclastes. The mechanism of NO reduction was studied with 180 labeled water in organisms containing copper or cytochrome cd1 nitrite reductases. It was found that 180 from water was incorporated into both the product and the substrate, producing N2130 and N180. This exchange reaction reached equilibrium rapidly and could be inhibited by electron donors added to the crude cell extract. These results suggest that an enzyme nitrosyl complex exists during reduction of NO, similar to that found in the reduction of nitrite. The findings that N20 is the major product of nitrite reductase and that 180 exchange during NO reduction, raise questions on the hypothesis that N20 is formed via the direct attack of the second nitrite. To study the relationship between the N02' and NO reduction, mutants deficient in nitrite reduction (N ir‘) were obtained with Tn5 mutagenesis in Pseudomonas fluorescens which contains a cytochrome cd1 nitrite reductase, and in Pseudomonas sp. G- 179 which contains a copper nitrite reductase. Mutants in the nitrite reductase structural gene from both strains showed not only a loss of nitrite reductase activities, but also a reduction in NO reductase activities. In P. fluorescens, the 180 exchange reaction was abolished in all Nir' mutants. These results suggest that although reduction of N O is distinct from the nitrite reduction, mutation in the nitrite reduction step had physiological and /or genetic effects on NO reduction. By isolating Tn5 Nir' mutant, a copper nitrite reductase gene was isolated from Pseudomonas sp. G-17 9 and showed strong homology with other denitrifiers that contains the copper-type nitrite reductase. Analysis of the upstream region of this gene indicates that its expression may be under the control of FNR and 0'54. The role of FN R is further supported by studies with an FNR‘ mutant from P. aeruginosa , which showed no growth on nitrate, nitrite and nitrous oxide. in the hope that all these years' efi‘ort will help understand somethings in the nature iv ACKNOWLEDGMENTS Without the support of my parents, Li Xiuqiong and Ye Rongtao, during those difficult years of my childhood, I would never have developed interests in science. My exceptional college English teacher gave me encouragement and inspiration to pursue an educational opportunity in the US. My wife and best friend, Mary Mei Mao, contributed tremendously to my personal and academic lives. During the course of my research, Aruna Alahari, Mark Coyne, Jim Cole, Inez Taro-Suarez, Dave Harris, and Marcos Fries have provided me with much needed help and suggestion. I enjoyed the company of Cathy McGowan in that small corner of the lab. I also enjoyed those good discussions with Rob Sanford and Joan chee-Sanford and working in the same lab with J ong-ok Ka and Jorge Santo Domingo. I would like to thank Drs. Larry Snyder, Michael Thomashow and Michael Bagdasarian, who have kindly served on my guidance committee. Dr. Bruce A. Averill have provided me with excellent advises for my research. Finally, Dr. James M. Tiedje has been an excellent advisor and I have benefited greatly in many ways from his guidance. TABLE OF CONTENTS List of tables ...................................................................................................... ix List of figures .................................................................................................... xii Chapter One: Dissimilatory reduction of nitrite and nitric oxide by denitrifying bacteria, a review .................................................... 1 Introduction ............................................................................................... 2 Establishment of NO as an intermediate of denitrification .................... 5 N O is the major product of dissimilatory reduction of nitrite ...... 5 Purification of NO reductases ........................................................ 8 Reduction of NO is energy-conserving ......................................... 10 Mechanism of N 0 reduction ......................................................... 11 N09; and NO reduction are two distinct processes .................... 12 Cytochrome cd1 nitrite reductases ......................................................... 13 Cu-type nitrite reductases ...................................................................... 14 Genetics of nitrite reduction ................................................................... 17 Regulation of gene expression ............................................................... 21 Regulation by oxygen ................................................................... 21 Regulation by substrate ............................................................... 24 Conclusion ................................................................................................ 25 References ................................................................................................ 27 Chapter Two: Fe(II) enhanced N O and N20 production by cytochrome cd1 nitrite reductase from Pseudomonas stutzeri J M300 ........ 39 Abstract .................................................................................................... 40 Introduction .............................................................................................. 41 Materials and Methods ............................................................................ 43 Results ...................................................................................................... 44 Discussion ................................................................................................. 47 References ................................................................................................ 57 Chapter Three: H2180 isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria ............................................................... 59 Addendum to Chapter Three: A study on the reversibility of 130 exchange during reduction of NO in Alcaligenes eutrophus .................. 64 Chapter Four: Mutants of Pseudomonas fluorescens deficient in dissimilatory nitrite reduction are also altered in nitric oxide reduction ................................................................ 68 Chapter Five: Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. G- 17 9 with a copper nitrite reductase ........................................................................ 74 Abstract ................................................................................................... 75 Introduction ............................................................................................. 76 Materials and Methods ............................................................................ 78 Results ...................................................................................................... 81 Discussion ................................................................................................. 84 References ................................................................................................ 96 Chapter Six: Characterization of the structural gene for a copper-containing nitrite reductase and its homology to other denitrifiers .................................................. 102 Abstract .................................................................................................. 103 Introduction ............................................................................................ 104 Materials and Methods .......................................................................... 106 Results .................................................................................................... 107 Discussion. .............................................................................................. 110 References .............................................................................................. 124 viii List of Tables Table Page Chapter One 1 Characteristics of NO reductases ......................................................... 9 2 Characteristics of genes involved in nitrite reduction ........................ 20 3 Putative regulatory features of genes involved in denitrification ...... 23 Chapter Two 1 Conversion of nitric oxide or nitrite to nitrous mode in different assay conditions ................................................................. 49 2 Production of nitric oxide and nitrous oxide by nitrite reductase at the presence of different chelators ........................................................ 5O 3 Effect of Fe(II) on the production of nitric oxide and nitrous oxide from nitrite by Cu—containing nitrite reductase from A cycloclastes .......... 51 ix Isotope study on Fe(II)-enhanced production of N20 by nitrite reductase ................................................................................................. 52 Chapter Three Percent exchange with H2130 during dissimilatory reduction of NO and nitrite by whole cells of denitrifying bacteria containing either heme cd1 or copper nitrite reductases ................................................... 61 Exchange with H2130 during reduction of NO by crude extracts of R. sphaerodes forma sp. denitrificans in the presence of added electron donor or mediator .................................................................................... 61 N itrosyl transfer from 15N O to 14N3' in reaction containing H2180 and R. sphaeroides crude extract ........................................................... 61 Chapter Four Characteristics of Nir- and Nos- mutants from P. fluorescens AK-15..70 Rates of N02‘ and NO reduction and extents of 180 exchange by wild type and mutant strains of P. fluorescens AK- 15 ................................... 71 Chapter Five Growth characteristics of Tn5 mutants of Pseudomonas sp. strain G- 179 deficient in denitrification pathway ................................................ 88 Remaining nitrate and accumulation of nitrite following anaerobic growth of Tn5 mutants of Pseudomonas sp. G-17 9 on nitrate .............. 89 Activities of N 02‘ and N O reductases in wild type and mutants of Pseudomonas sp. G-179 ........................................................................... 90 Chapter Six Regulatory features of genes involved in denitrification ..................... 119 A summary of Western and Southern blots of different denitrifiers .............................................................................. 120 xi List of Figures Figure Page Chapter One 1 Denitrification pathway in gram-negative bacteria ............................... 3 2 Gene organization of the operon containing cd1 nitrite reductase ....... 18 Chapter Two 1 Fe(Ilienhanced N O and N20 production by purified nitrite reductase from P. stutzeri J M300 ........................................................................... 54 2 Production of NO and N20 from N02- by the nitrite reductase at the presence of different concetration of Fe(II) ............................................ 55 3 Effects of pH on the conversion of NO to N20 by Fe(II) (panel A) or N02' to NO and N20 by nitrite reductase (panel B) .............................. 56 Chapter Three xii Production of 18NO and N213O during reduction of NO by whole cells of Alcaligenes eutrophus in reactions containing H2130 ........................... 67 Chapter Four Western blot of proteins from wild type and mutant strains ................ 71 The time course of 180 exchange in resting cells of the YT101(A) and YT25 1 1 (B) mutant strains ..................................................................... 71 Chapter Five Southern analyses of Tn5 insertions from Nir- mutant strains ............ 95 Physical mapping of the Tn5 insertion sites in RTCZZ and RTC23 ..... 94 Western blot of proteins from wild-type and mutant strains ............... 93 Growth curve and evolution of N20 and NO during anaerobic growth on nitrate in wild type RTCOl and mutant strain RTCOB ................... 92 Chapter Six Physical mapping of the 1.9 kb EcoRI and BamHI fragment ............. 118 xiii Nucleotide sequence of the 1.9 kb EcoRI and Barn HI fragment and the predicted amino acid sequence of the Cu-type nitrite reductase ........ 119 Comparison of the amino acide sequences of Cu—dNirs from Pseudomonas sp. G-17 9 and Achromobacter cycloclastes (AC) ........... 121 Comparison of the copper binding region for Cu-dNirs from Pseudomonas sp. G-179 and Achromobacter cycloclastes. ................... 122 Hydropathy index of the encoded nitrite reductase by the method of Goldman, et a1 ( ) or Kyte and Doolittle ( ........... ) .................... 123 xiv Chapter One Dissimilatory Reduction of Nitrite and Nitric oxide by Denitrifying Bacteria a review ix) Introduction Denitrification is the dissimilatory reduction of nitrate to nitrogen gases. It is carried out by a variety of bacteria that occupy a wide range of natural habitats, including soil, water, foods and digestive tract (78). These organisms are facultative and prefer oxygen as their electron acceptor, but in the absence of oxygen they can obtain energy from electron transport phosphorylation coupled to the denitrification. Besides the traditional concern over the loss of fertilizer as a result of denitrification, another great emphasis has been on the negative impact of NO and N20 evolution, which has been found to contribute to ozone destruction and global warming(11,34,51). At the same time, denitrification has been very useful in the treatment of waste discharges. Utilization of nitrate as the terminal electron acceptor may often be a better way to degrade contaminants by bacteria under oxygen limiting conditions, such as in the aquifers (33). A growing number of denitrifiers with these properties are being discovered( 14,52). Four enzymatic steps are required to convert nitrate to dinitrogen gas (Figure 1). In gram-negative bacteria, nitrate reductase is bound to the cytoplasmic membrane with its active site facing the cytoplasm (61). Nitrate is reduced at the cytoplasmic site of the membrane and therefore has to be transported inside the cell. However, nitrite reductase is located in the periplasmic space. It is believed that import of nitrate and export of nitrite is carried out via an antiport system. There are two major types of dissimilatory nitrite reductases: those containing l - N Periplasmic N NO N20 ‘ 2 /\ Nii’ w m Nar Nor 7 U Figure 1. Denitrification pathway in gram-negative bacteria. Nar, nitrate reductase; Nir, nitrite reductase; Nor, nitric oxide reductase; Nos, nitrous oxide reductase. cytochrome cd1 (cd1-dNirs) and those containing copper ( Cu-dNirs) in the active site (10,30,54). The major product of nitrite reduction by nitrite reductase is nitric oxide, which is subsequently reduced to nitrous oxide by the nitric oxide reductase. Nitric oxide reductases purified so far are membrane-bound(8,12,29). In organisms like P. aureofaciens and P.chlororaphis, N 20 formation is the last step of denitrification (16). Loss of N20 reduction ability can also be found in some soil isolates that have been cultured for many generations in laboratories. Reduction of N20 is accomplished by nitrous oxide reductase which is located in the periplasmic space. Nitrous oxide reductase is usually isolated under anaerobic conditions and exposure to oxygen inactivates most of the known enzymes (30,61). The focus of research on denitrification has been on the process of nitrite reduction to nitrous oxide for the following reasons. 1) Dissimilatory reduction of nitrite is the key point of divergence from assimilatory nitrogen metabolism because the products of dissimilatory nitrite reduction leads to formation of gases (Fig.1). 2) Evolution of NO and N 20 has significant environmental impact (34). 3) The mechanism of formation of the N=N bond in nitrous oxide was unknown (1), and whether NO is a free and obligate intermediate of denitrification has been controversial. During the past few years, a great deal of information has been accumulated to support the idea that nitric oxide is indeed an obligate intermediate in denitrification. Nitric oxide reductases have been successfully isolated (8,12,29), the crystal structure of the copper dNir from Achromobacter cycloclastes has been elucidated (23), and great progress has been made in the studies of genes involved in nitrite reduction from denitrifiers containing cd1 or Cu-dNirs (39,57,59,65). The genes involved in nitric oxide reduction in denitrifiers containing cd1 nitrite reductases has also been identified (5,6). These findings provide a much better understanding of denitrification and point to new directions for future research. Establishment of N O as an obligate intermediate of denitrification. Nitric oxide has been proposed as an intermediate in denitrification for several decades (16,50). However, there has been uncertainty as to the true role of N O in the reductive pathway. It was reported that there was a lack of scrambling between the N species from nitrite and added nitric oxide at high cell density in several denitrifiers (20). Unlike other intermediates in the pathway, NO is present only at very low steady state levels (4,26). No denitrifiers are able to grow on NO alone except Thiobacillus denitriflcans (35), probably due to the toxic nature of NO. The nitric oxide reduction system was poorly studied and the existence of enzymatic NO reduction was questionable. One group suggested that nitrous oxide could be formed from nitrite directly by a single enzyme, the nitrite reductase (1,67). NO is the major product of dissimilatory reduction of nitrite. Purified nitrite reductases studied so far produce NO as the major product and N20 as the minor one (7,43,69). The N20 produced can be abolished by chelators such as EDTA, suggesting chemical conversion of N O to N20 by a trace amount of Fe contamination(77). Shapleigh et al used Triton X-100 to inhibit NO reduction in crude extracts of several denitrifiers (55). In this system, 80-95% of the nitrite was recovered as NO. Addition of CHAPS- soluble extract with nitrite reductase but devoid of N 0 reductase restored the capacity to convert N02' to N 20. Production of NO also depends on the pH of the growth media (66). Between pH 6.5 and 7.0, reduction of nitrite or nitrate leads to accumulation of N O in in resting cells of P. aeruginosa. At pH above 7.5, only N 20 is detected. To quantify the amount of NO diffusible out of the cell during reduction of nitrite or nitrate, extracellular hemoglobin(Hb) was used to trap NO in several denitrifiers (24). About 2 N atoms in 3 were trapped irreversibly as NO. The rate of HbNO formation was approximately zero order at the diffusion controlled limits. Since the formation of HbNO was irreversible, the denitrification pathway was short-circuited. To better study the production of NO and its subsequent reduction to N20, Zafirou, et al, used gas sparging to measure the extracellular NO (74). This process permits kinetic measurements under partially reversible conditions. The yield of N 0 gas depends on the rate of gas flow in this system and the maximum scavengeable NO was estimated to be 78% with a simplified Michealis-Menten equation (4,74), in agreement with the Hb trapping experiment. All the above experiments demonstrated that NO can diffuse out of the cell and that a majority of the N atoms from nitrite or nitrate are found in NO in vivo before it is reduced to N 20. If NO is the intermediate during the reduction of nitrite to nitrous oxide, exogenous NO would have two effects: (i) increased accumulation of endogenous NO as the exogenous NO increased due to competition for NO reductase or even inhibition; and (ii) a nearly completely random combination (scrambling) between the exogenous N and endogenous N to form N20 during simultaneous reduction of nitrite and NO. These two phenomena were observed by Firestone et al (16) in 13N labelling experiments with P. aureofaciens and P. chlororaphis. Complete scrambling was also found in Paracoccus denitriflcans (25). Early observations that there was a lack of extensive scrambling between 14N and 15N with a high density of cells might have been due to lack of equilibration between the gas and liguid phases (25). The result of random scrambling is not consistent the hypothesis that the bulk of N20 is formed directly by the nucleophilic attack of a second nitrite molecule on the nitrosyl complex in the nitrite reductase(1,67). However, the above experiments do not rule out the possibility that some of the N 20 produced comes from the direct reduction of nitrite without the formation of N O. The ultimate proof that NO is the only product of nitrite reductase comes from the isolation of Nor' mutants which lack of the ability to convert NO to N20. These mutants were created by gene replacement by Zumft and his colleaques (5). No N20 was produced by the cd1 nitrite reductase from reduction of nitrite in these mutants and thus suggests that NO is the obligate intermediate in vivo. The Nor' mutant is conditionally lethal under anaerobic conditions in the presence of nitrate due to the accumulation of toxic NO. A double mutations in both nitrite and NO reduction rendered the bacteria again viable. To address the question of a low steady-state levels of NO, Gorestski, at el, (25,26) showed that a low level of NO allows NO to be an intermediate without reaching toxic steady-state levels. This is accomplished by a very low apparent Km of less than 10 uM and a higher Vmax of the nitric oxide reductase than the nitrite reductase. It is estimated that the Vmax for of NO uptake in five denitrifiers at low cell density condition exceeded the Vmax for nitrite uptake. Steady state concentration of NO under denitrifying conditions ranged from 1 to 65 nM. Once established, the amount of NO does not change with time and is independent of the initial concentration of nitrate or nitrite. This level was re-established after addition of exogenous NO or loss of NO by sparging. Formation of nitric oxide complex of cytochrome c' in cells of denitrifying bacteria may play a role in reducing the level of NO in conjunction with the NO reductase (73). Purification of NO reductase. Most of the NO reductase activity is found in the membrane fraction (28,77). Choices of suitable detergents are essential for purification of the enzyme. Grant, et al, used CHAPSO to extract NO reductase from the membrane fraction of Paracoccus halodenitrificans, resulting in the separation of nitrite and nitric oxide reductase activities (28). Cytochromes were found in the nitric oxide reductase fraction. NO reductase from P. stutzeri was purified with Triton X-100 (29), while NO reductase from Paracoccus denitriflcans was purified with dodecyl maltoside (8). A single purification scheme with octyl glucoside is also reported to purify the NO reductase from Paracoccus denitrificans(12) Characteristics of these two NO reductases are summarized in Table 1. They are both cytochrome complexes Table 1. Characteristics of NO Reductase P. stutzeri (28) Pa. denitrifican (8,12) MW(kDa) 38,17 37.18.45.29 Heme types b,c b,c Specific Activity 12.7 11 Km < 1 uM Iron/M r 6 5.2-6.1 pH optimum 4.8 Absorption maxima 420.5,522.5,552.5 420.5,552.2,558.8 10 containing heme b (associated with 37 or 38 kDa subunit) and heme c (associated with the 17 or 18 kDa subunit). Each NO reductase molecule contains two heme groups in both organisms, but there are 6 Fe molecules in the enzyme isolated from P. stutzeri . The number of nonheme Fe molecules in the enzyme from Pa. denitriflcans is around 2.2 to 3.1. The role of these heme or nonheme iron atoms is unknown. The activity of NO reductase from P. stutzeri was markedly enhanced by the addition of lipids, such as soybean lipids or detergents ( e.g. n-octyl-b-d- glucopyranoside ) (29). The enzyme activity is inhibited by cyanide with a Ki of 2.6 mM. NO reductase can be inhibited by addition of high concentration of NO. Inhibition of NO reduction activity by nitrate and nitrite was also been reported (50). Reduction of NO is energy-conserving. As the intermediate of denitrification, one important function is to serve as the electron acceptor and conserve energy. Proton translocation unique to NO reduction under denitrifying conditions has been shown in a number of idenitrifiers (53). Values obtained were consistent with the expected ratios of 0.5 mol of H+lmol of NO for reduction of NO to N20 in Paracoccus denitriflcans (7). Antimycin A strongly inhibited the NO-dependent proton translocation, further suggesting that a proton electrochemical gradient is generated. NO reduction with inverted membrane vesicle preparations from P. denitrificans give a ratio of 0.75 Pi per pair of electrons, comparable to that 11 found in the reduction of nitrate (7). However, an NO-dependent Hi“- gradient was not observed in either P. aeruginosa or A. brasilense(66). This result challenges the significance of N 0 reduction for the purpose of energy generation in these two organisms. More experiments are needed to confirm these observations, because a small amount of 02 was present in the assay system as indicated by the authors (66). Mechanism of N 0 reduction. Our studies on the mechanism of NO reduction show(71): 1) exchange with H2180 , resulting in production of N218O; 2) competition of 180 exchange by availability of electrons ; 3) attacked of a N intermediate by nucleophilic compounds; 4) formation of N180 due to the reversible reactions before N O is reduced. These findings suggest that the formation of an enzyme nitrosyl, E-NO+, during dissimilatory reduction of NO, similar to the intermediate proposed for reduction of nitrite by nitrite reductase (21). The site of 180 exchange during NO reduction is presumed to be the NO reductase. The 180 exchange reaction during reduction of NO differs among different denitrifiers studied. Those with the heme cd1 -dNiRs exhibited relatively less exchange, while organisms with Cu-dNiRs gave generally higher extents of exchange but over a broader range of values. In two organism containing Cu-dNirs,P. aureofaciens and A. cycloclastes, very little 130 exchange was found during reduction of N02“ to N20, but a significant amount of exchange was observed during reduction of NO to N 20. This suggests that there are some mechanistic differences in the reduction of extracellular N O as compared to the reduction of the intracellular pool of NO. 12 The realization that NO, as a denitrification intermediate, can undergo 180 exchange was completely unexpected and raises questions about previous studies on the mechanism of N=N bond formation from nitrite (21,67). In these studies, 180 exchange was determined by measuring the 130 enrichment in N20 product using nitrite as the substrate. As a result, it is impossible to determine whether the observed 180 exchange occurred during reduction of N02“ to NO or reduction of NO to N20. In nitrosyl trapping experiments with azide or hydroxylamine and whole cell or crude extracts, it is also impossible to determine at which step the nucleophilic attack took place. It was shown that catalysis of nitrosyl transfer seemed to depend on N 0 when nitrite and azide were used, since removal of NO by CrSO4 eliminated the nitrosation reaction(27). It is possible that nucleophilic attack by azide or hydroxylamine occurred after formation of NO from nitrite in these previous experiments. Therefore, we conclude that the evidence does not support formation of the N=N bond by attack of a second nitrite molecule (67). Nitrite and nitric oxide reductions are two distinct processes. Purified cd1-dNirs do not have the ability to convert NO to N20 (43). In crude extracts, when Cu-type nitrite reductases were inhibited by the chelator, DDC, the ability to convert NO to N20 was not affected (54). In P. stutzeri, which contains a cd1-dNir, a normal rate of NO conversion was observed in Nir' mutants(75). These results indicate that nitrite and NO reductions are two distinct processes. However, this does not imply that these two steps do not influence each other. Tn5 Nir' mutants obtained from P. fluorescens AK—15 showed a significant decrease in the rate of NO reduction (72). Furthermore, the oxygen atom exchange with H2130 during 13 reduction of NO was abolished. This suggests that there is a functional or genetic interdependence of these two steps in this organism. In the case of P. stutzeri, two Nir' mutants have been found to have effects on the production of cytochrome c552 and soluble alpha-type cytochromes (75). Cytochrome cd1 Nitrite Reductase cd1-dNirs have been isolated from a large number of bacteria including Pseudomonas aeruginosa, Thiobacillus denitrificans, Paracoccus denitriflcans, Pseudomons stutzeri , Paracoccus halodenitrificans, and Alcaligenes faecalis (30). These enzymes are composed of two identical subunits with a molecular mass of 60 kDa, and each contains one heme c prosthetic group covalently linked to the polypeptide chain and one heme d1 moiety noncovalently associated with the protein. Heme 0 binding ligands (39,56,69) are located near the N- terminus of the protein. Antibodies raised against the dNir from P. aeruginosa cross-react strongly with those from P. fluorescens and Alcaligenes strains, which are commonly found in natural environments (10). Comparison of the amino acid sequences of nitrite reductases from P. aeruginosa and P. stutzeri strain Zobell reveals 56.4% identity (39). The heme 6 binding regions near the N-termini show strong homology (39,69). All dNir gene sequences reveal the existence of a signal peptide, in agreement with location of these enzymes in the periplasm (39.56.30). Incorperation of both heme c and heme (11 into the apoprotein is proposed to occur inside the periplasm (49). 14 The presence of heme d1 is unique in denitrifiers with cytochrome cd1 nitrite reductases. Chang and Wu proposed a porphyrindione (dioxoisobacteriochlorin) structure for this green colored chromophore (9). The apoprotein lacking the heme d1 could be reconstituted with the synthetic heme d1 and about 80% of enzyme activity could be restored, indicating the correct structural model of heme d1 and the key role of this heme in the conversion of nitrite to NO (69). The detailed kinetics of nitrite reduction by cytochrome cd1 was studied by Stopped-flow and rapid freezing EPR (57). The first step involved electron transfer from reduced d] to nitrite and dehydration, resulting in the binding of oxidized heme d1 to NO species. This step is very fast, being lost in the dead time of rapid mixing. In the second step, the electron is passed from the heme c to heme d 1 with a rate constant of 1 $4. When heme Cl] is NO-bound, the rate at which heme c can accept electrons from ascorbate is remarkably increased as compared to the oxidized enzyme and this electron transfer results in the formation of c+2d1+2-NO, which can be detected by EPR. The binding of NO to the reduced heme d] is very tight. Since this study was done at pH 8.0, the kinetic data under physiological conditions (pH 7.0) may be different. Cu-type nitrite reductases Denitrifiers with copper dNirs occur at the frequency of 32% among numerically dominant isolates from soil (10). These denitrifiers include species from Pseudomonas, Alcaligenes, Corynebacterium, Bacillus, Rhizobium, Agrobacterium, and Rhodobacter. Most of the Cu-dNirs cross- react with the polyclonal antibodies raised against copper dNir from A. cycloclastes (10) or from R. sphaeroides fp. denitriflcans (47). These results 15 suggest that most Cu dNirs share substantial similarity. Cross-reaction is not found in some Bacillus and Rhizobium species( 10). The nitrite reductase from A. cycloclastes is best studied (15,23,36). The enzyme is a trimer with the molecular weight of 36 kDa per monomer and it has a total of six copper atoms per molecule (15,23). The amino acid sequence (15) and 2.3 A X-ray stucture (23) reveal that the two copper atoms in the monomer comprised of one type 1 copper site and one putative type 2 copper site, which are about 12.5 A apart. The type 2 copper is bound with nearly perfect tetrahedral geometry by residues not within a single monomer, but from each of two monomers of the the trimer. Amino acid residues 8-175 folds into domain I with a Greek-key B-barrel structure. Domain I contains the type 1 copper held by ligands of Cy3136, Hisl45, Met150, and Hi395. Residues from 175-340 fold into the domain II which is also a B-barrel similar to the type 1 copper binding domain. The type 2 copper binding ligands are comprised of His 100 and His 135 from the domain I of one subunit and His306 from domain II of the second. It has been reported that the active site of Cu dNirs is the type 2 copper, since only type 1 c0pper was detected in the copper dNirs isolated from Pseudomonas aureofaciens (76) and Alcaligenes xylosoxidans (46) and the nitrosyl complex formed upon the addition of NO was found at the type 1 site (62). However, the following evidence strongly suggests that type 2 copper site is required for enzymatic activity and it may even be the active site of the enzyme. (i). Nitrite binds to the type 2 copper site, not to the type 1 Cu. This is observed with competition experiments between nitrite and azide (Hulse, C. and B. Averill, submitted for publication ) and with studies of 2.3 A X-ray 16 stucture of the enzyme (23). (ii). Type 2 Cu can be removed from the enzyme and reconstituted, and p0protein with very small amount of type 2 Cu has very low activity (Libby, E and B. Averill, submitted for publication). The reconstituted enzymes showed increased activity as the ratio of type 2 copper atoms per enzyme molecule increased. (iii) Ascorbate oxidase has a type 2 Cu in its active site and its location is very similar to the dNir from Achrombacter cycloclastes(23). (iv). The amino acid sequence of the copper dNir from Pseudomonas aureofaciens reveals type 2 copper binding site (22). It is possible that the type 2 copper is weakly held by its ligands and thus can be removed from the enzyme molecule during purification. It appears that the role of type I Cu is to accept electrons from its physiological donor and pass them to the type II copper active site, similar to the relationship between the heme c and heme d1 in the cytochrome cd1 enzyme. The reduced form of azurin or pseudoazurin, the blue copper protein, is found to serve as an effective physiological electron donor for copper dNirs from A. faecalis strain S-6 (36), Achromobacter cycloclastes (45) and P. aureofaciens (76). This small blue copper protein has molecular weight of 12 to 15 kDa and has one Cu atom per molecule. Three absorption maxima are found in the oxidized form: 453, 595, and 750 nm and only one peak at 278 nm is found when reduced. The EPR spectrum is typical of those containing type I Cu. The blue protein can be reduced by various reducing agents including dithionite, ascorbate, cysteine, 2-mercaptoethanol, dithiothreitol, glutathione, and hydroquinone. The reduced protein is autooxidized very slowly. The electron transfer from blue protein to dNir was studied (42). When the concentration of reduced pseudoazurin was 17 higher than that of oxidized dNir, the reduction of the latter occurred in two steps: a burst phase followed by steady-state kinetics. However, when the concentration of pseudoazurine to dNir was 1:1, only the burst kinetics was observed. Inhibitors of Cu-dNirs include metal chelators such as diethyldithiocarbamate (DDC), thioglycollate, o-phenanthroline, 8- hydroxyquinoline, ethylene diamine tetraacetic acid (13) and sulfhydryl group inhibitors such as N-ethylmaleimide and p-chloromercuribenzene sulfonate or p-chloromercuribenzoate (36). Electron transport chain inhibitors, KCN and CO, also inhibit the Cu-dNir activity. Genetics of nitrite reduction Characterization of genes involved. An operon containing the cd1-dNir structural gene (nirS) is found in P. stutzeri (39,59), P. aeruginosa (56) and P. fluorescens (72)(Fig. 2). This operon starts with 18 \I/ Anaerobic TI'GAC....ATCAA —— S T B M C P. stutzeri l S M * C P. fluorescens P. aerugmosa Fig. 2. Gene organization of the operon containing cd1 nitrite reductase. S: nitrite reductase structural gene; T: tetraheme; B: cytochrome C552 M: cytochrome C551; C: another monoheme. l9 nirS. Immediately downstream are nirM , encoding the cytochrome c551, and nirC (ORF5) encoding an unknown heme protein in P. aeruginosa and P. fluorescens. In P. stutzeri nirT, encoding an unknown tetra-heme protein, and nirB , encoding cytochrome c552, are interchelated between nirS and nirM (39). This difference in gene organization raises questions with regards to the role of cytochrome C551 and cytochrome C552 in nitrite reduction among different denitrifiers. In P. aeruginosa and P. fluorescens, the location of the cytochrome cs5l gene is consistent with the in vitro results showing that reduced cytochrome c551 can serve as electron donor to nitrite reductase._ However, in P. stutzeri, cytochrome 0551 is not regulated by anaerobic condition. There is a 320 base pair intergenic region with multiple inverted repeats between the nirB and nirM. Thus cytochrome c551 may belong to a separate operon and its exclusive role in nitrite reduction is questionable in P. stutzeri (39). Instead, cytochrome c552 is produced under anaerobic conditions. The location of its structural gene (nirB) in the nir operon implies its role as an electron carrier for nitrite reduction. Since mutation in nirT leads to a defect in electron donation but has no effect on the production of active nitrite reductase, the authors propose that the hydrophobic portion at the mature N-terminus of the nirT protein might anchor the cytochrome in the membrane and allow electron flow between the respiratory chain and a periplasmic acceptor. Electrons could pass directly from NirT to NirS, or via cytochrome C55,. The function of NirC is unknown, although it is also has a heme c binding motif. Tn5 insertion in this region resulted in production of a defective nitrite reductase in P. fluorescens, suggesting Table 2. characteristics 20 of genes involved in nitrite reduction genes Protein Function heme c M r motifs nirS reductase nitrite reduction 1 59,532 nirM C551 e’ transport 1 8,563 nirC(ORF5) - - 1 9,202 nirT - e‘ transport 4 19,738 nirB 0552 e’ transport 2 28,180 nirD ? heme (1] synthesis - - 21 that this region, or gene(s) downstream, are important for the production of active nitrite reductase (72). Possible function of cytochromes encoded by nir operons is summarized in Table 2. Apart from those in the nir operon, gene(s) responsible for heme d1 synthesis (nirD) has been identified. In P. stutzeri, two mutations were found the region containing nirD, leading to the production of an inactive nitrite reductase due to a lack of heme d1 prosthetic group. Cosmid- mapping and Southern hybridization revealed a close linkage of the genes nirS ,nosZ and nor(6). Structural gene for Cu-dNir (nirA) has been isolated from Pseudomonas. sp. G-179 (Ye,et al, submitted for publication) and P. aureofaciens (65). The deduced amino acid sequences appears homologous to that of Achromobacter cycloclastes. Regulation of gene expression. Regulation by oxygen. Although E. coli and other enteric bacteria can not denitrify, they can respire nitrate and nitrite under anaerobic conditions. The fnr gene is essential for the expression of genes involved in fumarate and nitrate reduction (38,60) under anaerobic conditions. A conserved symmetrical sequence, TTGATN4ATCAA, the FNR box, is located upstream of the FNR-dependent genes and operons and plays an important role in regulation of gene expression. Under low oxygen tension, the FNR protein acts as a transcriptional activator by binding to the FNR box. A similar regulatory mechanism was found for the anaerobic 22 metabolism of arginine, nitrate and nitrite in P. aeruginosa(17). With chemical mutagenesis, van Hartingsveldt et al, isolated a mutant which results in the inability of P. aeruginosa to dissimilate nitrate and nitrite (64). It was found later that this strain also lacks the ability to metabolize arginine under anaerobic condition (17). The gene is thus renamed as our for anaerobic regulation of arginine deiminase and nitrate reduction. Haas and his colleaques showed that anr encodes an FNR-like protein that acts on the consensus FNR box to regulate the gene expression under anaerobic condition in P. aeruginosa (17,19). The arcDABC operon responsible for enzymes in the arginine deiminase pathway of P. aeruginosa was rendered virtually noninducible by a deletion or an insertion in the -40 FNR binding site and by a mutation in the anr gene. Anaerobic induction requires the FNR box in cis and anr protein in trans. This are operon can be expressed at a low level in E.coli under anaerobic conditions. Introduction of an anr+ plasmid had a larger positive effect. The FNR-dependent promoter containing the consensus -40 sequence from E. coli was expressed well in P. aeruginosa and was regulated by oxygen limitation. anr and fnr proteins have 51% sequence identity, and several amino acid residues known to be essential for FNR function are strictly conserved in anr. Thus, ANR and FNR appear to have similar functions. 23 Table 3. Putative regulatory sequences from denitrifying bacteria.° Strains Genes fnr box ntrA box P. sp. strain G~179 Cu-nir T'I‘GAT....ATCAA Tl‘QQAGCAAAC ATQQI GTflAGC CGAGGTTQQT P. aeruginosa (56)” cd1 ~nir UNC CGGQAGTTCCCGACGQA AAGGGAGCGCC TCGCA P. stutzeri JM300 (59) ed] -nir TICAT....GTCAA TTGAC....ATCAA UN P. stutzeri Zobell (59) ed] -nir 'I'ICAT....ATCAA T'I‘GAT....GTCAA UN P. stutzeri Zobell (65) nos UN GWCCCTGAGCQQG P. aeruginosa (31) azu T'ICAC....ATCAG GCQECACATCT GTQQT Alcaligenes denitrificans (31) azu TTGAT....GTCAA CAgflCATGTGCC'I‘GQQG Alcaligenes faecalis S-6(70) Peudo-aw TTGAT....ATCAA GTCLQCGTGTTGAGLEC “ References are given in the parenthesis followoing the strain names. Abbreviations are nir: nitrite reductase gene; nos: nitrous oxide reductase gene; azu: azurin gene. 5 The presence of possible regulatory regions were identified in this work based on published results. ° UN=Unknown. 24 Putative FNR boxes have been found in the upstream region of the promoter of dNir structural gene from P. stutzeri (59) and Pseudomonas sp. G-179 (Ye, et al, submitted for publication). This box is also found in the upstream region of the gene encoding the pseudo-azurin, which is believed to be the physiological donor of the Cu-dNir in Alcaligenes faecalis S-6 (70). The presence of FNR boxes and the pleotrophic effects of theanr' mutation on nitrate and nitrite dissimilation strongly suggest that the our protein regulates the genes involved in denitrification under oxygen limited conditions. However, the interaction between the anr protein and the operator region for nitrite reduction or the denitrification pathway has not been characterized. Another regulatory protein ntrA, which encodes O54 has been found to be involved in diverse physiological processes, including nitrogen fixation and nitrate assimilation (37). The binding site for 0'54 usually has the GG and GC doublet separated by 10 bp. Analysis of the promoter regions of some genes involved in denitrification or anaerobic metabolism reveals putative ntrA boxes (Table 3). It has been found in genes for hemecd1 or Cu-dNirs, nitrous oxide reductase, pseudo-azurin, and azurin. These lines of evidence suggest that 654 may facilitate the recognition of RNA polymerase to promoters involved in denitrification. Regulation by substrates. Substrates for the denitrification pathway, such as nitrate, nitrite and N20 are required for the full expression of enzymatic activities. This is further supported at the gene level. Hirouki, et al, introduced the xylE gene which encodes catechol 2,3-dioxygenase 25 (C230), into the nir operon containing the nitrite reductase gene and promoter region, and studied gene expression under different conditions by measuring C230 enzyme activity (3). When cells were grown on arginine anaerobically in the absence of nitrate, the promoter activity of the operon was approximately one-fifth of that under anaerobic denitrifying conditions with nitrate as the electron acceptor. This experiment suggests that substrates activate the transcription of the genes involved in denitrification. Conclusions Evidence from several experimental approaches have now shown that nitric oxide is an intermediate in the denitrification pathway. Key evidence is that N or- mutants accumulate NO as the only product of nitrite reduction. NO reductases are cytochromes and contains heme c and b moieties. The mechanism of NO reduction is proposed to involve a nitrosyl complex based on the 180 exchange studies. Cu nitrite reductases may require both type 1 and type 2 Cu for their activities. Type 1 Cu is bound within a monomer whereas type 2 Cu is bound between two monomers. Regulation of nitrite reduction by oxygen may be under the control of an FNR protein and 0'54. This is supported by FNR‘ mutants which are inactive in denitrification and FNR boxes present upstream of the promoter of the nitrite reductase genes studied. Many areas of nitrite and nitric oxide reduction remain as possible focuses for study. Although NO reductases have been isolated and characterized from two organisms containing cd1 nitrite reductases, they have not been isolated from organisms with Cu-type nitrite reductases. It 26 is still unclear whether all the NO reductases are similar. The mechanism of NO reduction is poorly understood. The major role of N 0 reduction (detoxification or/and energy generation) in denitrification pathway remained to be clarified. In recent years, gene regulation has been one of major foci of research of prokaryotic genetics. However, very little information is available on regulation of denitrification.lt is of particular interest since it regulated by oxygen. The roles of fnr and (554 -like proteins need further investigation. Another aspect of gene regulation is the possible factors involved in signal transduction. Finally, studies on the components involved in electron transport including electron donors for specific steps and those further up in the cascade needs more emphasis. 27 References l. Averill, BA. and J.M. Tiedje. 1982. The chemical mechanism of microbial denitrification-- a hypothesis. FEBS Lett. 138:8-11. 2. Arai, H., Y. Sanbongi, Y. Igarashi and T. Kodama. 1990 Cloning and sequencing of the gene encoding cytochrome c-551 from Pseudomnonas aeruginosa. 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The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue copper protein from Escherichia coli cells. J. Bacteriol. 169:5648-5652. 71. Ye, aw, I. Taro-Suarez, J.M. Tiedje, and BA. Averill. 1991. H2130 isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria: evidence for an electrophilic nitrosyl during reduction of nitric oxide. J. Biol. Chem. 266: 12848-12851. 72. Ye, R.W, A. Arunakumari, B.A.Averill, and J.M. Tiedje. 1992. Mutants of Pseudomonas fluorescens deficient in dissimilatory nitrite reduction are also altered in nitric oxide reduction. J. Bacteriol. 174:2560-2564 73.) Mat (‘3 O *tJ Ba Ne 38 73. Yoshimura, T, H. Iwasaki, S. Shidara, S. Suzuki, A. Nakahara, and T. Matsubara. 1988. Nitric oxide complex of cytochrome c' in cells of denitrifying bacteria. J. Biochem 103:1016-1019 74. Zafirou, 0.0., (2.8. Hanley and G. Snyder. 1989. Nitric oxide and nitrous oxide production and cycling during dissimilatory nitrite reduction by Pseudomonas perfectomarina. J. of Biol. Chem. 264:5694-5699. 75. Zumft, W.G., H. Korner, S. Lochelt, A. Viebrock and K. Frunzke. 1988. Defects in cytochrome cd1-dependent nitrite respiration of transposon Tn5- induced mutants from Pseudomonas stutzeri. Arch. Microbial. 149:492-498. 76. Zumft, W.G., D.J. Gotzmann and PM.H. Kroneck. 1987. Me 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureafacies. Eur. J. Biochem 168:301-307. 77. Zumft, W.G. and K. Frunzke. 1982. Discrimination of ascorbate- dependent nanenzymatic , membrame-baund reduction of nitric oxide in denitrifying Pseudomonas perfectromarinus. Biochim. Biophys. Acta. 681:459-468. 78. Zumft, W.G. 1991. The denitrifying prokaryotes. p. 554-582. In: A. Balows, at e1 (ed.), The prokaryotes, second edition. Springer-Verlag, Inc., New York. Chapter Two Enhanced Efi‘ect of Fe(II) on Nitric Oxide and Nitrous Oxide Production by Cytochrome cd1 Nitrite Reductase from P. stutzeri JM300. Abstract We examined the effects of Fe(II) on the production of NO and N20 from nitrite under routine assay conditions for nitrite reductase from Pseudomonas stutzeri JM300. The major product of the enzymatic reduction of nitrite by the purified nitrite reductase was N O and a minor product was N 20. The enzyme did not appear to have the ability to convert N O to N 20. However, with the addition of 10 uM Fe (II), as much as 30% of the product was N20. Fe(II) at this concentration also enhanced NO production. This dual catalytic capability of Fe (II) led to an increase in the rate of nitrite reduction. The enhanced NO and N20 production was specific for Fe(II) and could not be obtained with other metal ions. Fe chelators diminished the production of N 20 by nitrite reductase in the presence of Fe (II). EDTA and NTA had little effect on NO production, but when Fe(II) was added NO production increased markedly. The Cu- containing dissimilatory nitrite reductase from Achromobacter cycloclastes also showed a similar increase in both NO and N20 production with the addition of Fe(II). Under the assay conditions of this experiment, the increased amount of N20 production from nitrite by 10 uM Fe(II) could not be accounted for by the chemical reduction of NO from the gas phase. Instead, the role of Fe(II) may be to intercept NO directly from the enzyme and efficiently reduce it to N20 . 41 Introduction The denitrification pathway has been established as : N03’ -> N 02‘ -- > NO and N20 --> N2. The key ecological step in denitrification is the dissimilatory reduction of nitrite since the subseqent products are unavailable to the biota. Two types of dissimilatory nitrite reductases in denitrifying bacteria: one type contains cytochromec and d1 and the other contains copper(4,9). Organisms containg the cytochrome cd1 nitrite reductase are more frequently isolated from nature, whereas Cu-type nitrite reductases are found in orgaisms that exhibit more phylogenic diversity and occupy a wider range of ecological niches (4). The product of nitrite reduction, NO, exists at very low steady-levels during denitrification (1). Nitric oxide reductases have been isolated from P. stutzeri strain Zobell (8) and Paracoccus denitrificans (3,5). Mutations in the structural genes for nitric oxide reductase in P.stutzeri Zobell generate only NO from nitrite and Nor“ mutants can not grown on nitrite due to the toxicity of NO (2). 130 exchange studies of N=N formation during reduction of NO indicates the existance of an enzyme-nitrosyl complex, similar to that found in the reduction of nitrite (13). Assay systems for nitrite or nitric oxide reductases in vitra include ascorbate - PMS, NADH-FMN, or NADH - PMS. Zumft and Frunzke (14) found that NO can be converted to N20 nonenzymatically by Fe(II) in the assay systems with ascorbate. Chelators for Fe(II) eliminate this nanenzymatic activity and allow the distinction between the enzymatic and chemical conversion of NO in the assay systems with membrane fractions 42 containing nitric oxide reductase. These findings lead these authors to suggest that the presence of adventitious iron and the ubiquitous use of ascorbate might have contributed to the nanenzymatic conversion of NO to N20 in previous work. However, their experiments were done with higher concentrations of Fe(II) ( 1.0 mM ) and of NO (41.1 umol in 12 ml flasks) than normally present in enzymatic assays for nitrite reductase. While their study was focused on characterizing the nanenzymatic reaction of Fe (ID-ascorbate with NO and the assay for nitric oxide reductase in membrane fractions, the effect of Fe(II) on N20 production in assays for nitrite reductase was not characterized in detail. In this experiment we firrther investigated the effects of Fe(II) on the production of both NO and N20 in assay systems for nitrite reductases without ascorbate. Under these conditions gas phase N O was not reduced to N20, but low concentration of Fe (II) increased both N O and N20 production from nitrite. Materials and Methods Enzymatic assay. The heme-type nitrite reductase (>95% pure) was purified as previously described from Pseudomonas stutzeri JM 300 (12). The assay for nitrite reductase activity was routinely carried out in an 8- ml serum bottle with a 1 ml solution containing 50 mM HEPES (pH 7.3), 4 mM NADH , 0.12 mM PMS and 1.0 mM NaN02 and 30 ug of enzyme. All water used was from reverse osmosis and Millicue treatment. Anaerobic conditions were established by repeatedly evacuating and flushing the gas phase with argon. Assay mixtures were incubated at 25°C with constant shaking ( 100 rpm ) with the serum bottles laid on their sides to ensure equilibration of dissolved gases between the liquid and gas phase. N O and N 20 was analyzed by gas chromatography (10). Standards for N 20 were purchased from Matheson (New Jersey, USA). NO standards were freshly prepared from pure NO. Fresh solutions of FeSO4 or FeCl were prepared in degased water. Fe(II) was added after PMS and NADH were present in the reaction mixture. For assays of the chemical reduction of NO, the same assay conditions were used except that 1.0 umol of NO was used as the substrate. Results were presented as average of duplicates. Isotope experiment. Na15N 02 (99% 15N) was purchased from Cambridge Isotope Laboratory (Massachusetts, USA) . Gas samples were analyzed with an HP 5985 gas chromatography/mass spectrometry system equipped with a Porapak Q column (13). The mass spectrometer was operated using electron impact and selective ion monitoring. Results Stimulatory efl'ects of Fe (II) on NO and N20 production. Without the addition of Fe (II), purified nitrite reductase produced mainly NO from nitrite ( Figure 1A). N20 was a minor product and made up no more than 10 % of the total nitrogen converted . With the addition of 10 uM of Fe(II), N20 production rate increased markedly and reached up to 30% of the total nitrogen converted. The rate of NO production was also increased, especially in the early stages of the reaction. As a result, the presence of Fe (II) approximately doubled the rate of nitrite reduction (Figure 1 B). The enhanced effects of Fe(II) on both NO and N20 productin could be observed at Fe(II) concentrations from 1 to 100 uM, with 10 uM being the optimum concentration for total N-gas production (Figure 2). At 1 mM, the enhanced effect of Fe(II) was reduced for both gases. Other metal ions tested individually were MnClg, CuSO4, ZnSO4, CaClg, MgSO4, and CoC12 at 10 or 20 nM. They showed no effect on NO or N20 production under the standard assay condition. Chemical conversion of NO to N20. The nanenzymatic conversion of NO to N20 depends on the concentration of both NO and Fe(II) (14). Since 1.0 umol of nitrite was used in the enzymatic reaction, 1.0 umol NO was used to evaluate the chemical conversion of NO added to the gas phase. The combination of PMS, NADH, and nitrite produced a very trace amount of N20 after 30 min without the addition of Fe(II) ( Table 1), indicating that NO 45 was fairly stable under these assay conditions. In contrast to the reduction of nitrite by nitrite reductase, the addition of 10 uM of Fe(II) did not markedly enhance N20 production from NO. The nanenzymatic NO reduction catalyzed by Fe (II) was inhibited at low pH (Figure 3 A). The optimal conversion was observed at pH 8 . At pH values higher than 9, the chemical conversion was slow. The pH optimum for nitrite reductase activity was around pH 7 (Figure 3 B). At pH 8 or higher, the enzymatic activity dropped sharply. The Fe(II) enhanced NO and N20 production from nitrite paralleled the pH profile of the enzyme and was different from that of the nanenzymatic N20 production. Efl'ect of chelators. A11 iron chelators inhibited N20 production by nitrite reductase in the presence of 10 uM Fe(II) (Table 3). DDC, a specific chelator for Cu, also diminished the N20 production. Specific Fe(II) chelators 2,2'-bipyridy1, o-phenathroline, 8-hydroxyquinoline and EGTA inhibited NO production as well. EDTA and NTA did not have any effects on NO production. However, combinations of EDTA and Fe(II) or NTA and Fe(II) has a stimulatory effects on NO production. The Fe(II) enhanced NO and N20 production from nitrite was not unique to heme-type nitrite reductase. This phenomenon was also observed with a Cu-type nitrite reductase purified from A. cycloclastes (Table 3). If Cu was removed by the Cu-specific chelator DC, the presence of Fe(II) catalyzed no NO production. 46 Isotope experiment . The chemical reduction of NO by 10 uM Fe (II) did not appear to account for the marked increase of N20 production from 1 umol of nitrite in the presence of 10 uM of Fe (II) and purified nitrite reductase (Table 1). To further test whether the chemical reduction of N20 under standard assay conditions was due to the chemical reduction of NO that was released to the gas phase and then equilibrated back to the solution, 14NO was added in the gas phase and used as a tracer. In this experiment, 357 nmol of 14NO was added to serum bottle 1 min before the start of the reaction with 1.0 umol of Na15N02. All the reaction vials were under constant shaking. Chemical reduction of nitric oxide from the gas phase will be a random process, producing l4N15NO proportional to the amount of 14N and 15N. In the experiment with 10 uM of Fe(II), 33% of the headspace was 14N and 77% was 15N0 of the total nitric oxide in the gas phase after 30 min of incubation. As a result, chemical reduction of nitric oxide in the gas phase should yield at least 33 % of 14N out of the total amount of nitrogen in nitrous oxide produced. The experiment yielded only about 5 %, inconstant with the above calculation. A similar result was obtained with 200 uM Fe(II) except that more 14N0 from the gas phase was converted to nitrous oxide. This result further indicates that most of N20 produced from nitrite by nitrite reductase under standard assay conditions plus Fe(II) was not due to the reduction of NO from the gas phase. 47 Discussion Zumft and Frunzke showed the catalytic ability of Fe(II) to reduce NO to N20 with PMS/ascorbate system in the absence of any enzyme (14). Our study extends information on the special catalytic role of Fe(II) by showing that both N O and N20 production from nitrite are enhanced when Fe(II) was added to both heme c,d1 and Cu nitrite reductases. Under our assay conditions (PMS/NADH, 1 umol of NO), little NO was reduced to N20 in the absence of enzyme. The amount of Fe(II) need for stimulatory effect on NO and N20 production from nitrite by nitrite reductases was 10 uM, which is equivalent to approximately 40 mol of Fe(II) per mol of enzyme. In the chemical reduction system studied by Zumft and Frunzke, reduction of NO to N20 increases as Fe(II) concentration increases, ranging from 50 uM to 1 mM. However, lower concentration of Fe(II) did not have a high capacity to convert added NO to N20 in the standard assay systems used in this experiment (Table 1). Thus it could not account for the marked increase of N20 produced by the enzyme when Fe(II) was present at least at concentrations below 200 nM. This is further supported by isotope experiment in which a random scrambling between the 14N and 15N from the gas phase was not observed ( Table 4). These lines of evidence suggests that the majority of the N20 produced in the presence of Fe(II) in the assay systems with nitrite and nitrite reductase was not due to the chemical reduction of NO released to the gas phase. One simple interpretation is that there is a lack of equilibration between the gas phase and the solution in the assay condition. Such a possibility has been proposed during the reduction of NO with high cell density (7). However, it is also possible that Fe(II) 48 molecules has a very high affinity for N O and thus is able to intercept its release into the gas phase. Direct channeling of NO from the nitrite reductase to Fe(II) molecules in the solution may increase the efficiency of NO reduction to N20. The possible catalytic mechanism responsible for the dual role of Fe(II) is unclear. The Fe(II) enhanced effect on NO production may be different from that on N20 production since EDTA and NTA chelators inhibited the later but not the former. Fe(II) may increase the efficiency of delivering electrons to nitrite reductase to enhance the NO production. Fe(II) may also able to relieve any inhibitory effect of NO by remove the NO from the enzyme. The complexes of EDTA-Fe(II) and NTA-Fe(II) may even make Fe(II) more available for these purposes, while inhibiting its catalytic ability to convert NO to N20. 49 Table 1. Conversion of nitric oxide or nitrite to nitrous oxide in different assay conditions.“ Assay mixture N20 (nmole-N) PMS + NADH + nitrite + NO PMS + NADH + enzyme + NO PMS + NADH + enzyme + Fe + NO PMS + NADH + enzyme + EDTA + NO PMS + NADH + enzyme + nitrite PMS + NADH + enzyme + Fe + nitrite finesse ‘1 Results presented were obtained after 30 min incubation. The amount of nitric oxide and nitrite used in the assay system were 1.0 umole. EDTA and Fe (II) used were at 1.0 mM and 10 uM, respectively. Results were average of duplicates. Table 2. Production of nitric oxide and nitrous oxide from nitrite by nitrite reductase in the presence of different chelators.“ Assay mixture NO N20 (nmol-N) Control 17 0.2 + Fe 50 11.4 EDTA 13 0 EDTA 4» Fe 110 0.3 EGTA 3 0 EGTA + Fe 5 0 NTA 28 0 NTA + Fe 123 1.4 DDC 23 0 DDC + Fe 31 0 Bipyridyl 3 0 Bipyridyl+ Fe 2 0 o-phenanthroline 3 0 o-phenanthroline + Fe 1 0 8-hydroxyquinoline 2 0 8-hydroxyquinoline + Fe 2 0 (1 Concentration for chelators was 1.0 mM. The Fe (II) concentration was 10 nM. NO or N20 was measured after 2 min of reaction. Reactions were carried out according to standard assay conditions. 51 Table 3. Effect of Fe (II) on the production of nitric oxide and nitrous oxide from nitrite by the Cu-nitrite reductase of A. cycloclastes“ (nmole) 2 min 30 min Assay mixture NO N20 Nap" NO N20 N r Control 106 0.4 106 873 32 909 + Fe 442 72 514 629 356 985 +DDC 0.5 0 0.5 4 0 4 +DDC + Fe 1.6 0 1.6 7 0 7 “Assay mixtures contained 1.0 mM of DDC or 10 uM of Fe(II) if used. 5 NT = Total amount of nitrogen. Table 4. Isotope study on Fe(II)-enhanced production of N 20 by nitrite reductase.“ Fe (II) 1‘4NO 15NO % l4N0 14N14NO 14N15NO 15N15NO %14N (uM) (nmol) (nmol) 20 355 713 33% 0.8 3.6 as 5% 200 350 620 37% 1.5 12.6 ' 124 9% ‘1 Different NO and N20 species are presented as nmol produced in 30 min. 357 nmole of 14NO was added to the reaction mixture 1 min before the addition of 1 mM Na15N02 (99% 15N) to start the reaction. Percent 14NO or 14N15NO was calculated based on the total amount of nitric oxide or nitrous oxide. Figure legends Figure 1. Fe (II) enhanced N0 and N20 production by purified nitrite reductase from P. stutzeri JM 300. Panel A: evolution of N0 and N20 during reduction of N02'. Panel B: total amount of nitrogen production calculated from panel A. The reaction was carried out under the standard assay conditions with 1 umol of nitrite. The Fe (II) concentration was 10 uM if used. Figure 2. Production of N0 and N20 from N02' by nitrite reductase in the presence of different concentrations of Fe(II). The reaction was carried out under the standard assay condition with 1 umol of nitrite. Results were obtained after 2 min of reaction. Figure 3. Effect of pH on the conversion NO to N20 by Fe(II) without nitrite reductase (panel A) or N02' to N0 and N20 by nitrite reductase (panel B). Reactions were carried out under the standard assay conditions in HEPES buffer adjusted to the indicated pH values. Results were obtained after 30 min of reaction. —0— NO, + F9 —O— NO, - Fe + N20, +Fe —0— N20, -Fe A 1000 800‘ 242:5 36:20am no EsoE< q 4 u q u q 0 m m m 30 20 10 +Fe -Fe 3O 20 10 800“ 600- 400- 2 no “ESE... :38. 200‘ Time (min) E fill Z/////////. _..|.| 7////////////////////////A _.. 7%//////////// l. .g E N0 Cl N20 nu n0 _ nu nu nu nU nu nu Ru Afi n6 9g 4| 2.385 3268a mo 250:3. 100 1000 1 10 0A 0 Fe(II) Concentration (uM) <—‘.— 2mMFe —'O-— 2uMFe A 12 4 4 4 20- q 0 4 80 60 -‘ £355 223 38:2 2 330 03.2 no cozoauom —O— NO, +Fe + NO, -Fe N20, -Fe —0— N20, +Fe \ .Ii a . m .m 242:5 32:55 no EsoE< 12 10 pH 57 1. Betlach, MR. and J.M. Tiedje. 1981. Kinetic explanation for accumulation of nitrite , nitric oxide, and nitrous oxide during bacterial denitrification. Appl. Environ. Microbiol. 42:1074-1084. 2. Braun, C., and W. Zumft. 1991. Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide. J. Biol. Chem. 266:22785-22788. 3. Carr, G.J. and SJ. Ferguson. 1990. The nitric oxide reductase of Paracuccus denitrificans. Biochem J. 269:423-430. 4. Coyne, M.S., A. Arunakumari, B.A. Averill, and J .M. Tiedje. 1989. Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria. Appl. Environ. Microbiol. 55:2924-2931. 5. Demastia, M., T. Turk, and T.C. Hollocher. 1991. Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characteristics. J. Biol. Chem. 266:10899-10905. 6. Firestone, M.K., R.B. Firestone and J .M. Tiedje. 1979. Nitric oxide as an intermediate in denitrification: evidence from nitrogen-13 isotope exchange. Biochem. Biophys. Res. Commun. 91:10-16. 7. Goretski J. and T.C. Hollocher. 1990. The kinetic and isotopic competence of nitric oxide as an intermediate in denitrification. J. Biol. Chem. 265:889- 895. 8. Heiss, B., K. Frunzke and W. Zumft. 1989. Formation of the N-N band from nitric oxide by a membrane-bound cytochrome be complex of nitrate- respiring (denitrifying) Pseudomonas stutzeri. J. Bacteriol. 171:3288-3297. 58 9. Hochstein, LL and GA. Tomlinson. 1988. The enzymes associated with denitrification. Annu. Rev. Microbiol. 42:231-261. 10. Kaspar, H. and J.M. ’I‘iedie. 1980. Response of electro-capture detector to hydrogen, oxygen, nitrogen, carbon dioxide, nitric oxide and nitrous oxide. J. Chromatograph. 193:142-147 11. Kim, C.H. and T.C. Hollocher. 1984. Catalysis of nitrosyl transfer reaction by a dissimilatory nitrite reductase (cytochrome c,d1). J. Biol. Chem 259:2092-2099 12. Weeg-Aerssens, E, W. Wu, R.W. Ye, J M. Tiedje, and C.K. Chang. 1991. Purification of cytochrome cd1 nitrite reductase from Pseudomonas stutzeri JM 300 and reconstitution with native and synthetic heme (11. J. Biol. Chem. 266:7496-7502 13. Ye, R.W., I. Taro-Suarez, J.M. 'Iiedje, and BA. Averill. 1991. H2130 isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria: evidence for an electrophilic nitrosyl during reduction of nitric oxide. J. Biol. Chem. 266: 12848-12851. 14. Zumft, W.G. and K. Frunzke. 1982. Discrimination of ascorbate- dependent nanenzymatic and enzymatic, membrane reduction of nitric oxide in denitrifying Pseudomonas perfectomarinus. Biochim. Biophy. Act. 681:459-468. Chapter Three H2130 isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria 59 60 HZ‘BO Isotope Exchange Studies on the Mechanism of Reduction of Nitric Oxide and Nitrite to Nitrous Oxide by Denitrifying Bacteria EVIDENCE FOR AN ELECTROPHILIC NITROSYL DURING REDUCTION OF NITRIC OXIDE‘ (Received for publication. January 18. 1991) Rick W. Yet. Inez Toro-Suarez§. James M. Tiediefll. and Bruce A. Averill“ From the tDcpartment of Microbiology and Public Health and the EDepartment of C rap and Soil Sciences. Michigan State University. East Lansing. Michigan 48824 and the iDcportmcnt of Chemistry, University a] Virginia. Charlattesuillc, Virginia 2290) Reduction of NO and NO; by whole cells of eight strains of denitrifying bacteria known to contain either heme cd. or capper-containing nitrite reductases (NiRs) has been examined in the presence of H,"O. All organisms containing heme cd. NiRs exhibited rela- tively large extents of exchange between NO; and H;"O (39-100%), as monitored by the I‘0 content of product N 30. Organisms containing copper NiRs gave highly variable results, with Achromobacter cyclo- clastes and Pseudomonas aureofaciens exhibiting no “0 incorporation and Rhodopseudomonas splice- roides and Alcaligenes entropllus exhibiting complete exchange between N 0; and H,“O. Organisms contain- ing heme ed. N iRs exhibited significant but lower lev- els of exchange between NO and H,"O than between NO; and H,“O, while organisms containing copper NiRs gave significantly higher amounts of “O incor- poration than observed for the heme ed. organisms. These results demonstrate the existence of an NO- derivcd species capable of undergoing O-atom ex- change with H,"0 during the reduction of NO. Trap- ping experiments with "NO. "NE. and crude extracts of R. sphaeroides support the electrophilic nature of this intermediate and suggest its formulation as an enzyme nitrosyl. E-NO‘, analogous to that observed during reduction of NO; The observation of lower levels of "O incorporation with NO; than with NO as substrate for A. cycloclastes and P. aureofaciens indi- cates that. for these organisms at least. a sequential pathway involving free NO as an intermediate is sig- nificantly less important than a direct pathway in which N30 is formed via reaction of two NO; ions on a single enzyme. ‘This research was supported by National Science Foundation Grants CHE-8607681 and DMB-89174‘Z7 (to B. A. A. and J. M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "odlvrtiscmcnt" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I To whom correspondence should be addressed, Tm ion nan. no 1100100er (‘luulsrn Vol 266, .\u It) lulu 0' Jul\ 15 pp IJMn-llfi‘rl, HIM ( 1991 h\ The American Soc-cu 1m Hmrhcmnln and “circular timings, Inc Printed us l. S A The mechanism of microbial denitrification remains a con- troversial subject, despite a wealth of detailed studies on both intact bacteria and isolated enzymes (1-4). It is now generally accepted that denitrifying bacteria possess a nitric oxide reductase activity that is distinct from the nitrite reductase (NiR)' activity. The latter are typically soluble enzymes that are rather easily purified and have been shown to be of two distinct types: a cytochrome cd.-conraining dimer of ~60-era subunits and a copper-containing enzyme that is more vari- able in both subunit size and degree of oligomerization (l, 5). The membrane-bound nature of the nitric oxide reductase activities has hindered their purification and characterization. but recently nitric oxide reductases have been purified to apparent homogeneity from two organisms (6-8) and shown to contain both heme b and c prosthetic groups (7, 8). Virtually all workers in the field now agree that at least a significant portion of the total nitrogen flux occurs via a stepwise pathway with NO as an intermediate (Equation 1. where NoR is nitric oxide reductase), v- N N0; -. N0: —"'—R. no °R N10-9N; ill rather than via the direct pathway previously proposed (Equa- tion 2). in which two N05 ions are reduced to N30 on a single enzyme (MR) (9). NiR N0; -0 NOS 'hX;0 —. ‘AN; (2) Indeed, quantitative studies of NO levels during denitrifica- tion by several denitrifiers have been interpreted as indicating that only the former pathway (Equation 1) is operative and that NO is a free obligatory intermediate in denitrification (IO-13). This conclusion is consistent with the observed lack of reduction of NO by isolated NiRs, with the fact that most isolated NiRs produce predominantly NO upon reduction of nitrite, and with the fact that mutants lacking either the heme cd. (14) or copper NiRs2 are still capable of reducing NO. It fails, however, to account for the following observations. (11 Purified NiRs do. in at least some cases, produce significant amounts of N20 that cannot be attributed to chemical reduc- tion (15-17); (ii) N0“; and reagents such as N; and H,"O that are known to react with a nitrosyl intermediate derived from N0; exhibit apparent competitive behavior (18. 19): (iii) the l‘0 content of N20 produced from nitrite is about half that of nitrosation products derived from the nitrosyl intermediate. suggesting that unlabeled oxygen from a second nitrite enters the reaction (19); and (iv) the magnitude of the "N isotope effect increases with increasing nitrite concentration, sug- gesting that two nitrite ions react with the enzyme prior to the first irreversible step (20, 21). Consequently, the relative importance of the routes shown in Equations 1 and 2 remains unclear. In this communication, we present evidence that the NO reductase exhibits a remarkable and previously unsuspected similarity to the N iRs in that an electrophilic species derived from N0 can be trapped during the reduction of NO. Further, 'The abbreviations used are: NiR. nitrite reductase; HEPES. ‘- (2-hydroxyethyl)-l-piperazineethancsulfonic acid. 2 R. Ye and J. M. Tiedje, unpublished results. ()1 Afcchaanm o/ licduction of NO and Nitrite by Denitri/icrs Tituu; 1 Percent crchangc tt'li’l H,"0 during dissimilator) reduction 0] NO and nitrite by whole cells a] dentin/mg bacteria containing either heme cd. (I -4) or copper (5—8) nitrite reductases Acetylene (0.2 atmosphere) was added to the gas phase to block nitrous oxide reductase activity. Cell density corresponded to a pro- tein concentration of 11 mg/ml. 1.0 umol of NO or nitrite was used as substrate. H,"O was present at the range of 843%. After comple- tion of the reaction (50 min). 0.1 ml of 10 N NaOH was added to the reaction vial. Data are means of three replicates f ram two independent experiments. Substrate Strain Nitrite NO 1. P. denitri'licans ATCC 19367 59 z 5 11.3 :- 1.2 2. P. aeruginosa FAQ 1 76 z 7 19.0 t 0.2 3. P. stutzeri JM300 58 z 14 4 a 1 4. P. fluorescent AK-15 39 z 1 15.0 t 1.2 5. A. eutrophus ATCC 17699 94 a 5 84 z 6 6. A. cycloclastes ATCC 21921 4 t 2 30 z 7 7. P. aureolociens ATCC 13985 6.0 .t 0.2 37 t 14 8. R. aphaeroider forma sp. denitrilicans 90 z 15 61 z 19 we demonstrate via comparison of the amount of "O incor- porated into N30 from either NO; or NO that. in certain bacteria at least. reduction of NO; may not proceed entirely according to the stepwise pathway shown in Equation 1. MATERIALS AND METHODS Bacterial Strains—Pseudomonas aeruginosa PAOl was from B. W. Holloway. Monash University. Clayton. Australia; Rhodopseudomo- no: sphaeroides forma sp. denitri/icans was from T. Satoh; and Pseudomonas stutzeri JM 300 was from J. Ingraham. Pseudomonas fluorescens AK-15 was a soil isolate obtained in this laboratory.’ The rest of the strains used were from ATCC. Characterization of copper- type or cytochrome ed. nitrite reductases in some of these strains has been described (22); classification of Alcaligenes eutrophus ATCC 17699 as a copper NiR-containing organism was performed using NJVodimethyl dithiocarbamate as described (22). Sample Preparation—Cultures were grown anaerobically in 3% tryptic soy broth (Sigma) containing 0.15% potassium nitrate in 110- ml serum bottles. P. aeruginosa PAOl was grown overnight at 37 ‘C. and the rest of the cultures were grown at 30 'C. Cells were harvested. washed. and resuspended in tryptic soy broth in an 8-ml serum bottle. 111cc bottles were made anaerobic by flushing with argon. and nitrite or NO was added to start the reaction; suspensions were maintained at room temperature. 100 ul of 10 N NaOi-l was added to stop the reaction and absorb C0,. Crude extracts were prepared by sonication followed by centrifugation at 10.000 x g for 30 min to remove cell Isotopes and Their Analyst's—“NO was prepared by mixing 1 ml of 100 mu 11:50.. 1 ml of 100 mu KI. and 1 ml of 299 mu Na"NO. (99.9 atom ”Nil in a 25-ml serum bottle (23). Gas chromatography] mass spectrometry measurements and the calculation of the extent of "0 exchange were performed as described (19. 23). RESULTS AND DISCUSSION Exchange with H,"O during Nitrite Reduction—The results obtained for four denitrifiers known to contain heme cd. NiRs and four denitrifiers known to contain copper NiRs are pre- sented in Table 1. column 1. As expected based upon previous worlt demonstrating the existence of an electrophilic nitrosyl intermediate during reduction of nitrite by the heme cd. NiRs (18. 19. 23—25). organisms containing such enzymes exhibited relatively large amounts of “0 incorporation from H;"O (extents of exchange ranging from 39 to 80%) during reduc- tion of NO; to N30. In fact. strain 2 exhibited significantly more than 50% exchange. This is significant because our original hypothesis regarding the direct pathway (9) (Equa- ' A. Arunakumari and J. M. Tiedje. unpublished results. 12849 tion 2) postulated that the N—N bond was formed by attack of a second NO; upon an electrOphilic nitrosyl intermediate. E-NO‘. derived from the first nitrite. which is known to undergo facile I‘O exchange by a reversible hydration/dehy- dration process (23-25). Shearer and Kohl (21) have shown that the NiR from P. stutzeri JMBOO is a “sticky” enzyme and that NO; is committed to reduction once bound. Hence. I‘O- labeled NO; does not accumulate and “O incorporations of >50% are not consistent with the direct pathway (Equation 2). unless the NiR in organism 2 differs substantially from that in P. stutzeri J M 300 (organism 3). The organisms containing copper NiRs exhibited signifi- cantly different behavior in two cases (6 and 7). where essen- tially no “0 incorporation into No product was observed. This result is consistent with previous work on the Achro- mobacter cycloclastes system. which showed undetectable amounts of "0 exchange (26). In contrast. R. sphaeroides exhibited virtually complete exchange between H;“O and product N10. suggesting the presence of an electrophilic ni- trosyl intermediate in these organisms that differs dramati- cally in its reactivity with II,“O from the other organisms containing a copper NiR. Such behavior is perhaps not sur- prising. given the extreme variability in subunit size and immunoreactivity observed with copper NiRocontaining or- ganisms (22). Exchange with Hf'O during Reduction of NO—As shown in column 2 of Table I. substantial amounts of l‘O were also observed in N30 produced by reduction of NO. The extent of "O incorporation observed with NO tended to be lower than that observed with N0; for the same organism (except for 6 ‘ and 7. see below) but was well above background levels for all but one case. The four organisms with copper NiRs gave TABLE II Exchange with H.“O during reduction 0] N0 by crude extracts of R sphoer'oides forma sp. denitrificans in the presence of added electron donor or mediator 0.5 umol of NO and 4.24% of H,"‘0 were used in the reaction mixtures. which contained cell extracts equivalent to a protein con- centration of 2.4 mg/ml in 30 mat HEPES buffer. pH 7.0. and 10 mM EDTA. Nitrous oxide species were measured after the reaction was completed (180 min). Conditions otherwise as in Table I. PMS. phenazine methosulfate. Sample N."O N.“O Exchange nmol nmol S Crude extract 23.0 a 0:1 477 a 0.3 100 Crude extract + PMS 23.2 a. 0.3 477 1 0.3 100 (‘0 5'“) Crude extract + NADH 13 a 1.2 487 z 1 56 (4 m“) Crude extract + NADH 3.2 a 0.3 497 a 0.3 9 (4 mm) + PMS (40 an) TABLE 111 Nitrosyl trons/er from “NO to “N; in reaction containing H30 and R. sphaeroides crude extracts Reaction mixtures contained 2 nmol of "NO and 1.0 mat NlNa: all other conditions were as in Table II. Reactions were stopped after 30 min. Percent nitrosation was calculated based on the ratio of "-“N,o and total amount of nitrous oxide produced. Data are for duplicate experiments at 1 mu azide. but similar results were obtained with duplicates run at both 2.5 and 5 mu azide as well. Isotope Amount Exchange Nitrosation nmol % % "'"Nf'O 0.74. 0.88 “-“N."0 20. 24 so. 79 13. 14 "‘“Ng'b 5.1. 5.8 "-"N.“o 134. 136 82. 87 62 IO :N O r , is” ___, r 15.0 __, E_lsm° __L. (35.1%; n - '1 0f 1‘ “2°” ‘ ism- ls.l5.ll° l £.m(-’ r-uzog .2". “‘2” ”2° Sensual ta at o E , is” bis" ___, I,tsmo z t,itntsDZ u - .15 . u) l r no 01' “in on 11‘! .z 2 E- A) "a”luo 20. l ozn°. 4‘20 15 ll .2 o 501ch significantly higher extents of “0 exchange Clo-84%) than did those known to contain a heme ed. NiR (449%). but the origin of this difference is unclear since an NO reductase has yet to be purified from any of the former. The data in Table I. column 2. demonstrate the presence of an NO-derived species capable of undergoing O-atom ex- change with Hg“0 during the reduction of NO. which would not be expected a priori to proceed via an electrophilic species. The overall reaction can be written as 2!? 4» 2e' 4» 2N0 - MD + H,0 (3) indicating that water is produced during the reaction (presum- ably by protonation and dehydration of a hyponitrite level species containing two N atoms. such as N303'). suggesting a possible route for “O incorporation if the final dehydration step were reversible. As a control experiment. cells of Pseu- domonas aureofaciens were grown on NO; suspended in me- dium containing 10% Hfi‘O. and incubated anaerobically for 5 h at room temperature with 0.1 ml of N10 (8.8 nmol) in an 8-ml bottle. The "0 content of the N,0 was measured and did not differ from the natural abundance. Thus, the observed "0 incorporation during reduction of NO must occur prior to reduction to the N30 leveL This conclusion is also supported by the data in Table II. which demonstrate that for R. sphaeroides at least the extent of exchange with H;“O decreased as the concentration of electron donor/mediator was increased. This suggests strongly that "0 exchange occurs via a relatively oxidized nitrogen intermediate. Trapping with Aside during NO Reduction—If an electro- philic NO-derived species is indeed present during reduction of NO. it might be expected to react with nucleophiles other than H:"‘O. For example. N; and NH20H have been reported to trap the electrophilic nitrosyl produced by the heme cd. Mechanism of Reduction of N0 and Nitrite by Denitri/ters MES (19. ‘20. 213-25). Thus. crude extracts of R. sphaeroides were treated with l‘NO in the presence of “N5 and H,‘°O; the amounts of the various isotopically labeled forms of N20 formed are given in Table lll. It is clear that substantial (~l4%) amounts of a nitrosation producr m"N10 were ob- served even at the relatively low azide concentration used (1 mM) and that both the nitrosation product and NO reduction product exhibited comparable “0 incorporation. Implications for the Mechanism of N0 Reduction—The H;"O exchange and N3 trapping results presented above strongly imply the presence of an electrophilic mononitrogen intermediate during reduction of N O. The simplest such spe- cies is an enzyme nitrosyl. E-NO'. analogous to that observed for the heme cd1 N iRs. This is a most unexpected result. since it is not obvious why an oxidized NO species should be an intermediate in its reduction to N10. We note that similar results observed earlier for P. stutzeri J M 300 in the presence of NO and Hfi‘O or “NO and “NH10l-l (19) are also com- pletely consistent with the results reported here (although they were initially attributed to an N0 complex of the heme cd. N iii in this organism). Two possible tentative explanations for the data are shown in Schemes l and 2. In Scheme 1. the "0 exchange and nucleophilic trapping occur via a species that is not on the catalytic pathway for NO reduction but is rather on an oxi- dized “shunt." Given the redox potentials for synthetic heme nitrosyls (27-29). it might not be surprising if electron trans- fer to another center on the enzyme or elsewhere were to generate a transient oxidized species that. as shown. has nothing to do with catalysis. The other extreme is represented in Scheme 2. in which the E-NO’ species is an obligatory intermediate, possibly reacting via a hypothetical enzyme~ bound NO; with a second E-NO’ in a fashion analogous to that postulated by us earlier for the reduction of N0; by MR (9). Available data do not permit us to distinguish between these alternatives or the many possible variants thereof. Implications for the Pathway of Denitrification—Examina- tion of the data in columns 1 and 2 of Table 1 reveals that. in most cases. our results are fully compatible with NO as an obligatory intermediate in denitrification. ie. the sequential pathway shown in Equation 1. That is. for organisms 1-5 and 8. the extent of “O incorporation observed for NO; as sub- strate is greater than that observed for N0. reflecting the fact that for these organisms l‘0 exchange can occur at either the NO; _. NO or NO _. N30 steps. These data do not. however. constitute proof that the bulk of the nitrogen flux proceeds via Equation 1 rather than Equation 2. although they are not inconsistent with this view. in contrast. the data for organisms 6 and 7 (A. cycloclastes and P. aureofaciensl are not compatible with the view that NO is an obligatory free intermediate. because the amount of “‘0 exchange observed with NO; as substrate is far less than with NO. (If the situation shown in Equation 1 were to obtain. the amount of u‘0 incorporated into N20 derived from N0; would always have to be at least equal to that in N20 derived from N0.) Thus. for these organisms at least. the sequential pathway of Equation 1 appears to be significantly less important than a direct path- way as indicated in Equation 2. REFERENCES l. Hochstein. L. l.. and Tomlinson. C. A. (19.51% Annu. Rev. Micro. biol. 42. 231-261 . Ferguson. 5. J. (1937) Trends Biochem. Sci. 12. 354-337 . Tiedje. J. M. (1983) in Biology of Anaerobic Microorganism (Zehnder. A. J. 8.. edl pp. 179-243. John Wiley & Sons. Inc.. New York 4. Zumft. W, C., Viebrock. .-\.. and Kurner. H. (1985) The Nitrogen and Sulphur Cycles (Cole. J. A. and Ferguson. 5.. eds! pp. 245— “IO 11. 12. 13. 14. 15. 16. 17. 63 Mechanism of Reduction of NO and Nitrite by [)enttri/iers 279. Cambridge University Press. New York . Payne. W. J. (1981) Denitrification. pp. 79-89. John Wiley & Sons. Mo. New York . Hoglen. J., and Hollocher. T. C. (1989) J. Biol Chem. 269. 7556— 7563 . Carr. C. J., and Ferguson. 5. J. (1990) Biochem. J. 269. 423429 . Heiss, 8.. Franzlre. K.. and Zumft. W. G. (1989) J. Bacteriol. 171. 3288—3292 . Averill. B. A.. and Tiedje. J. M. (1982) FEBS Lerr. 138. 8-11 . Carr. G. M.. Page. M. D.. and Ferguson. S. J. (1989) Eur. J. Biochem 179. 683-692 Zafiriou. O. C., Hanley. Q. 8.. and Snyder. G. (1989) J. Biol. Chem. 264. 5696-5699 Goretski. J., and Hollocher. T. C. (1990) J. Biol Chem 265. 889—895 Goretslti. J., Zaliriou. O. C., and Hollocher. T. C. (1990) J. Biol. Chem 265. 11535-11538 Zumft. W. G.. Déhler. K.. Korner, PL. Lochelt, S.. Viebrock. A.. and Frunzke. K. (1988) Arch MicrobioL 149. 492-498 Betlach. M. R.. and Tiedje. J. M. (1981) Appl. Environ. Microbiol. 42. 1074-1084 Wharton. D. C., and Weintraub. S. T. (1980) Biochem Biophys. Res. Commun. 97. 236-242 Bessieres. P.. and Henry. Y. (198‘) Biochimie 66. 313-318 18. 19. 20. 21. 22. 23. 24. 28. 29. 12851 Weeg-Aerssens. B., Tiedje. J. M.. and AVCf'lll. 15. A «1987) J Am Chem Soc. 109. 7‘2H—7‘215 Weeg-Aerssens. B., Tiedje. J. M.. and Averill. B. A. 11938) J. Am. Chem. Soc. 110. 6851-6856 Bryan. T. A.. Shearer. C., Skeeters. J. l... and Kohl. D. (1983) J. Biol. Chem. 258. 8613-8617 Shearer. C., and Kohl. D. (1988) J. Biol. Chem. 263. 13231- 13245 Coyne, M. S.. Arunakumari. A.. Averill. B. A.. and Tied)e. J. .\1. (1989) Appl. Environ. Microbiol. 53. 2924-2931 Aerssens. B., Tiedje. J. M.. and Averill. B. A. (1986) J. Biol Chem. 261. 9652-9656 Kim. C., and Hollocher. T. C. (1984) J. Biol. Chem. 259. 2092- 2099 . Garber. A. E.. and Hollocher. T. C. (1982) J. Biol. Chem. 257. 8091-8097 . Hulse, C. l... Tiedje. J. M.. and Averill. B. A. (1989) J. Am. Chem. Soc. 1 1 1. 2322-2323 . Olson. L. W., Schaeper. D.. Lancon. D.. and Kadish. K. M. (1952) J. Am. Chem. Soc. 104. 2042-2044 Fujita. B., and Fajer. J. (1983) J. Am. Chem. Soc. 105. 6743- 67(5 Barley. M. PL. Takeuchi. K. J., and Meyer. T. J. (1986) J. Am. Chem. Soc. 108. 5876-5885 Addt etch exch dem Two excl acu Alec des< up. amt firm 64 Addendum to Chapter Two -- A study on the reversibility of 180 exchange during reduction of NO. One. of the important features of a nitrosyl complex is its exchange of oxygen with H20. This exchange activity has been well demonstrated during the reduction of N02: As indicated in Chapter Two, the finding of 130 exchange during the reduction of N O to N20 was completely unexpected. We suggested that an enzyme nitrosyl complex may also form during the reduction of NO, similar to that which is involved in the reduction of N02: One of the characteristics of 130 exchange during the reduction of N02‘ is the reversibility of the reaction, i.e. the formation of N1302' in the reaction with H2130. To test whether NO will also be released from the enzyme after exchange with H20, we used H2130 as a tracer to measure the accumulation of 15le0 in the gas phase during reduction 15NC by Alcaligenes eutrophus. Experimental conditions were the same as described in Chapter Two except that time points were taken in this experiment. Results are presented as percent 130 in the total amount of nitric oxide or nitrous oxide present in the system at the time of measurement ( Figure 1). Indeed, accumulation of 15N130 was observed in the gas phase (Figure 1). The amount of 15N180 increased in a linear fashion as the reaction progressed. At late stage of reaction, the nitric oxide contained 5.2% 18O, which is equal to the amount of H2130 used (5.2%). This suggests that all oxygen atoms in nitric oxide that remained in the gas phase had been replaced by the oxygen atom 65 from water. Release of l5N130 to the gas phase implies that 180 exchange occurred before the committed step of N =N bond formation and further supports the hypothesis that an enzyme-nitrosyl complex (E-NO+) is formed during dissimilatory reduction of NO. The N 20 contained about 2.6% 18O in an early stage of reaction, indicating that the extent of exchange was above 50% and that the exchange reaction is very fast. The result found for P. fluorescensAK-15 during the reduction of nitric oxide also supports the rapid equilibration of 130 into nitrous oxide (see Chapter Three). However, we could not detect a pool of N130 with this organism. Thus P. fluorescens AK-15 could be considered to have a ‘sticky’ NO reductase, while Alcaligenes eutrophus had a ‘loose’ one. Another difference between these two organism is that the extent of exchange in P. fluorescens was about 25% (see Chapter Three), while in Alcaligenes eutrophus, it reaches above 80%. P. fluorescens AK-15 contains a heme c,d1 nitrite reductase, while A. eutrophus has a Cu nitrite reductase. In summary, denitrification of NO has the following features. i) 130 from H2130 can be incorporated into the product, nitrous oxide; ii) a pool of N180 can be detected in the gas phase at least in some organisms; iii) the incorporation of 180 into nitrous oxide is very fast and it reaches equilibrium rapidly; iv) abundance of electron donor abolishes the 130 reaction in vitro; A possible explanation of mechanism of NO reduction that is consistent with these features is shown in scheme I and II. The nitrosyl complex is possibly formed by exc F01 rex Th 66 by the NO reductase: E-NO+, which can undergo an oxygen exchange reaction with H20 (supported by features i and ii). Formation of nitrosyl complex and exchange with H20 are reversible, leading to the release of the substrate from the enzyme. The exchange reaction likely occurs before the electron accepting step(s) and the formation of N=N bond, based on features ii and iv. Available data do not permit us to draw the conclusion as to whether E-NO+ is obligatory for N=N bond formation and thus other routes may also possible. Figure 1. Accumulation of 18O labelled products during dissimilatory reduction of 15N0 in Alcaligenes eutrophus. The reaction mixture contained 5.2% H2130 and 1.5 umol of 15NO in 1m] final volume of TSB. 67 1.0 GEES 258 2.9.: Z on. 6. A 0 O 0 n p — p — 0.0 + nitrous oxide ""43"“ nitric oxide 28.3 was 252 mu. 5 O. 4 39.2 s b E38“. 40 Time (min) Chapter Four Mutants of Pseudomonas fluorescens deficient in dissimilatory nitrite reduction are also altered in nitric oxide reduction 68 92. (L ()9 JOURNAL OF Bac'rriumcm'. Apr. W92. p. 2560-1564 00213193/92/082560-0530200/0 Copyright C 1992. American Society for Microbiology Vol. 174. No 25' Mutants of Pseudomonas fluorescens Deficient in Dissimilatory Nitrite Reduction Are Also Altered in Nitric Oxide Reduction RICK W. YE.’ ALAHARI ARUNAKUMARI,"21 BRUCE A. AVERILL.’ AND JAMES M. TlEDJEm' Department of Microbiolog' and Public Health| and Department of Crop and Soil Science. 2 Michigan State University. East Lansing, Michigan 48824-1325: and Department of Chemistry. University of Virginia. Charlottcn'ille, Virginia 229013 Received 25 November 1991/Accepted 6 February 1992 Five Tn5 mutants of Pseudomonas fluorescens AK-IS deficient in dissimilatory reduction of nitrite were isolated and characterized. Two insertions occurred inside the nitrite reductase structural gene (nirS) and resulted in no detectable nitrite reductase protein on a Western immunoblot. One mutant had Tn5 inserted inside nirC, the third gene in the same uperon, and produced a defective nitrite reductase protein. Two other mutants had insertions outside of this nir operon and also produced defective proteins. All of the Nir" mutants characterized showed not only loss of nitrite reductase activity but also a significant decrease in nitric oxide reductase activity. When cells were incubated with "NO in H,“O, about 25% of the oxygen found in nitrous oxide exchanged with “,0. The extent of exchange remained constant throughout the reaction, indicating the incorporation of "0 from H,"O reached equilibrium rapidly. In all nitrite reduction-deficient mutants, less than 4% of the “0 exchange was found, suggesting that the hydration and dehydration step was altered. These results indicate that the factors involved in dissimilatory reduction of nitrite influenced the subsequent NO reduction in this organism. Dissimilatory reduction of nitrite is the key step in the denitrification pathway; it is the point of divergence from assimilatory nitrogen metabolism (24). There are two types of nitrite reductases: one contains the cytochromes cd,, and the other contains copper (6, 14, 20). Organisms containing the cytochrome ed, nitrite reductases are more frequently isolated from nature, whereas Cuotype nitrite reductases are found in organisms that exhibit more phylogenic diversity and occupy a wider range of ecological niches (6, 9). Nitric oxide is the major product of nitrite reduction by purified nitrite reductases, and nitrous oxide is a minor product (4, 14, 17, 26). NO is generally accepted as one of the free and obligatory intermediates in the denitrification pathway (2, 5, 8. 10, 14, 20, 28), and NO reductases have been purified from Pseudomonas stutzeri Zobell (13) and Paracoccus denitrificans (4, 7). We recently showed that many denitrifiers containing Cu or ed, nitrite reductases are capable of undergoing O—atom exchange with H,"O during the reduction of NO to N20 (27) and that a labeled interme- diate can be trapped with azide. These results suggest that a nitrosyl complex is formed during the reaction. it was also shown that the extent of the O-atom exchange reaction depended on the availability of electrons. The dependence of nitrosyl transfer upon the presence of N0 during reduction of NO,” has been demonstrated (11). Tn5 was used by Zumft et al. (29) to generate mutants deficient in dissimilatory nitrite reduction (Nir’) in P. stutzeri Zobell. All of the mutants isolated possessed normal NO reduction activity, indicating that NO reduction and nitrite reduction are distinct. The nitrite reductase gene (nirS) from Pseudomonas aeruginosa (21) and from two strains of P. stutzeri (Zobell [15] and JM 300 [23]) has been cloned. nirM encoding cytochrome c”, and m'rC encoding a ‘ Corresponding author. ‘lPresent address: Department of Biochemistry and Microbiol- Ogy, Cook College, Rutgers State University of New Jersey. New Brunswick, NJ 08903. heme protein (also named ORFS in P. stutzeri) are located immediately downstream of the nitrite reductase gene in P. aeruginosa (1. 15. 19). This is not the case in P. stutzeri (15), where nirT encoding a tetraheme protein and nirB encoding cytochrome c...$2 are located between the nirS and nirM genes. We used Tn5 to generate Nir‘ mutants in Pseudomonas fluorescens and found that, in contrast to P. stutzeri Zobell, all five Nir‘ mutants characterized not only lacked the ability to reduce nitrite but also showed a decreased ability to reduce NO. Furthermore, the extent of WO exchange with H2"O during reduction of ”NO to '5"’N:O was reduced to background levels in the mutants. These results suggest that the dissimilatory reductions of NO,“ and NO are linked in this organism. MATERIALS AND METHODS Bacterial strains. P. fluoresccns AK-lS is a gram-negative rod isolated from Capac loam soil from the Kellogg Biolog- ical Station. Kalamazoo County, Mich. This strain was identified based on its denitrifying ability, production of fluorescent pigments on King medium, growth at 4°C. lack of growth at 37°C. and fatty acid profiles. This strain was chosen for these studies because P. fluorescens strains are the most prevalent denitrifiers in nature and this strain has a high frequency of Tn5 mutagenesis. Bacteria were grown at 25°C in tryptic soy broth (TSB; Difco Laboratories) supple- mented with 0.15% K1903. A rifampin-resistant clone. YT101, was isolated from a spontaneous mutation. Conjugation and isolation of mutants. nu; rifampinoresis- tant strain of wild-type P. fluorescent, YT101, was used as the recipient, and Escherichia coli S-17 carrying the pSUbZOZl plasmid containing Tn5 was used as the donor (22). Mating was carried out at 25°C overnight. Exconjugants were selected on TSB plates supplemented with rifampin (50 rig/ml) and kanamycin (50 gig/ml). Colonies were replica plated onto TSB plates containing 0.15% KNO, and grown 70 VOL. 171. 1992 overnight in an anaerobic glovebox. Plates were then taken out of the anaerobic glovcbox, and a piece of Whatman no. 42 filter paper was laid on top of each plate and covered with a thin layer of cooled agar. N: gas bubbles appeared over denitrifying colonies in 30 to 60 min. If a thick top agar layer was used without the Whatman paper. it took more than 12 h before N2 gas bubbles appeared and they were more difficult to distinguish. Colonies that did not produce gas bubbles were picked and further tested for their ability to produce bubbles by growth in culture tubes containing inverted Durham tubes. Growth conditions and activity assays. Strain YT101 was grown in the presence of rifampin (SO jig/ml). Mutant Strains were grown in the presence of both kanamycin (50 pig/ml) and rifampin (50 jig/ml). Anaerobic cells were grown over- night at 25‘C in serum bottles containing 100 ml of T58 and 0.15% KN03. They were harvested by centrifuging at 8.000 x g for 15 min and were washed twice with 50 mM N-L’» hydroxyethylpiperazineN‘-2-ethancsulfonic acid (HEPES) bufier (pH 7.3). Crude extracts were prepared by sonication and subsequent centrifugation at 10,000 x g for 20 min at PC. The supernatant was assayed for nitrite and NO reduc- tase activities by measuring the rate of NO and N20 appear. ance. The reaction mixture for NO reduction by the crude extract included 10 mM EDTA, 40 MM phenazine methOSuIv fate (PMS), 4 mM NADH, and 50 mM HEPES (pl-l 7.3). To assay the NO reduction activities in whole cells, anaerobi- cally grown cells were suspended in 1 ml of TSB in an 8-ml serum bottle with an anaerobic atmosphere created by repeated evaculation and filling with argon. NO was injected to start the reaction. All reaction vials were shaken on their sides at 100 rpm to facilitate the rate of gas-liguid transfer. NO and N10 were detected by gas chromatography with “Ni electron capture detectors (16). The protein concentra- tion of the crude extract was determined by the bicincho— ninic acid (BCA) protein assay reagent (Pierce Chemical Co.). The amount of protein in whole cells was eStimated with the Folin and Ciocalteu phenol reagent (Sigma) after alkaline lysis with 1 N NaOI-i (12). DNA sequencing. To determine the Tn5 insertion site in the Nir’ mutants, subclones in pUC18 containing the left in- verted repeat region of Tn5 were sequenced with a primer made to a region near the end of the inverted repeat. DNA was sequenced by the didcoxy method with a sequencing kit (U.S. Biochemical Corp.). Western and Southern blots. Western immunoblots were developed with antibodies against nitrite reductase from P. aeruginosa as described previously (6). Genomic DNA isolation, restriction enzyme digestion, electrophoresis, and Southern blotting were done as described by Maniatis et al. (18). Plasmid pSUP2021 containing Tn5 was isolated by alkaline lysis (18) and labeled by nick translation (Boehringer Mannheim). The nitrite reductase gene from P. aenigi‘nosa in a 3.5-kb EcaRl fragment in pEMBL18 was provided by M. C. Silverstrini (21). Isotope analysis. lsNO preparation, gas chromatography- mass spectrometry measurements, and calculation of "‘0 exchange were performed as described previously (25, 27). RESULTS Isolation of Nir" and Nos“ mutants. Deficiency in any step of denitrification leads to a lack of N2 formation. The top agar method used previously to isolate denitrifying deficient mutants in P. stutzeri (29) did not work well with I’. fluorescens AKoIS, perhaps because of the slower rate of N2 \lTRIIIZ REDUCTION-DEFICIENT I’ FLUURIQSCEA'S MUIANTS 35(il TABLE 1. Characteristics of Nir' and Nos~ mutants from P. flimrrsccns AK-IS Accumulation Bubble formation from: Accumulation Strain of No.3 from of N_-O from NO." NO,’ N_.O plus NO.‘ NO." Y‘TlOl — + + + + - YT25” + - ++ - YT2471 + — + + - \“1‘3221 + - + ., _ W31 + - + + - YT4221 + - ++ — YT25” - - - + + ‘i'Tls - - - + + " Accumulation of NO: ' and N:O was tested after overnight growth. “ Four additional mutants with the same phenotype were also isolated. production in strain AK-IS. Whatman filter paper applied on t0p of colonies before a thin layer of tap agar was added proved to be more eflicient in trapping the N2 gas. Mutants that could not produce bubbles were categorized as Nir’ (deficient in NO,‘ reduction) or Nos' (deficient in N20 reducrion) based on the characteristics summarized in Table 1. The frequencies of Mr” and Nos’ phenorypes were about 0.032 and 0.024%, respectively. No nitric oxide reduction- dcficient (Nor’) mutants were found. All Nir' and Nos" mutants could grow with NO,’ as the electron acceptor, but they could not produce N: bubbles because of a block in either nitrite reduction or N20 reduction. Mutants deficient in nitrite reduction produced bubbles in the presence of both NO,‘ and N=O, suggesting that these mutants could convert N20 to N2. Nitrate was included in this test because P. fluorescens AK-IS could not grow well on N10 alone. After overnight growth on nitrate, Nir" mutants accumulated N02: whereas Nos” mutants accumulated N20. Two Nos' mutants (YTIS and YT25) were used for control studies. Physical characterization of Nir‘ mutants. To detect Tn5 in these mutants, Tn5 was labeled and used to probe the genomic DNA from the wild-type and mutant strains. When genomic DNA was cut with EcoRI and BamHI, two bands, which varied in sizes in difl'erent mutants, hybridized to the Tn5 probe, suggesting that single copies of Tn5 had been inserted into difl'erent positions in the genome (data nOt shown). One of the EcoRI and BamI-II fragments of each mutant strain with the intact neo gene of Tn5 was subcloned in pUC19 by selection for resistance to kanamycin. Sub- clones from YT2511, YT2471, and YT3221 hybridized to a common 12-kb EcoRI DNA fragment, which also hybridized to structural genes of nitrite reduCtases from P. aeruginosa (21) and P. stutzeri J M300 (23). suggesting that all three Tn5 insertion sites were clustered in this nir operon. By obtaining DNA sequences from regions flanking the Tn5 insert in pUCl9 subclones and comparing them with published nir operon sequences (15, 21, 23), we found that Tn5 was inserted near the middle of the nitrite reductase gene in YT2511 and near the carboxyl terminus in YT2471. In YT3221, Tn5 was located near the heme binding site in nirC. the third gene in the operon. Subclone fragments from mutants YT4221 and YT31 did not hybridize to the 12vkb EcoRI genomic DNA fragment containing the nitrite reduc- tase gene and had no homology with each other. Biochemical characterization of Nir" mutants. Crude cell extracts of all Nir" mutants showed no nitrite reductase activity (Table 2). When a Western bIOt was developed with antibodies against the nitrite reductase, mutants YT4221, 71 2502 YE ET AL. TABLE 2. Rates of NO; and NO rcducrion and extents of ”0 exchange by wild~type and mutant strains of P. fluorescent AK-IS‘ nmol of N mg" min'| Strain Crude extract I‘0 Exchange‘ (“H _ Cells (+NOI vNO,‘ +NO YTIOI 110 17.4 38.2 25.5 : 1.3 YTZSII 0" 6.4 10.1 1.3 2 0.4 man 0 8.2 8.0 1.5 t. 0.5 YT3221 0 7.1 12.8 1.3 e 1.3 YT31 0 7.7 10.4 1.2 YT4221 0 8.4 17.6 3.9 t 2.5 YT25 ND” ND 37.6 23.3 3 6 4 YTIS ND ND 39.4 23.7 - S 0 ‘ Data are means of duplicate samples for crude extracts ad triplicate samples for whole cells. The nitrite reductase activity assay mixture had about 0.04 mg of protein and 1 nmol of nitrite. The NO reductase assay contained 0.08 mg of prorein and 2 nmol of NO. The same amount of NO was used in the whole-cell assay. ‘ Reaction mixtures contained 4 mg of whole-cell protein. 5.14% H:“O. and 1.5 nmol of uNO in an B-ntl serum bottle with 1 ml of TSB solution. The reaction was stopped with 0.1m! of NaOH (10 N) after 40 min. The data are obtained from triplicate samples. ‘ The detection limit for specific activity was about 0.13 nmol tag" min". ‘ ND. nOt determined. YT31, and m221 showed a positive band corresponding in size to the wild-type nitrite reductase protein, indicating that defective proteins were made (Fig. 1). However, mutants YT2511 and YT2471 showed no such band. This was con- sistent with the physical characterization in that these two mutants had Tn5 inserted inside nirS. The fact that a defective protein was made by YT3221 suggested that nirC 70kt!- ......A q m, FIG. 1. Western blot of proteins from wild-type and mutant strains. The blot was developed with polyclonal antibodies raised against the nitrite reductase purified from P. aeruginosa. Samples of 5 ug of protein were used per blot for all strains. Lanes 1 through 6 contained strains YT4221. W31. YT3221. YTZSII. YT101. and YT2471. respectively. I. l1.\r'rt.ttt(ii.. 6N< A ’ *6 “30¢ i M ’3 '5 2004 (E: —-O-— M '2 : + M i ”‘3 3 X n e Y—f V Y Y Y o u 03 I o g e we 8 y E 2 ‘z' ‘ w 400. i '0 200. ’2 1 e . - 'T’f—t o 501 3? ‘0‘ C —O— "101 e. . —o— was" a ,0. s . W s ”1 X -t m 10‘ 0 v 3 T T 0 10 3 an N ” N Tlme(min) FIG. 2. The time course of “0 exchange in resting cells of the YT101 (A) and YT2511 (8) mutant strains. The extent of exchange (C) was calculated based on the data in panels A and B. Data are means of duplicate samples. The amounts of cells were equivalent to 1 mg of protein for the wild type and 4 mg of protein for YT2511. In YT2511. the ”"SN,“O species could not be detected until after 20 min. or a gene(s) further downstream is essential for the produc- tion of active nitrite reductase. The NO reduction activity in resting cells in Nir' mutants of P. fluorescens AK-IS was reduced by about three- to fourfold relative to that of the Rif' wild-type strain YT101. When crude cell extracts were assayed in the presence of artificial electron donors (PMS and NADH), the NO reduc- tion activity was reduced by about two- to threefold (Table 2). The NO reduction activity in Nos‘ mutants was net altered. Thus, there is a consistent reduction in NO reduc- tion activity only in Nir' Strains. "O-exchange studies to probe the mechanism of NO reduc- tion. To study whether mutations in nir genes also afl'ected the mechanism of nitric oxide reduction, the extent of oxygen atom exchange during the reduction of uNO in resting cells was measured. The ratio of ”'”N,"O (rule. 48) to the total amount of nitrous oxide produced reflects the extent of exchange (27); this was about 25% in the YT101 and Nos‘ mutants (YT25 and YTIS) (Table 2). However, in Mr" mutants the extent of exchange was reduced to back- ground levels except in YT4221, which was slightly above the background. The percentage of exchange remained con- stant at 25% throughout the course of reduction of NO to N30, indicating that the hydration-dehydration reaction rap- ‘ FL p; 2?. 72 VOL. 174. 1W! idly reached cqwlthrtum (Fig. 2). Since Nir‘ mutants had a reduced rate of NO reducuon. four times more cells from mutant YT2511 were used in the time course study. Only a very small amount of "'“N:“‘O was observed. suggesting that the decreased "‘0 exchange for YT2511 (Table 2) reflects an intrinsic dierrence in mechanism rather than simply a difference in the rate at which isompie equilibrium was reached. DISCUSSION Among the five characterized Nir‘ mutants. three had Tn5 inserted in the air operon containing the nitrite reductase structural gene (nirS). Insertion of TrLS into the nirS genes in YT2511 and YT2471 resulted in no nitrite reducmse activity and the absence of identifiable nitrite reductase protein in a Western blot (Fig. l). Insertions in the m'rC genes and two other regions. however. produced defective nitrite reductase proteins. suggeSting that three or more genes are involved in the production of an intact nitrite reductase. These may include genes involved in the synthesis of the unique heme d. chromOphore or for assembling or processing of the polypeptide. All five Nir" mutants isolated from this Strain of P. fluorescens showed a decrease in the rate of NO reduction and a lack of “0 exchange with H2150 during reduction of NO (Table 2). The decrease in NO reduction rate was about 3- to 4-fold in whole cells. but all mutants except YT4221 showed at least a lO-fold reduction in “‘0 exchange. suggest- ing that there is a genetic and/or mechanistic relationship between the dissimilatory reduction of NO,’ and that of NO. This relationship cannOt, however. be due simply to mutations in the regulatory region of the genome: YTZSII and YT2471 had TrLS inserted inside the nitrite reductase structural gene. yet they had the same decrease in NO reduction and "‘0 exchange as the others. Further. Tn5 mutants deficient in N20 reduction (Nos’) exhibited normal rates of NO reduCtion and “’0 exchange. indicating that the Tn5 insertion event per se does not result in the observed efl’ects on NO reduction. Possible explanations for these results include the follow- ing. (i) In normal cells in vivo. a substantial portion of the NO to N20 flux is catalyzed by the nitrite reductase. such that Nir" mutants exhibit reduced NO reduction levels with diflerent "0 exchange charaCteristics due to the functional NO reductase. Although definitive data with P. fluorescens have not yet been obtained. studies on P. stutzeri have shown that purified nitrite reductase is incapable of reducing N0 either by itself or in the presence of NO,“ (17). Furthermore. quantitative studies of NO concentrations during reduction of NO,” are consistent with the bulk of the nitrogen flux of denitrification occurring via NO (28). Thus. this explanation seems highly improbable. (ii) Production of N10 from NO,‘ is a two-step process involving NO produc« tion by the nitrite reductase and subsequent NO reduction to Nzo by NO reductase. These two enzymes may associate with each other in vivo to channel products from one to another. Loss of functional nitrite reductase may lead to disruption of this association. resulting in some loss of NO reductase activity and the absence ofWSO exchange. (iii) A third possibility is that a functional nitrite reductase or functional nitrite reduction system is necessary for full expression of NO reductase activity at the functional or genetic level. (iv) Similarly. a functional nitrite reduCtase might be necessary for the synthesis of one or more electron NITRITE REDCC'T1()\-Dlil:l(‘lll.\'T I’ ll.U'()/x‘l..§(.'l..'\'$ MUTANTS 2563 transfer proteins. such as cytochromes. that are specufic eIeCtron donors for the NO reduCtase. The results of Zumft et al. (29) with TrLi-indueed mutants of P. stutzeri Zobell may be consistent with the last inter- pretation. Although they found no significant changes in NO reduction activity. they did find two Nir' mutants that had significant changes in the amount of cytochrome c552 and/or alpha-peak c-type cytochrome. and they suggested that these gnochromes exhibited a functional and/or regulatory interdependence. However. P. fluorescens AK-LS and P. stutzeri Zobell difl'er both physiologically and genetically. For example. the former does not grow efficiently on N20 alone. This phenomenon is often observed in P. aeruginosa (14). The structure of the nir Operon in P. fluorescens AK-IS consists of ntrS followed immediately by nirM and nirC. similiar to the nir Operon in P. aeruginosa (unpublished result5). The data. however. do not allow us to distinguish explanations ii. iii. and iv. ACKNOWLEDGMENTS We thank Inez Toro-Suarez f0r her assistance in the gas chroma- tography-mass speCtrometry analysis and Joann Palma for helping in the isolation of TM mutants. This research is supported by National Science Foundation grant OMB-8917427 to E.A.A. and J.M.T. ADDENDUM During the reviewing process for this paper. a Nor“ mutant of another species was isolated by gene replacement (3). This mutant blocks the denitrification pathway at nitric oxide. indicating that N20 cannot be formed from nitrite direCtly via the nitrite reductase and thus providing more convincing evidence to reject explanation i. The Nor“ mutant isolated is conditionally lethal. which explains why no Nor“ mutants were found in this study. REFERENCES 1. Arai. B., Y. Sanbongi. Y. Igarashi. and T. Kodama. 1990. Cloning and sequencing of the gene encoding cytochrome c-SSl from Pseudomoms aeruginosa. FEBS Lett. 261:196-198. . Betlach. M. P... and J. M. Tiedje. 1981. Kinetic explanation for accumulation of nitrite. nitric oxide. and nitrous oxide during bacterial denitrification. Appl. Environ. Microbiol. 42:1074— 1084. 3. Braun, C., and W. G. Zumft. 1991. 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Nitric oxide and nitrous oxide production and cycling during dissimilatory nitrite reduction by Pseudomonas perfecrumamia. J. Biol. Chem. 264:5694-5699. . Zumft. W. C., H. Korner, S. Lochelt, A. Viebrock. and K. Frunzke. 1988. Defects in cytochrome cd.-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomo- nas stutzeri. Arch. Microbiol. 149:492-“18. Chapter Five Characterization of Th5 Mutants Deficient in Dissimilatory Nitrite Reduction in Pseudomonas sp. strain G-l79 with 3 Copper Nitrite Reductase 7li 75 Abstract. Tn5 was used to generate mutants that were deficient in dissimilatory reduction of nitrite in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide, but not on nitrite (Nir'). The two mutants isolated in this group accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot. Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for the nitrite reductase. The third type of mutant grew on nitrate, but not on nitrite or nitrous oxide (N20). It accumulated significant amounts of nitrite, NO, and N20 during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid (DDC), which inhibited nitrite reductase activity in the wild type, did not affect N 0 reductase activity, indicating that nitrite reductase did not participate in N 0 reduction. NO reductase activity in Nir‘ mutants was lower than the wild type when they were grown on nitrate, but was the same as the wild type when grown on nitrous oxide. These results suggest that the reduction of NO and N20 were carried out by two distinct processes and that mutants in nitrite reduction reduced NO reductase activity following anaerobic growth with nitrate. 76 Introduction Denitrification is the dissimilatory reduction of nitrate to nitrogen gases by the pathway of: N03' -> N02‘ -->NO --> N20 -->N2. The key step in denitrification is the reduction of nitrite by nitrite reductases, since it is the point of divergence from assimilation. Based on the characteristics of active sites, two major types of dissimilatory nitrite reductases are found: those containing the cytochrome c and d 1 and those containing copper (17). Cu-type nitrite reductases from many denitrifiers have homology to each other as shown by cross-reactivity in immunoblots (8, 24). Studies of the Cu- type nitrite reductase from Achromobacter cycloclastes reveals that the enzyme is a trimer with three type I and three type II coppers (10,13). Type I copper is bound within a single monomer, while type II copper is held by residues from each of two monomers of the trimer. Evidence from the crystal structure suggests that nitrite is bound to the type-II copper site (13). Pseudoazurin is the physiological electron donor (20, 21, 35). Recently, convincing evidence has accumulated to support the hypothesis that NO is a free and obligate intermediate at least in denitrifiers containing cytochrome cd1 nitrite reductases. Purified enzymes produce nitric oxide as the major product and can not convert NO to N20 (5, 22, 31). Isotope and kinetics experiments indicate that NO is kinetically competent and obligatory for N20 formation (3, 11, 14, 15). An insertional mutation in the structural gene of NO reductase in P. stutzeri Zobell strain yielded N O as the only detectable product of N02' reduction, thus showing that there is no other physiological alternative for N20 77 formation from nitrite (4). Furthermore, NO reductases have been purified from two organisms, P. stutzeri Zobell and Paracoccus denitrificans (6, 9 ,16). Some uncertainty, however, still exists as to whether there might also be a direct N 02' to N 20 pathway for denitrifiers containing copper nitrite reductases. It has been found that small amount of N20 can be formed from N0 via a "NO-rebound" pathway by the purified copper nitrite reductase from Achromobacter cycloclastes (18), and 18O isotope studies on A cycloclastes and P. aureofaciens showed lower level of 130 exchange occurred with NOz' as substrate versus N O (33). All genetic information on denitrifiers comes from strains that contain the heme cd1 nitrite reductase and none from strains with the copper enzyme. Because strains containing the copper type make up over one-third of isolates, represent a wider phylogenetic distribution(8), and because the enzyme has different mechanistic features than the heme type, it is worthwhile to obtain and compare genotypic and phenotypic information on this group of organisms as well. For the heme type strains, some genes involved in nitrite reduction have been identified. In P. stutzeri (19) one operon contains the structural gene for nitrite reductase (nirS), a tetraheme protein (nirT), cytochrome C552 (nirM), cytochrome 0551 (nirB ) and another monoheme protein (nirC); while in both P. fluorescens AK-15 (34) and P. aeruginosa (1), this Operon contains nirS, nirB and nirC, without nirT and nirM. Nir' mutants from P. stutzeri showed a normal NO reductase activity (36). In contrast, similar mutants isolated from P. fluorescens AK-15 showed not only a loss of nitrite reductase activity, but also a reduction in NO reductase activity and 180 exchange in the NO to N20 step (34). 78 In this paper, we report the isolation and characterization of three groups of mutants deficient in dissimilatory reduction of nitrite in a strain containing a Cu-type nitrite reductase, as well as the effects of Nir' mutants on NO reduction. Materials and Methods Bacterial strains. Pseudomonas sp. strain G-179 was from Gamble’s collection (12). It was isolated by this lab from a pampa agricultural soil from the Parana Experimental Station, Argentina. This strain contains a Cu-type nitrite reductase as demonstrated by diethyldithiocarbamic acid (DDC) inhibition and immuno-reaction to polyclonal antibodies against the nitrite reductase from A. cycloclastes (8). This strain was selected because it was Kans, could produce exconjugants at a reasonable frequency, and formed distinct colonies on N 02' plates in an anaerobic glove box. A rifampin-resistant clone, RTCO 1, was obtained from a spontaneous mutation. Conjugation and isolation of mutants. Transposon mutagenesis by conjugation was carried out by using the rifampin-resistant strain, RTCOl, of wild type Pseudomonas sp. G-179, as the recipient; and Escherichia coli S-17, carrying the pSUP2021 plasmid containing Tn5 , as the donor (33). Mating was carried out at 25°C overnight. Exconjugants were selected on tryptic soy broth (TSB. Difco Laboratories, Detroit, Mich) agar plates supplemented with rifampin (50ug/ml) and kanamycin (50 ug/ml). 7'9 Colonies were replica plated onto TSB agar plates with 10 mM KNOz and grown overnight in an anaerobic glovebox. Those clones that showed no growth were selected from the master plates and further tested for their ability to grow anaerobically on broth supplemented with nitrate, nitrite, or nitrous oxide as the terminal electron acceptors. Growth conditions. The growth medium contained TSB (30 g /L), 2 M of CuSO4, and 30 mM of potassium nitrate, if used. For growth on nitrous oxide, TSB was saturated with nitrous oxide before inoculation and then the gas phase was again filled with nitrous oxide. Strain RTCOl was grown in the presence of rifampin (50 ug/ml) and mutant strains were grown in the presence of both rifampin (50 ug/ml) and kanamycin (50 ug/ml). An inoculum from fresh plates was first transferred to 10 ml of growth medium and grown anaerobically overnight; this was used to inoculate a sealed bottle containing 120 ml growth medium. Cells were harvested after 27 h of anaerobic growth at 25°C with constant shaking (100 rpm). Cells were washed several times with 50 mM HEPES buffer (pH 7.0) until no nitrite was present. Cells not immediately used were stored at - 70°C. Measurement of nitrate, nitrate, NO and N20. Nitrate and nitrite were determined by HPLC using an anion exchange column (7 ). NO and N20 were measured with gas chromatography (34). Crude extract preparation and activity assays. Cells were disrupted by sonication. The supernatant after centrifugation (13,000 x g for 30 min ) was assayed for N02' and NO reductase activities. Assays for nitrite and a) nitric oxide reductase were carried out in 8-ml serum bottles with 1 ml solution as described earlier (8, 34). For the nitrite reductase assay, the rate of evolution of both NO and N20 was measured. For the nitric oxide reductase assay, the rate of N 20 evolution was measured. For rate of nitrous oxide reduction in whole cells, the rate of N20 disappearance was measured. Western and Southern blots. Western blots were developed with antibodies raised against the nitrite reductase from A. cycloclastes as described previously (8). Genomic DNA isolation, restriction enzyme digestion, electrophoresis, and Southern blotting were done as described by Maniatis, et al (23). Tn5 was labelled with a random primer kit (Boehringer Mannheim, Germany). 81 Results Isolation of mutants deficient in nitrite reduction. Transoonjugants that did not grow anaerobically on TSB agar plates supplemented with potassium nitrite were screened and further tested for growth and bubble formation in tubes with TSB medium containing potassium nitrate or nitrous oxide. Three types of mutants were isolated. The first group of mutants, as represented by RTCO7 and RTC 14, showed a lack of growth not only on nitrite, but also on nitrate and N20 (Table 1). The second type of mutants (RTC22 and RT023) were deficient only in nitrite reduction and grew under anaerobic conditions with nitrate and nitrous made as the terminal electron acceptor (Table 1). These two Nir' mutants accumulated nitrite (Table 2). Mutant strain RTCO3 represented the third type of mutant; it grew on nitrate, but not on nitrite or nitrous oxide. It accumulated nitrite after overnight growth (Table 2). Physical characterization of Tn5 mutants. To detect the presence of Tn5, genomic DNA from mutant strains of the first group of mutants and RTCO3 was digested with EcoRI and BamHI, which has a recognition site inside Tn5. The Southern blot was probed with labelled Tn5. Bands from these different mutant strains varied in size, suggesting different locations of Tn5 (data not shown). Genomic DNA digested with EcoRI from both Nir‘ mutant strains, RTCZZ and RTCZ3, showed a band equal in size in a Southern blot probed with Tn5 (Fig. 1). BamHI digestion resulted in two bands and the combined 82 size of these two bands was also the same between these two mutants, indicating that the two Tn5 insertions were clustered in the same region. The EcoRI and BamHI DNA fragment containing the neo gene of the Tn5 and a flanking genomic region (Fig.1, lanes 5 and 6) was subcloned in pUC 19 by screening for kan’ and used as a probe to isolate the corresponding wild type EcoRI and BamHI fragment. The resulting clone, pYTC18, contains a 1.9 kb fragment, in which both Tn5 insertion sites were located by mapping ( Fig.2). Sequencing results indicated that this 1.9 kb fragment contains the structural gene of the copper nitrite reductase (32). Biochemical characterization of N in Tn5 mutants. The wild type strain had nitrite reductase activity that was abolished upon the addition of the copper chelator DDC (Table 3), which is characteristic of Cu-type nitrite reductases. Both Nir' mutants lacked nitrite reductase activity. Mutant strain RTCO3 had a slightly higher nitrite reductase activity than did the wild type. In a Western immuno-blot developed with polyclonal antibodies against the Cu-type nitrite reductase, both wild type clone (RTCO 1) and RTCOB showed positive nitrite reductase bands, while no such bands were found in RTCZZ and RT023 (Fig.3). Presence of two bands in wild type and RTCO3 may be due to degradation, two different start sites or two different processing sites for signal peptide. Addition of DDC did not affect NO reductase activity in the wild type, suggesting that nitrite reductase did not participate in the NO reduction (Table 3). However, the NO reductase activities in Nir' strains RTCZZ and RTC23 were lower relative to the wild type when cells were grown on nitrate. When the cells were grown on N20, this difference was not 83 detected, although the NO reductase activity of all strains, including wild type, was lower than that of the nitrate grown cells. In mutant strain RTC03, the N 0 reductase activity was similar to that found in the wild type. There was essentially no difference in the ability to convert N20 to N2 among wild type, RTC22, and RTC23. However, this ability was lost in mutant RTC03, which is consistant with the result that significant amounts of N20 accumulated during anaerobic growth on nitrate (Fig. 4). The wild type strain, RCT01, produced very low levels of NO during anaerobic growth on nitrate (Fig. 4). In contrast, much higher levels of NO as well as N20 were observed at late stages of growth in mutant strain RTCO3. This mutant also showed a much slower growth rate and lower yield. Discussion: Both Nir’ mutants RTC22 and RTC23 showed a lack of nitrite reductase protein bands on a Western blot (Fig. 3), suggesting that T115 insertions in the Nir' mutants were located in the operon containing the nitrite reductase structural gene. We sequenced the 1.9 kb DNA fragment that contained both Tn5 insertion sites (32). This fragment revealed an open reading frame which has 80% identity and 89% similarity in amino acid sequence to the nitrite reductase isolated from Achromobacter cycloclastes (10). Amino acid residues responsible for type I and type II copper binding were also conserved (32). When the 1.9 kb fragment was used as a probe, it hybridized to most other denitrifiers with Cu-type nitrite reductases (32). We have previously suggested that N O is an intermediate of denitrification in some denitrifiers containing copper nitrite reductases (33). We also suggested that there is a possible alternative route for N20 formation without the production of N O for P. aureofaciens and Achromobacter cycloclastes, since 180 exchange occurred during reduction of NO to N20, but not of N02‘ to NO (33). This alternative, if it exists, appears to be a minor one in Pseudomonas sp. strain G-179. Reduction of nitrite and nitric oxide were carried out by two different processes, and nitrite reductase does not seem to participate in the reduction of NO, since inhibition of nitrite reductase by DDC had no effect on NO reduction (Table 3). This is consistant with the results previously obtained in other denitrifiers (28,29). Furthermore, when cells were grown 85 on N20, the rates of NO reduction were similar between the wild type and mutants (Table 3). As a result, there is no evidence to support that nitrite reductase can directly reduce N 02‘ to N 20 without formation of N O in this strain. The lower NO reductase activity in Nir' mutants (RTC22 and RTCZ3) relative to the wild type when grown on nitrate may be due to the level of expression. This explanation is supported by the results that NO reductase activity from these mutant cells was similar to the wild type when grown on N20 as the only terminal electron acceptor. When cells were grown under denitrifying conditions with nitrate, most nitrogen was converted to N2 via NO and therefore, NO reductase activity should be fully expressed. In contrast, the availability of NO was very limited in the two Nir’ mutants. Other explanations we provided (34), such as close functional and/or genetic relationship between N02' and NO reduction, can not be excluded. Cells grown on N20 from both wild type and Nir' mutant strains have lower N 0 reductase activity as compared to those grown on nitrate (Table 3). This suggests that the level of expression of N 0 reductase is higher when grown on nitrate than on N20. We also observed a lower rate of NO reduction in Nir' mutant relative to the wild type of P. fluorescens AK-15, which contains a cytochrome c,d1 nitrite reductase (34). However, P. fluorescens AK-15 did not grow well on N 20 alone. Thus, we do not know whether the explanation provided here can apply to this organism. NO is normally found in very low concentrations during denitrification (3,14). The wild type strain of Pseudomonas sp. G-179 also produced a typical low level of NO (Fig. 4). In contrast, mutant RTCO3 86 generated a larger amount of NO at late stages of growth. This mutant also accumulated nitrite (Table 2) and N20 and had a slower growth rate (Fig.4), even though it had the capacity to convert N02" to N O and NO to N20 (Table 3). One possible explanation is that the coupling process between energy conservation and the N02‘ or NO reduction steps was blocked, resulting in slower growth rate and slower conversion of these two intermediates (Table 2). Precedent for such a lesion in energy coupling may occur in Chromobacterium violaceum, since growth yield results indicate that denitrification is not coupled to growth (2). The accumulation of intermediates in this mutant could be due to several factors including a kinetic effect resulting from a general inhibition (reduced growth in this case) to sequential Michaelis-Menten reactions (which has been modeled and verifed for denitrifiers (3)) and a partial inhibition of NO reductase by nitrite, which has been shown in membrane fractions (29). A deficiency in N20 reduction ( Table 3) led to the accumulation of N20. It is also possible that the mutation may affect a factor common to several steps, such as the involvement of copper. The group I mutants (such as RCT07 and RTC 14) that were deficient in growth on all intermediates in the denitrification pathway may have resulted from Tn5 insertion into regions responsible for gene regulation or electron transport chain components unique to denitrification (Table 1). Tn5 insertion occurred in different sites in 10 of these mutants (data not shown) and this class was obtained at a high frequency. This suggests that there are many factors other than those involved in the direct catalytic steps of the pathway that are essential to the process of denitrification. 87 ACKNOWLEDGMENTS We thank Terry Barrett, Jamie Dewitt, and Darcy Herman for their help in isolation and characterization of Tn5 mutants. We also thank Cathy McGowan for her comments on the manuscript. This research was supported by National Science Foundation grant DMB-8917427 to B.A.A. and J .M.T. Table 1. Growth characteristics of Tn5 mutants of Pseudomonas sp. strain G-179 deficient in denitrification.“ Strains + NO3‘ + NOz' + N20 RTCOl + + + RTCO7 b - - - RTC14 - - - RTCO3 c + - - RTC22 + - + RTC23 + - + a Growth on nitrate and nitrite was tested on plates and in broth under anaerobic conditions. Growth on nitrous oxide was tested in tubes with 10- ml TSB and 1 atmosphere nitrous oxide. 5 Nine additional mutants of this type were isolated. c One additional mutant of this type was isolated. Table 2. Remaining nitrate and accumulation of nitrite following anaerobic growth of Tn5 mutants of Pseudomonas sp. strain G-179 on nitrate medium.“ Strains Nitrite Nitrate ( mM ) ( mM ) RTCOl NDb 0.8 RTCO3 12.5iO.4 15.7i1.0 RTCZZ 25.7i3.7 3.2114 RTC23 26.9i2.6 2.0i2.4 0 Cells were grown in a sealed serum bottle with 120 ml TSB supplemented with 30 mM potassium nitrate. Cultures were shaken constantly and harvested after 27 h of growth. Data are the averages and standard deviation from triplicates. b ND=not detectable. The detection limit was 1 uM. Table 3. Activities of N02“, NO, and N20 reduction in wild type and mutant strains of Pseudomonas sp. G479.“ nmol produced or consumed - min'1 - mg protein-1 Strains NOz‘ ->NOb NO ->N20 N20->N2 b + N03' 5 + N20 0 RTCOl 82:3.5 28.2i2.2 5.7:00 44.8iO.9 RTCOl + DDC 0 26.3i1.6 ND d ND RTCO3 118i1.2 24.8:09 ND 03:03 RTC22 0 15.7.4505 60:20.4 36.7il.4 RTC23 0 13.8il.7 7.64.0.9 37.7i0.6 a Crude extracts were used to assay the appearance of product(s) of nitrite and NO reductases. Whole cells were used to assay for nitrous oxide disappearance of nitrous oxide was measured. Initial substrate amounts were 5 mole nitrite and 2 nmole NO. EDTA(5mM) or DDC(5mM) was added in assays for NO reduction. 5 Anaerobic growth on nitrate. C Anaerobic growth on nitrous oxide. d ND = Not determined 91 Figure legends FIG.1 Southern analyses of Tn5 insertions from Nir' mutant strains. The blot was probed with 32F labeled HpaI fragment of Tn5. Lane 1, wild type DNA digested with EcoRI and BamHI. DNA in lanes 2 (RTC23) and 3 (RT022) were digested with EcoRI, 4 (RT023) and 5 (RTCZZ) with BamHI, 6 (RT023) and 7 (RTC22) with both EcoRI and BamHI. FIG. 2. Physical mapping of Tn5 insertion sites of mutant strains RTCZZ and RTCZB in the 1.9 kb EcoRI and BamHI fragment. A, Ach; E, EcoRI; X, anI; B, BamHI; S, SalI. Triangle represents Tn5. Arrows indicate the orientation of Tn5 with respect to its SalI and BamHI sites. FIG. 3. Western blot of proteins from wild-type and mutant strains. The blot was developed with polyclonal antibodies raised against the nitrite reductase purified from A. cycloclastes. Lanes 1 through 4 were wild type, RTCZZ, RTCZ3 and RTC03, respectively. FIG. 4. Growth curve and evolution of N20 and N 0 during anaerobic growth on nitrate in wild type RTCOl and mutant strain RTCO3. Optical density was measured at 600 nm. Cells were grown under anaerobic conditions in sealed 160-ml serum bottles with 100 ml TSB and 30mM of KNO3. The gas phase was argon. Data are averages of triplicates. Nitric Oxide ( umole) Nitrous Oxide ( umole ) O.D. 40 , —c1— RTC03 20- 10. Oil-=15 Wt 20 ——Cl-—- RTCO3 —O— RTCOl 10- 0H 1%. —"U'_ RTCO3 —-.-— RTCOI _ 30 40 Time ( hours) Figure 4 1 . C, o it) ,o u l V‘. . ...(-¢ . O C P. 0.. . . .2 L. r ._........ .3 .. .. . .. , o A. ,. . . . . .l . c . . .r .a 154.. s ... . I v . I o .\ .u. . q . .74 I . . .... s . . . . “hf...” ‘ afir. . .. . .-~¢ u’. ' ' ‘ '3' fix“; .1 i I Fig. ll BA 94 23 22 +—>— WV Fig. 2 0.4 kb 6.5- 2.3— 2.0— Figure l References l. Arai, B., Y. Sanbongi, Y. Igarashi and T. Kodama. 1990. Cloning and sequencing of the gene encoding cytochrome c-551 from Pseudomonas aeruginosa. FEBS Lett. 261:196-198 2. Bazylinski, D. A., E. Palome, N. A. Blakemore, and R. P. Blakemore. 1986. Denitrification by Chromobacterium violaceum. Appl. Environ. Microbiol. 52:696-699. 3. Betlach,M.R. and J.M.Tiedie. 1981. Kinetic explanation for accumulation of nitrite, nitric oxide, and nitrous oxide during bacterial denitrification. Appl. Environ. Microbiol. 42:1074-1084. 4. Braun, C.and W.G.Zumft.. 1991. Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide. J. Biol. Chem. 266:22785-22788. 5. Carr. G.J., M.D. Page and S.J. Ferguson. 1989. The energy-conserving nitric-oxide-reductase system in Paracoccus denitrificans. Eur. J. Biochem. 179:683-692 6. Carr, G.J. and S.J. Ferguson. 1990. The nitric oxide reductase of Paracuccus denitrificans. Biochem J. 269:423-430. 97 7. Christensen, S. and J.M. Tiedje. 1988. Sub-parts-per-billion nitrate methods: use of an N20-producing denitrifier to convert N 03' or 15NO3‘ to N20. Appl. Environ. Microbiol. 54:1409-1413. 8. Coyne, M.S., A. Arunakumari, BA. Averill, and J.M. Tiedje. 1989. Immunological identification and distribution of dissimilatory heme Cd] and nonheme copper nitrite reductases in denitrifying bacteria. Appl. Environ. Microbiol. 55:2924-2931. 9. Demastia, M., T. Turk, and T.C. Hollocher. 1991. Nitric oxide reductase. Purification from Paracoccus denitriflcans with use of a single column and some characteristics. J. Biol. Chem. 266:10899-10905. 10. Fenderson,F.F., S. Kumar, E.'l‘. Adman, M.-Y.Liu, W.J. Payne, and J. LeGall. 1991. Amino acid sequence of nitrite reductase: a copper protein from Achromobacter cycloclastes. Biochem. 30:7180-7185. 11. Firestone, M.K., R.B. Firestone and J.M. Tiedje. 197 9. Nitric oxide as an intermediate in denitrification: evidence from nitrogen-13 isotope exchange. Biochem. Biophys. Res. Commun. 91:10-16. 12. Gamble, T.N., M. R. Betlach and J. M. Tiedje. 1977. Numerically dominant denitrifying bacteria from world soils. Appl. Environ. Microbiol. 33:926—939. 98 13. Godden, J. W., S. Turley, D.C. Teller, E. T. Adman, M.Y. Liu, W.J. Payne, and J. LeGall. 1991. The 2.3 angstrom X-ray structure of nitrite ' reductase from Achromobacter cycloclastes. Science. 253:438-442. 14. Goretski, J ., O. C. Zafiriou, and T. C. Hollocher. (1990) Steady-state nitric oxide concentrations during denitrification. J. Biol. Chem. 265:11535- 11538 15. Goretski J. and T.C. Hollocher. 1990. The kinetic and isotopic competence of nitric oxide as an intermediate in denitrification. J. Biol. Chem. 265:889-895. 16. Heiss, B., K. Frunzke and W. Zumft. 1989. Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome be complex of nitrate- respiring (denitrifying) Pseudomonas stutzeri. J. Bacteriol. 171:3288-3297. 17. Hochstein, LI. and GA. Tomlinson. 1988. The enzymes associated with denitrification. Annu. Rev. Microbiol. 42:231-261. 18. Jackson, M.A., J.M. Tiedje, and BA. Averill. 1991. 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Maniatis T., E.F. Fritsch, and J. Sambrook. 1982. Molecular cloning: A laboratory manual. Cold Spring Harbor Press. Cold Spring Harbor, NY. 24. Michalski, W.P. and D.J.D. Nicholas. 1988. Immunological patterns of distribution of bacterial denitrifying enzymes. Phytochemistry. 27:2451- 2456. 25. Nordling M., S. Young, B.G. Karlsson and LG. Lundberg. 1990. The structural gene for cytochrome c551 from Pseudomonas aeruginosa-The nucleotide sequence shows a location downstream of the nitrite reductase gene. FEBS Lett. 259:230-232. 1(1) 26. Payne, W.J. 1981. Denitrification. John Wiley & Sons, Inc., New York. 27. Silvestrini, M.C., C.L. Galeotti, NL Gervais, E. Schinina, D. Barra, F. Bossa and M. Brunori. 1989. Nitrite reductase from Pseudomonas aeruginosa: sequence of the gene and the protein. FEBS Letter 254:33-38 28. Shapleigh, J.P. and W.J. Payne. 1985. Differentiation of c, d1 cytochrome and copper nitrite reductase production in denitrifiers. FEMS Microbiol. Lett. 26:275-279. 29. Shapleigh, J.P., KJ.P. Davies, and W. J. Payne. 1987. Detergent inhibition of nitric oxide reductase activity. Biochim. Biophys. Acta. 911:334- 340. 30. Smith, B.G. and JM. Tiedje. (1992) Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria. Appl. Environ. Microbiol.58:376-384. 31. Weeg-Aerssens, E, W. Wu, R.W. Ye, J.M. Tiedje, and C.K. Chang. 1991. Purification of cytochrome c,d1 nitrite reductase from Pseudomonas stutzeri JM 300 and reconstitution with native and synthetic heme d1. J. Biol. Chem. 266:7496—7502 32. Ye, R.W., M. R. Fries 1, S. B. B, B. A. Averill, and J. M. Tiedje. Characterization of a copper nitrite reductase gene and its homology with other denitrifiers. Appl. Environ. Microbiol. (submitted for publication) 101 33. Ye, R.W., I. Tom-Suarez, J.M. Tiedje, and BA. Averill. 1991. H2180 isotope exchange studies on the mechansim of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria: evidence for an electrophilic nitrosyl during reduction of nitric oxide. J. Biol. Chem. 266:12838-12851. 34. Ye, R.W., AArunakumari, BA. Averill and J .M. Tiedje. 1992. Mutants of Pseudomonas fluorescens AK-15 deficient in dissimilatory nitrite reduction are also altered in nitric oxide reduction. J. Bacteriol.174:2560- 2564. 35. Zumft, W.G., D.J. Gotzmann and P.M.H. Kroneck. 1987. Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens. Eur. J. Biochem 168:301-307. 36. Zumft, W.G., H. Korner, S. Lochelt, A. Viebrock and K. Frunzke.1988. Defects in cytochrome cd1-dependent nitrite respiration of transposon Tn5- induced mutants from Pseudomonas stutzeri. Arch. Microbiol. 149:492-498. Chapter Six Characterization of the Structural Gene for a Copper Containing Nitrite Reductase and its Homology to Other Denihifiers RICK w. YE 1, MARCOS R. FRIES 1, SERGUEI B. BEZBORODNIKOVl, BRUCE A. AVERILLZ, and JAMES M. TIEDJE 1* Departments of Microbiology and Public Health and of Crop and Soil Sciences, Michigan State University, East Lansing, Michigan 48824 1; and Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901 2 102 103 Abstract. A copper-containing nitrite reductase gene (nirA) from Pseudomonas sp. G-179 was found in a 1.9 kb EcoRI and BamHI DNA fragment. The coding region contained information for a polypeptide of 379 amino acids. The encoded protein had 78% identity and 88% similarity in amino acid sequence to the nitrite reductase purified from Achromobacter cycloclastes . Ligands for type 1 and type 2 copper binding sites were conserved. Analysis of the promoter region revealed two tandem putative ntrA boxes, suggesting that a 654-like factor is needed for its expression. Upstream from the promoter, one putative fnr box was found, indicating that a FNR-like protein may be involved in regulation of the nitrite reductase gene under anaerobic conditions. When the structural gene was used to study the homology among different denitrifiers, it showed positive signals with DNA from17 out of 18 gram-negative denitrifiers containing copper nitrite reductases. Except for Pseudomonas stutzeri JM300, all denitrifiers that contain heme c,d1 nitrite reductases showed no or weak signals with this probe. Thus this structural gene may be useful as a probe to detect denitrifiers with copper nitrite reductases . 104 Introduction Denitrification is the dissimilatory reduction of nitrate to nitrogen gases. The key environmental step in denitrification is the reduction of nitrite by nitrite reductases since this enzyme converts a mineral form of nitrogen to a gaseous form, NO, which can not be assimilated by the biosphere. Based on the characteristics of active sites, two major types of dissimilatory nitrite reductases are known (9,22): those containing the cytochrome c and d1 (c,d1 -dNirs) and those containing copper (Cu-dNirs). Cu-dNirs from many denitrifiers exhibit homology to each other as shown by cross-reactivity in immunoblots (2, 18). Studies of the Cu-dNir from Achromobacter cycloclastes have revealed that the enzyme is a trimer with three type 1 and three type 2 coppers (5,8). Type 1 copper is bound within a single monomer, while type 2 copper is held by residues from each of two monomers of the trimer. Evidence from the crystal structure shows that nitrite is bound to the type 2 copper site (5), implying that type 2 copper is essential for enzyme activity. Another copper containing protein, pseudoazurin, is the physiological electron donor for the Cu-nitrite reductase in vitro (12,13, 30). Denitrifiers grow using aerobic respiration in aerobic environments but can shift to using nitrogen oxides as electron acceptors under anaerobic conditions. How these genes involved in denitrification are regulated is poorly understood. In Escherichia coli and other enteric bacteria, the fnr (fumarate and nitrate reductases) gene is a positive regulator for the expression of genes involved in fumarate and nitrate respiration as well as 105 other anaerobic processes (see reviews, ref. 11 and 25). It is generally accepted that under anaerobic conditions, the FNR protein acts as a transcriptional activator by binding to its binding site, the FNR box, upstream of the promoter. FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa has been demonstrated (6). In P. stutzeri JM300, two FNR boxes have been found upstream of the promoter in the operon containing the nitrite reductase structural gene, suggesting the existence of a similar type of regulation (23). Another regulator, the ntrA gene product 654, is involved in diverse physiological processes (14). Both fnr and ntrA boxes have been found in the upstream region of the promoter of azurin and pseudoazurin genes in several denitrifiers (10) and upstream of the nifA promoter of Azorhizobium caulinodans (19). Recently, research efforts have been devoted to isolating denitrifiers that degrade environmental contaminants and to studying the ecology of denitrifiers in soil. The use of gene probes to identify and monitor populations of denitrifiers in natural environments and enrichments offers several advantages over the conventional culture-dependent methods (20,23). The majority of the available genetic information on denitrifiers comes from strains that contain the cd1 -dNirs. Because strains containing the copper type make up over one-third of the studied isolates and represent a wider phylogenetic distribution (2), and because the enzyme has different mechanistic features than does the c,d1-dNir, it is important to obtain genotypic, phenotypic, and ecological information on this group of organisms as well. Probes for c,d1-dNirs alone will not meet the needs for 106 population studies. Hence, it is important to evaluate whether there are suitable probes for Cu-dNir containing denitrifiers. In this paper, we report the primary structure of a Cu-dNir structural gene. The upstream and the promoter region have been analyzed for possible regulatory mechanisms. We also report on the homology of this gene to the DNA of other denitrifiers and find it specific for most of Cu—type denitrifiers. Materials and Methods Strains and growth conditions. Pseudomonas sp. strain G~179 was isolated from a pampa agricultural soil from the Parana Experimental Station, Argentina (7). This strain contains a Cu-type nitrite reductase as demonstrated by diethyldithiocarbamic acid (DDC) inhibition and immuno- reaction to the polyclonal antibodies raised against the Cu-type nitrite reductase from A. cycloclastes (2). The growth medium contained half- strength tryptic soy broth (TSB), 2 uM of CuSO4, and 5 mM of potassium nitrate, if used. Cells not immediately used were stored at -70°C. Southern blots and sequencing. Genomic DNA isolation, restriction enzyme digestion, electrophoresis, and Southern blotting were done as described by Maniatis, et al (16). DNA fragments were isolated from agrose gel with a Geneclean kit (Bio 101, La Jola, California) and was labelled with 32P with a random primer kit (Boehringer Mannheim, Germany). After hybridization, Southern blots were washed three times at room temperature with following solution: 2 x SSC/0.1% SDS, 0.5 x SSC/0.1 % SDS 107 and 0.1 x SSC/0.1%. The final wash was carried out at 50°C with 0.1% SSC/1% SDS. All DNA sequencing reactions were carried out by the di- deoxy method using the Sequenase kit (U.S. Biochemical Corp., Cleveland, Ohio). Both strands were sequenced. Analysis of DNA sequence was performed with the GCG sequence analysis program (4) Results Isolation of the nitrite reductase gene. We have previously found that two Tn5 Nir' mutants of Pseudomonas sp. G-17 9, RTC22 and RTC23, had no nitrite reductase activity and showed no nitrite reductase protein band in Western blots (29). The Tn5 insertion sites in these two mutants were clustered in the same EcoRI and BamHI fragment. Genomic DNA from these two mutants was digested with EcoRI and BamHI and the DNA fragments containing the neo gene of the Tn5 plus the flanking genomic region were subcloned in pUC19 by selection on kanamycin plates (50 ug/ml). Subclones were then used as probes to isolate the corresponding wild type EcoRI and BamHI fragment. Several positive clones were obtained and they all possessed a 1.9 kb EcoRI and BamHI fragment as shown in Fig. 1. Results from DNA sequencing revealed an open reading frame corresponding to a copper nitrite reductase transcribed in the direction of EcoRI to BamHI (Fig. 2). The Tn5 in both mutants was found inside the structural gene (29). The sequence has been placed in Genebank with the accession No.M97294. Properties of the derived protein product. When the deduced amino acid sequence was compared to the amino acid sequence of the Cu-dNir 108 from Achromobacter cycloclastes (5), 78% identity and 88 % of similarity were found (Fig.3). Studies on the crystal structure and amino acid sequence of the dNir from Achromobacter cycloclastes, have revealed both type 1 and type 2 copper binding sites (5,8). Amino acids responsible for copper binding in the Cu-dNir from A. cycloclastes and the corresponding region of the Pseudomonas sp. G-179 sequence are compared in Fig. 4. All of the ligands to both copper sites are strictly conserved and the amino acid sequences in these regions exhibit a very high degree of homology. This suggests that the Cu—dNir from Pseudomonas sp. G-179 contains both type 1 and type 2 copper. Due to the presence of two methionines among the first 12 amino acids, there are two possible start sites . The first 32 amino acids showed strong hydrophobicity (Fig. 5), a feature of typical signal peptides. It has been shown by immunogold labelling and electron microscopy that nitrite reductases from two Cu-dNir containing organisms and two c,d1-dNir containing organisms are located in the periplasmic space in gram- negative bacteria (3). Presence of signal peptide is consistant with this observation. The first amino acid of the mature protein is likely to be Glu- 32, since it is proceeded by an Ala-X—Ala processing site, another feature of signal peptides. The mature protein had low hydrophobicity, suggesting that the enzyme is soluble (Fig.5) Characterization of the promoter region. The protein FNR has been found to be an important activator for anaerobic metabolism in E.coli and other organisms. The consensus sequence to which the FNR protein binds is TTGATN4ATCAA (10,19,24). The five nucleotides in the first and second 109 halves and the spacing of four nucleotides between are highly conserved. Such an FNR box, is found upstream of the promoter in the copper nitrite reductase gene of Pseudomonas sp G-179; it is located 364 bp from the assumed start codon. The sequence was (289)flG_A_IGAAAAT_CAA_(303) (Fig. 2). The ntrA gene, which encodes 054, has been foundtoflbe a regulatory factor in nitrogen metabolism and many other physiological processes (14). The ntrA box has the consensus sequence of CTGGYAYRN4TTGCA (1). The highly conserved features include GG and GC doublets separated by 10 bp. Two such consensus sequences were found, (580) TTQQAGCAAACATGQT (595 ) and (623)GTCIQAGCCGAGGTTGQT(639) (Fig. 2) . In the first ntrA-like box the GG and GC doublet was separated by 9 bp instead of 10. Two ntrA-like boxes can also be seen in the operon containing the c,d1 -dNir gene from Pseudomonas aeruginosa and upstream of genes involved in denitrification (Table 1). Homology of the Cu-nitrite reductase gene to DNA from other denitrifiers. To test the homology of the nitrite reductase gene with DNA from other denitrifiers and evaluate its potential as a probe for denitrifiers, the 1.2 kb an I and BamHI fragment was used. Southern blots containing DNA from different strains of denitrifiers were probed with this labelled fragment. Among all 18 Cu-dNir-containing and gram-negative denitrifiers, 17 have moderate to strong homology with this probe and only Alcaligenes eutrophus gave a weak signal (Table 2). Among the c,d1 -dNir- containing and gram-negative denitrifiers, only Pseudomonas stutzeri J M 300 showed a strong band with the Cu probe. This band did not correspond 110 to the band hybridized by the c,d1 -dNir probe isolated from this strain (23). Two the c,d1-dNir containing denitrifiers had weak hybridization with the Cu gene probe and six had no signal. A gram-positive denitrifier, Bacillus azotoformans , showed strong hybridization to this probe, but a second strain of Bacillus gave no hybridization. Overall, the results obtained with Southern blots were consistant with the results obtained with Western blots (Table 2). Discussion: The amino acid sequence of the copper nitrite reductase from Achromobacter cycloclastes determined by Fenderson, et al (5), was highly homologous with the derived sequence we found for Pseudomonas sp. G- 179. The ligands responsible for binding both types of copper present in the former are conserved (Fig. 3), indicating that the copper nitrite reductase from Pseudomonas sp. G-179 also contains both type 1 and type 2 copper. As a result, a similar model of protein structure for this Cu-dNir can be proposed. The type 1 Cu would be coordinated by four ligands from domain I of the same monomer: C-175, H-184, M-189, and H-134. The type 2 Cu would be coordinated by H-139 and H-174 from domain I of one molecule and H-345 from domain II of the second (Fig. 4). Cu-dNirs isolated from Alcaligenes sp. and P. aureofaciens have been reported to contain only type 1 copper based on their EPR spectra (17,30). Therefore it has been suggested that type 1 copper is the active center and that the type 2 copper may not be required for enzymatic activity. However, recent protein crystal structure studies suggest that type 2 copper is the binding site for the substrate, nitrite (8). It has also been observed 111 that type 2 copper can be removed from the enzyme, and when reconstituted with copper the enzymatic activity is restored proportional to the amount of type 2 copper added ( E. Libby and BA. Averill, submitted for publication). These results indicate that the type 2 copper is essential for the enzymatic activity, but that it may be easily lost during the purification process. The gene product of ntrA, C54, confers specificity on core polymerase for diverse physiological functions, including nitrogen fixation, catabolism of toluene and xylene, and nitrate and nitrite assimilation (14). Analysis of DNA sequence in the promoter regions of some genes involved in denitrification or anaerobic metabolism reveals the existence of putative ntrA boxes (Table 2). This box was found upstream of the N20 reductase gene from P. stutzeri Zobell (26), the cd1-dNir gene from P. aeruginosa (21) and the pseudo-azurin gene from Alcaligenes faecalis S-6 (27), which is the physiological electron donor for Cu-type nitrite reductase in vitro (12). In the copper-nitrite reductase gene from Pseudomonas sp. G-179, we noted two tandem ntrA-like boxes were found (Fig. 2 and Table 2). These results suggest that the ntrA protein may involved in the regulation of promoters involved in denitrification under anaerobic conditions. However, involvement of G54 has not been tested in NtrA' mutants. Upstream of the promoter region in Pseudomonas. sp. G-179, an fnr box was also found, suggesting an FNR-analog in this organism may play the role of activator (Fig. 2 and Table 1). It has been shown that a mutation in a fnr-like gene results in loss of nitrate and nitrite dissimilation in P. aeruginosa, which contains a c,d1-dNir (6). Two fnr boxes were found upstream of the promoter of the nitrite reductase gene in P. stutzeri (23). 112 The fnr box is also found upstream of the azurin and pseudoazurin genes (Table 1). Preliminary experiments with an FNR'(ANR') mutant of P. aeruginosa PAOl, provided by Dieter Haas (6) suggest the involvement of this regulatory mechanism in denitrification (unpublished data). As a result, it is very likely that an FNR-like protein (ANR) plays an global role in regulation of denitrification under anaerobic conditions. One hypothetical model for the regulation of Cu-nitrite reductase from. Pseudomonas sp. G-179 is that under anaerobic conditions, an FNR-like protein activates transcription by contacting the RNA polymerase-promoter complexes. This process could be mediated by formation of a DNA loop since the fnr binding site is more than 300 bp from the promoter. Such a loop formation mechanism has been found in regulation of the glnA promoter, which is controlled by NtrC and O54 (28). There are two putative ntrA boxes in the promoter region and this may explain the existence of two protein bands with slightly different molecular weights that we observed in Western blots (29). Obviously, mutants that are deficient in FNR and NtrA needs to be further characterized. Further analysis of the promoter region is also required to elucidate the mode of regulation. When the 1.2 kb anI and BamHI fragment containing the nitrite reductase was used as a probe, it hybridized to most of the gram-negative, Cu-dNir containing denitrifiers tested and not to those containing cd1 dNir except for P. stutzeri JM300. As a result, this probe may be useful in identifying this group of denitrifiers. The c,d1 -dNir genes have been used as probes (15,23), but they are primarily limited in c,d1 -dNir containing denitrifiers. The suite of both Cu- and heme-type nitrite reductases gene 113 probes may be useful to reveal ecological features of these two groups of organisms. ACKNOWLEDGMENTS We thank James R. Cole for his help in the analysis of the DNA sequence. This research was supported by National Science Foundation grant DMB-8917427 to B.A.A. and J .M.T. 114 Table 1. Putative regulatory sequences from denitrifying bacteria.“ Strains Gene fnr box ntrA box P. sp. strain G~179 Cu-nir TTGAT....ATCAA T'IEQAGCAAAC ATQQI GTQQAGCCGAGGTTQQT P. aeruginosa (21) 5 ed] -nir UN c CGQQAGTTCCCGACQQA AAGGGAGCGCC TCGCA P. stutzeri JM300 (23) cd1 ~nir T'DGAT....GTCAA TTGAC....ATCAA UN P. stung-i Zobell (23) ed] -nir TTGAT....ATCAA TTGAT....GTCAA UN P. stutzeri Zobell (26) nos UN GTQQAACCCTGAGCQQG P. aeruginosa (10) azu T'I‘GAC....ATCAG GCQQCACATCT GTQQT Alcaligenes denitrificans (10) am TTGAT....GTCAA CAECATGTGCCTGQQG Alcaligenes faecalis S-6 (27) Peudo-azu T'I‘GAT....ATCAA GTQQCGTGTTGAGQQC a References are given in the parenthesis following the strain names. Abbreviations are nir: nitrite reductase gene; nos: nitrous oxide reductase gene; azu: azurin gene. 5 The presence of possible regulatory regions were identified in this work based on published results. c UN=Unknown or inadequate information 115 Table 2. A summary of hybridization results by Western and Southern blots of different denitrifiers. My“ Strains nir type cd1- Cu nirAb (CU) Ps. sp. G-179 Cu - + + Achr cycloclastes ATCC 2192 Cu - + + Ps. aureofaciens ATCC 13985 Cu - + + Alcal. xyolsoxidans NCIB 11015 Cu - + + Alcal. eutrophus ATCC 17699 Cu - ND +/- Rhodops. sphaeroides f. sp denitrificans Cu - + + Corynebacterium nephridii ATCCll425 Cu - + + Bacillus azotoformans ATCC 29788 Cu - + + Ps. chlororaphis ATCC 43928 Cu - N D + Ps. fluorescens ATCC 17575 Cu N D ND + Agrobacterium tumefaciens A348 Cu - ND 4- Ps. type 11 G~107 Cu - + + Ps. type 11 G~163 Cu - + 4. Ps. type 11 G~188 Cu - + + Alcal. faecalis G-191 Cu - + + Alcal. faecalis G-41 Cu - + + Alcal. faecalis G—65 Cu - + + Bacillus sp. G~193 Cu - - - Ps. picketii PKOl Cu ND ND + Ps. aeruginosa PAOl heme + - - Ps. aeruginosa ATCC 10145 heme + - - 116 Ps. aeruginosa ATCC 19429 heme ND ND +/- Ps. aeruginosa ATCC 15692 ? 4.]. Ps. fluorescens ATCC 33512 heme + - - Ps. fluorescens AK—15 heme + - - Ps. stutzeri JM 300 heme + - + Ps. stutzeri ATCC 11607 heme + - +/- Ps. stutzeri ATCC 11405 heme 4» ND - Pa. halodenitrificans +/- Ps. type 2 G~83 N D - - - Tol 4 heme N D N D - Ps. sp KC ND ND ND - Non-denitrifiers: Agrobacterium tumefaciens A60 - Ps. mendocina - a Results of Coyne, et a1 (2). Some nir types were also determined by inhibition of activity after adding the Cu chelator, DDC. b The extent of hybridization in Southern blots with the 1.2 kb fragment containing the Cu-dNir gene is indicated by plus (+) or negative (-) signs. 117 Figure legends FIG. 1. Physical map of the 1.9 kb EcoRI and BamHI fragment. The open reading frame region for nirA is shaded and the arrow indicates the direction of transcription. FIG.2. Nucleotide sequence of the 1.9 kb EcoRI and BamHI fragment and the predicted amino acid sequence of the Cu-type nitrite reductase. Numbers given on the right are for nucleotides and on the left for amino acids. Possible regulatory regions (fnr box and ntrA box) and the putative ribosome-binding site (Shine-Dalgamo sequence, SD ) are underlined. FIG. 3. Comparison of the amino acid sequences of Cu-type nitrite reductases from Pseudomonas sp. G179 and Achromobacter cycloclastes (AC). FIG. 4. Comparison of the copper binding region for Cu-dNirs from Pseudomonas. sp. G-179 and Achromobacter cycloclastes (8). The ligands responsible for Cu are shown in bold and the number underneath indicates the type of Cu it binds. FIG.5 . Hydropathy index of the encoded nitrite reductase by the method of Goldman et al ( ) or Kyte and Doolittle ( ------- ) (4). The analyses were performed with the GCG program. The hyrophobic regions are above the horizontal zero line, whereas the regions of relatively hydrophilic nature are below the zero line. The amino acid numbers are the same as in Fig.2. 118 Acct Accl Accl l ' —{1f/J///://///////J////j///////77J EcoRI anl Sail BamHI _>. .L n' 0. 4 kb Figure 1 19 39 S9 79 99 119 139 179 199 219 239 119 AATTCGGCCGTCCCGGGGTAATGTCGTCAGGGCCGGGTGGGCCGTAGACGATTTCATACG CCCAAACTGCGAAACCGTAGCTGGCGACGGTACCAACGGCCACGACTCGCCAAATCCCAA AAGCGAGAACGACAAAGGTCAGCAGCTCGCGGCGCCGCTTTTGGCGGAAGGATGTCTCTT CCAGAGTTCCTATCGGCTCTGCCATGTTCACTTCCGGTCTTCAGCAAAATCTGAAATGCA AAAATGCACTAAATGCCTCAAATATAACCAAATCCGTCCATCACTCGCCIIQATGAAAAI fnr box CAAATTTAGTCGCGGTTCGCEGAAAAAATATGCGTAAAGATTAAAAAGCAGACACACATA AGTTGTGCCATGAGCGACGCACAACATTAAGGACAAAGCTGCTCATCACTCAAATAGATA CCAATAATCGTTTTTTTGCTCAGCTGCTGTACTCGCCTCCGATGAGCACAACTTCCTTTC CCTAACGCTTGAGTGTTCCCTCAAAAACAGCATTAATCAAGTGGCCGTGTGTATGTTCGT GTAATCTACCTATAGGCGAAAACTCTCGTTTTATTTGTGIIQQAGCAAACAIQCTCGCGA ntrA box ATGTCGATCTACGTTCAGGGCTQIQQAGCCGAGGITQQICCGCTTCTTGAAAAAQQQAQA ntrA box SD GACTTTCATCAGTGAACAGTTTCGGTTGACCCGCCGGAGCATGCTGGCCGGCGCAGCTGT M S E Q P R L T R R S M L A G A A V +1 CGCGGGAGCACTAGCGCCGGTCGTCACCTCTGTTGCACATGCCGAAGGCGGGGGTATCAA A G A L A P V V T S V A H A E G G G I K GACAAATTCTGCTGCGACAGCAGCAAACATCGCGACGCTGGAGCGCGTCAAGGTGGAACT T N S A A T A A N I A T L E R V K V E L GGTCAAGCCACCCTTTGTGCACGCCCACACCCAGAAGGCTCAGGGCGAGCCCAAGGTTCT V K P P F V H A H T Q K A E G E P K V V CGAGTTCAAGATCACCATCCAGGAAAAGAAGATCGTTGTCGACGACAAGGGGACCGAGGT E F K M T I Q E K K I V V D D K G T E V CCATGCGATGACGTTCGACGGATCTGTGCCGGGGCCGATGATGATCGTTCACCAGGATCA H A M T F D G S V P G P M M I V H Q D D TTATGTCGAACTGACCCTCGTCAATCCCGATACAAACGAATTGCAGCACAATATCCACTT Y V E L T L V N P D T N E L Q H N I D P CCATTCGGCGACGGGTGCGCTCGGTGGTGGAGCGCTGACCGTGGTCAATCCCGGTCACAC H S A T G A L G G G A L T V V N P G D T CGCCGTGCTCCCTTTCAAGGCCACCAAGGCGGGTGTTTTTGTCTATCACTGTGCCCCCGC A V L R F K A T K A G V F V Y H C A P A CGGCATGGTGCCGTGGCATGTCACTTCGGGCATCAATCGTGCGATCATGGTCCTCCCGCG G M V P W H V T S G M N G A I M V L P R TCACGGTCTCAAGGATCACAAGGGCCACGAGCTTGTTTACGACAAGGTCTACTATGTCGG D G L K D H K G H E L V Y D K V Y Y V G CGAGCACGACTTTTATGTCCCCAACGATGAGAACGGCAAGTTCAAGAAATACGAAAGCGC E Q D F Y V P K D E N G K F K K Y E S A CGGCGAGGCCTATCCGGATGTGCTCGAAGCCATGAAGACACTGACCCCGACCCATGTCGT G E A Y P D V L E A M K T L T P T H V V Figure 2 50 120 180 240 300 360 420 480 540 600 660 720 780 740 900 960 1020 1080 1140 1200 1260 1320 1380 1440 259 279 299 319 339 359 379 120 GTTCAACGGGGCGGTCGGCGCCCTGACGGGCGATAACGCCTTGCAGGCAAAGGTGGGCGA F N G A V G A L T G D N A L Q A K V G D TCGTGTCCTCATTCTTCATTCGCAAGCCAACAGGGATACGCCCCCGCACCTGATCCGGGG R V L I L H S Q A N R D T R P H L I G G GCATGGCGATTATGTCTGGGCTACCGGCAAGTTCGCCAACCCGCCGGAACTCGATCAGGA H G D Y V W A T G K P A N P P E L D Q E AACCTCGTTCATTCCCGGAGGTGCTGCCGGGGCGGCTTACTACACGTTCCAGCAGCCCGG T W F I P G G A A G A A Y Y T P Q Q P G TATCTATCCGTATGTAAACCACAATCTGATCGAGGCGTTCGAACTCGGCGCGGCCGGCCA I Y A Y V N H N L I E A F E L G A A G H CTTCAAGGTGACGGGCGACTGGAACGACGACCHCATCACAGCCGTGGTTTCGCCGACCTC F K V T G D W N D D L M T A V V S P T S GGGTTGACGGTGTCGGCCCGGCGCCGGATCAGGCGCCGGGCCACATCCTTATGCCGGCCG G t GGGGAGGCCGGAAGGATCGGAGGATC 1886 1500 1560 1620 1680 1740 1800 1860 Nit-179 Percent G179 AC 40 90 51 140 101 190 151 240 201 290 251 340 301 121 x Nix-AC Similarity: 88.496 Percent Identity: 78.466 MSEQFRLTRRSMLAGAAVAGALAPVVTSVAHAEGGGIKT NSAATAANIATLERVKVELVKPPFVHAHTQKAEGEPKVVEFKMTIQEKKI ..:i.:.:l.ll.llll:|ll||||l||.| I..:l:||||.|ll:l||: aagaapVDISTLPRVKVDLVKPPFVHAHDQVAKTGPRVVEFTMTIEEKKL VVDDKGTEVHAMTFDGSVPGPMMIVHQDDYVELTLVNPDTNELQHNIDFH l:| .ill:|llll:lll||l:|:||::l|||l |:l||||.l llllll VIDREGTEIHAMTFNGSVPGPLMVVHENDYVELRLINPDTNTLLHNIDFH SATGALGGGALTVVNPGDTAVLRFKATKAGVFVYHCAPAGMVPWHVTSGM .IIIIIIIIIII IIII:...IIII|II:IIIIIIIII.IIIIIIIIIII AATGALGGGALTQVNPGEETTLRFKATKPGVFVYHCAPEGMVPWHVTSGM NGAIMVLPRDGLKDHKGHELVYDKVYYVGEQDFYVPKDENGKFKKYESAG IIIIIIIIIIIIII.II:.I.III:IIIIIIIIIIIIII.I.:IIII.:I NGAIMVLPRDGLKDEKGQPLTYDKIYYVGEQDFYVPKDEAGNYKKYETPG EAYPDVLEAMKTLTPTHVVFNGAVGALTGDNALQAKVGDRVLILHSQANR III.I.:.II:IIIIII:IIIIII|IIIII:II I II:III::IIIIII EAYBDAVKAMRTLTPTHIVFNGAVGALTGDHALTAAVGERVLVVHSQANR DTRPHLIGGHGDYVWATGKFANPPELDQETWFIPGGAAGAAYYTFQQPGI llllllllllllllllllll lll:ll||ll:lll|.|||l:||l.|||: DTRPHLIGGHGDYVWATGKFRNPPDLDQETWLIPGGTAGAAFYTFRQPGV YAYVNHNLIEAFELGAAGHFKVTGDWNDDLMTAVVSPTS 378 Illllltlllllllllllllllll:ll|llll.lI.l.l YAYVNHNLIEAFELGAAGHFKVTGEWNDDLMTSVVKPAS 339 Figure 3 89 50 139 100 189 150 239 200 289 250 339 300 G—179 AC G-l79 AC D1 01 D2 DZ 132 93 337 298 122 LQHNIDFHSA 168 AGVFVYHCAPAGMVPWHVTSGMNGA ALLHNIDFHAA 129 PGVFVYHCAPEGMVPWHVTSGMNGA l 2 2 l 1 l PGIYAYVNHNLI PGVYAYVNHNLI 2 Fignm.4 123 u D u x0300: > 7? >7 . > 2. ink/H? o ...< 42... .E.<. <4: . :32: .s (can K. ..~ ( U f. 0 . woo no.2... 2 o. # } >b 7» > . . . 1.. 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The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue c0pper protein from Escherichia coli cells. J. Bacteriol. 16925648-5652. 128 28. Wedel, A, D. S. Weiss, D. Pophem, P. Droge, and S. Kustu. 1990. A bacterial enhancer functions to tether a transcriptional activator near a promoter. Science 248:486-489. 29. Ye, R.W, BA. Averill and J .M. Tiedje. 1992. Charaterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179 with a copper nitrite reductase. J. Bacteriol. (submitted for publication) 30. Zumft, W.G., D.J. Gotzmann and P.M.H. Kroneck 1987. Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens. Eur. J. Biochem 168:301-307. 129 Contributions by Other Authors Ms. Inez Toro-Suarez provided technical help for the GC/MS analysis throughout my research. Without her help it would have been impossible to complete the isotope analysis done in Chapter Two, Three and Four. Mr. Serguei B. Bezborodnikov help with the sequencing of the 1.9 kb fragment that contains the Cu-type nitrite reductase gene. Dr. Alahari Arunakumari isolated Pseudomonas fluorescens AK-15 and initiated the work on isolation of Tn5 mutants. The mutants represented in this thesis were ones that I isolated. Mr. Marcos R. Fries provided the information on the homology of Cu- type nitrite reductase gene to other organisms. We collaborated on this work. MICHIGAN STnTE UNIV. LIBRARIES ill[WWIWilli/11111111111IllIIWIWUIIIHIIWWI 31293008803623