Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA1
Author for correspondence: James P. Shapleigh. Tel: +1 607 255 8535. Fax: +1 607 255 3904. e-mail: jps2{at}cornell.edu
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ABSTRACT |
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Keywords: photosynthetic denitrifier, denitrification, nitrite reductase, blue copper protein
Abbreviations: MBP, maltose-binding protein
The GenBank accession number for the sequence determined in this work is AF339883.
a Present address: Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08540-1014, USA.
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INTRODUCTION |
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In addition to the genes required to encode the terminal nitrogen oxide reductases there are additional genes required for assembly and function of the terminal reductases. For example, the haem-containing nitrite reductase requires at least seven genes for the synthesis of the novel d1 haem (Zumft, 1997 ). Nitrate, nitric oxide and nitrous oxide reductases have all been shown to require additional proteins for synthesis of cofactors or assembly of the finished active complex (Zumft, 1997
). In most cases genes encoding accessory proteins are located in clusters of genes whose products are required for the assembly of a particular reductase.
Rhodobacter sphaeroides strain 2.4.3 expresses a copper-containing nitrite reductase encoded by nirK (Tosques et al., 1997 ). Analysis of chromosomal sequence downstream of nirK in R. sphaeroides 2.4.3 identified two ORFs encoding products found in other denitrifiers. One of the ORFs, which has been designated ppaZ, encodes a protein with significant similarity to pseudoazurins. Pseudoazurins are small periplasmic proteins that contain a single copper atom (Petratos et al., 1987
). Biochemical studies have suggested that pseudoazurin is a direct electron donor to nitrite reductase (Kukimoto et al., 1996
; Williams et al., 1995
). Recent genetic evidence has confirmed this suggestion (Koutny et al., 1999
). The other gene downstream of nirK encodes a product similar to NirV from Pseudomonas G-179 (Bedzyk et al., 1999
). In G-179, as in R. sphaeroides 2.4.3, the nirV gene is immediately downstream of the gene encoding nitrite reductase. The role of the nirV product is unknown.
The experiments described in this paper were designed to study the role of pseudoazurin and the nirV product during nitrogen oxide reduction in R. sphaeroides. Expression studies showed that pseudoazurin is expressed during anaerobic growth. However, the role of this protein is unclear because three of the four amino acid residues required for copper ligation are not conserved in the pseudoazurin encoded by 2.4.3. Inactivation of the gene encoding pseudoazurin did not cause any detectable phenotypic changes. Analysis of the expression of nirV indicated that it is cotranscribed with nirK and is therefore a member of the NnrR regulon, which includes nirK and the genes encoding nitric oxide reductase (Tosques et al., 1996 ). Inactivation of nirV also did not cause detectable phenotypic changes to cells cultured under low-oxygen conditions.
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METHODS |
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E. coli strains were grown in LB medium (Maniatis et al., 1982 ). Rhodobacter strains were grown in Sistroms medium at 30 °C (Leuking et al., 1978
). Nitrate was added to Rhodobacter cultures to a final concentration of 12 mM. Other culture conditions and amendments were as described previously (Tosques et al., 1997
). Procedures for growing wild-type and mutant R. sphaeroides microaerobically, which permits expression of genes regulated by NnrR, are described elsewhere (Tosques et al., 1996
).
Taxis assays were similar to previously described R. sphaeroides taxis assays (Gauden & Armitage, 1995 ). Cells for taxis assays were grown microaerobically in medium unamended with nitrate. After growth, cells were concentrated twofold in Sistroms medium by centrifugation and then diluted in Sistroms agar such that the final agar concentration in the cell suspension was 0·4%. Cells resuspended in agar were poured into Petri plates and a plug containing either 0·5 M nitrate or 0·36 M nitrite in 2% Sistroms agar was inserted in the centre of the plate. The plates were cooled for 510 min and then placed in an anaerobic jar and incubated under a N2 atmosphere.
DNA manipulation and sequencing.
Chromosomal DNA was isolated from 2.4.3 using the Puregene system (Gentra Systems). Plasmid isolations were done using the alkaline lysis method (Birnboim & Doly, 1979 ). Standard methods were used for restriction digests, agarose gel electrophoresis, and ligations. Southern hybridizations were carried out as described previously (Toffanin et al., 1996
). Transformations were done using TSS (Chung et al., 1989
). Plasmids were moved into 2.4.3 by conjugation. Biparental matings were carried out with E. coli S17-1 as the donor.
The 3·0 kb EcoRI fragment encoding nirV and ppaZ was originally isolated from a lambda library of 2.4.3 DNA (Fig. 1a). Some of this fragment has been sequenced previously (Tosques et al., 1997
). Both strands of the remaining unsequenced regions were sequenced at the Cornell University BioResource Center. Fragments were generated for sequencing using available restriction sites or by PCR. Sequence comparisons were carried out using the BLAST program (Altschul et al., 1990
) available at each of the URLs listed in the text. Preliminary sequence data were obtained from the DOE Joint Genome Institute (JGI) at http://www.jgi.doe.gov/.
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The ppaZlacZ construct was generated by ligating a 0·55 kb EcoRIStuI fragment into pUC19 (Fig. 1a). This fragment contained 426 bp upstream of the putative ppaZ translation start. This construct was digested with EcoRI and PstI to remove the insert, which was ligated, along with the lacZKnr cassette of pKOK6 digested with PstI, into pRK415 (Fig. 1
). The ppaZ1lacZ construct was generated by amplifying a DNA fragment with the upstream primer 5'-GCGAATTCGGAATTGATGCAGCGCAAC-3' and the downstream primer 5'-GCGGATCCTTGCGGTATCGACATG-3'. The amplicon was restricted with KpnI and BamHI and ligated with the pKOK6 lacZKnr cassette digested with BamHI into pRK415. The ppaZ2lacZ construct was generated using same strategy as for ppaZ1lacZ but was amplified with the upstream primer 5'-GCGGTACCGGGGCGGAATTGATGCAGCGAA-3' and the downstream primer 5'-GCGGATCCTTGCGGTATCGACATG-3'. The ppaZ3lacZ construct was generated by isolating a 650 bp BamHIPstI fragment containing approximately 200 bp upstream of the putative ppaZ translation start (Fig. 1a
). This fragment was ligated, along with the lacZKnr cassette of pKOK6 digested with PstI, into pRK415 digested with PstI and BamHI. The nirKlacZ construct has been previously described (Tosques et al., 1997
). All the fusions are transcriptional.
To disrupt nirV two fragments were amplified that contained nirV along with flanking DNA. The upstream region was amplified using the oligonucleotides 5'-GGCGAATTCGAGATGCGGATCATC-3' and 5'-GCGGATCCTTCCTGTTCGAGAACG-3'. The downstream fragment was amplified using the oligonucleotides 5'-GCCTTCATGGTCGATTTCATCC-3' and 5'-GCGAATTCCTTCCCGTCGTAAACC-3'. These two amplicons were purified, the upstream amplicon digested with PstI and BamHI and the downstream amplicon digested with EcoRI and BamHI. A BamHI-digested antibiotic-resistance cassette of pHP45 (Prentki & Krisch, 1984
), encoding streptomycin/spectinomycin resistance (Str/Spcr), was also isolated. These fragments were ligated into pSUP202 that had been digested with PstI and EcoRI. Cells were selected on media containing tetracycline, and streptomycin plus spectinomycin, and appropriate constructs were confirmed by restriction digest. The construct was transformed into E. coli S17-1. After conjugation of this plasmid into 2.4.3, exconjugants were isolated that were tetracycline sensitive (Tcs) and Str/Spcr. Inactivation of nirV was confirmed by Southern hybridization using genomic DNA isolated from the mutant probed with a digoxigenin-labelled fragment containing the enitre nirV ORF plus about 100 bp of flanking DNA. The NirV-deficient strain was designated strain R351. Inactivation of ppaZ was carried out by cloning a 2·8 kb EcoRI fragment, which contains ppaZ and flanking DNA, into the vector pJP5603. This was digested with StuI, which cuts at position 123 of the ppaZ ORF, and ligated with the streptomycin/spectinomycin-resistance cassette of pUI1638 (Eraso & Kaplan, 1994
) digested with EcoRV. After conjugation of this plasmid into 2.4.3, exconjugants that were Tcs and Str/Spcr were isolated. The ppaZ disruption was likewise confirmed via Southern hybridization of a labelled DNA fragment encompassing the entire ppaZ ORF to genomic DNA isolated from the mutant.
The gene encoding FnrL (fnrL) was inactivated by using the following oligonucleotides to amplify a fragment of the fnrL ORF using 2.4.3 chromosomal DNA as template: 5'-CGCGGTACCGACAAGATGGATTTCGTGG 3' and 5'-GCCGAATTCCGAAGTCTGTCACGATCAC-3'. These oligonucleotides were designed based on the fnrL sequence from R. sphaeroides 2.4.1 (Zeilstra-Ryalls & Kaplan, 1995 ). The amplicon was digested using EcoRI and KpnI, sites that were added by sequences on the oligonucleotides, and ligated into the suicide plasmid pJP5603 digested with the same enzymes. This construct was conjugated into 2.4.3 and kanamycin-resistant single crossovers were isolated.
The pMALppaZ construct was generated by amplifying a 385 bp fragment containing the bulk of ppaZ except the for the signal sequence. The oligonucleotides used for the amplification were 5'-CGGAATTCCATTACGAGATCGCG-3' and 5'-GCGGATCCATCAGTTGGCCTCGTAG-3'. The amplicon was digested with EcoRI and BamHI and ligated into the pMAL-C2 vector (New England Biolabs) digested with the same enzymes. The pMALnirV fusion was constructed by amplifying an 822 bp fragment containing all of nirV except the signal sequence. The oligonucleotides used for the amplification were 5'-CGGAATCTTCGGAGGCTTCGGCC-3' and 5'-CGCAGGTGCTGCAGAACTACGAG-3'. The amplicon was digested with EcoRI and PstI, and ligated into the pMAL-C2 vector digested with the same enzymes. Expression and purification of the fusions were carried out as described previously (Olesen et al., 1998 ).
Assays.
ß-Galactosidase activities were determined in duplicate on at least three independently grown cultures as previously described (Tosques et al., 1996 ). Cells removed from stoppered flasks were not kept anaerobic but were used immediately for assays. Samples were taken at various times during growth and the highest values obtained before the cells stopped growing were used to determine the reported values. Copper measurements were carried out using the protocol described by Felsenfeld (1960)
. MBPNirV and MBPPpaZ were purified from cells grown in medium containing 100 µM CuSO4. Nitrite reductase was assayed by measuring the rate of nitrite disappearance of washed whole cells incubated at 30 °C in Sistroms medium (Stewart & Parales, 1988
).
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RESULTS |
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Since the pseudoazurin encoded by 2.4.3 does not appear to be capable of copper binding, the gene has been designated ppaZ for pseudopseudoazurin. There is a ribosome-binding site located 8 bp upstream of the putative translation start. A region with similarity to the FnrL and NnrR binding sites is located 79 bp upstream of the putative translation start. The sequence of this region is 5'-TTGATGCAGCGCAA-3'. The TTGAT sequence is identical to half sites found in the FnrL consensus sequence (Spiro, 1994 ) and the CGCAA sequence is identical half sites found in the NnrR consensus sequence (Tosques et al., 1997
). A similar site has been observed upstream of hemN of R. sphaeroides 2.4.3, which is located downstream of the operon encoding nitric oxide reductase (T. B. Bartnikas & J. P. Shapleigh, unpublished).
Sequence analysis of R. sphaeroides strain 2.4.1
The genome sequence of the closely related 2.4.1 strain of R. sphaeroides has recently become available (http://www.jgi.doe.gov; follow the link to the Rhodobacter page. Examination of the available contigs indicates that 2.4.1 also contains a gene encoding a putative pseudoazurin (Fig. 1b). The deduced protein sequence is 88% identical to the ppaZ gene product, with the 2.4.1 product being one residue shorter because of a deletion in the hydrophobic stretch of the putative signal sequence (Fig. 2b
). Significantly, three of the residues required for copper ligation in other pseudoazurins are not conserved in the 2.4.1 sequence. In both 2.4.1 and 2.4.3 the residue at position 65, which in other pseudoazurins is a copper-binding histidine, is a phenylalanine encoded by the codon TTC. The switch from histidine, encoded by CAT or CAC, to phenylalanine requires a two-base change assuming usage of the more frequently used CAC codon. A single base change was required to make residues 103 and 106 encode serine and tyrosine, respectively, in both 2.4.1 and 2.4.3, instead of the cysteine and histidine found in the identical positions in other pseudoazurins. The conversion of residues 103 and 106 to hydroxyl-containing amino acids, which are potential metal-binding ligands, makes it possible that a metal could be bound to PpaZ. However, PpaZ is distinct from other pseudoazurins because it lacks a binding site for a type I copper centre. As in 2.4.3, the sole copper-binding residue remaining in the 2.4.1 pseudoazurin is the methionine at position 110. A potential FnrL or NnrR binding site is located 69 bp upstream of the putative translation start.
Examination of the region downstream of ppaZ in 2.4.1 revealed that there is no nirK or nirV as in 2.4.3. Instead there is an approximately 500 bp region that does not appear to contain any ORFs, followed by a region encoding a protein that is similar to slr1485 from Synechocystis sp. strain PCC 6803 (Fig. 1b). The region immediately upstream of nirK in 2.4.3 also encodes a protein with significant identity to slr1485. The slr1485 protein is related to proteins with phosphatidylinositol-4-phosphate 5-kinase activity (not shown). Alignment of the available sequence from within the slr1485 ORFs from the two strains shows there is >85% sequence identity until a few residues upstream of the termination codon (not shown). There is <35% sequence identity in the region immediately downstream of slr1485. A similar pattern is seen in the region encoding ppaZ. There is significant sequence identity throughout the ppaZ ORFs, but upon reaching the termination codon the identity of the sequences from the two strains decreases significantly. There is an approximately 220 bp region immediately upstream of the putative translation start of ppaZ in 2.4.3 that shares little identity with the same region in 2.4.1 (not shown). However, distal to this stretch there is a significant level of identity between the available sequences from the two strains (not shown). In 2.4.1 the region upstream of ppaZ is predicted to encode a protein with significant identity to the 2-isopropylmalate synthase from N. meningitidis. Assuming conservation of gene order in 2.4.1 and 2.4.3 the sequence analysis indicates that nirK, nirV and ppaZ appear to constitute the nitrite reductase gene cluster in 2.4.3.
Expression of nirV
The 210 bp gap between nirK and nirV could be sufficient to allow nirV to be regulated independently of nirK. To test this a nirVlacZ clone was made that included the entire intergenic region plus 198 bases of the 3' end of the nirK ORF. Expression of this construct in 2.4.3 was assessed microaerobically in medium with or without added nitrate. There was essentially no ß-galactosidase activity detectable under any culture condition (not shown). One explanation for this lack of expression is that nirK and nirV are cotranscribed. To test this a construct, designated nirVBHlacZ, was made that included the intergenic region, all of nirK and approximately 5·0 kb of DNA upstream of the nirK translation start. There was detectable ß-galactosidase expression using the nirVBHlacZ fusion, with expression of 482 Miller units of activity in cells grown in nitrate-amended medium under microaerobic conditions. Cells grown under identical conditions but in medium lacking nitrate had fivefold lower ß-galactosidase activity. This decrease in expression is consistent with a requirement for NnrR for expression. The importance of NnrR was confirmed by the lack of expression of the nirVBHlacZ construct in the NnrR-deficient strain R125. There was no detectable expression under aerobic conditions in medium lacking nitrate. These data are consistent with nirK and nirV being cotranscribed, suggesting that the physiological function of NirV is required when 2.4.3 uses nitrite as a terminal electron acceptor.
Expression of ppaZ
The presence of a possible NnrR or FnrL binding site upstream of the ppaZ ORF suggests that this gene will be preferentially expressed under low-oxygen conditions. This was tested by constructing a lacZ fusion, designated ppaZlacZ, that included about 400 bp upstream of the putative translation start. Maximal expression of ppaZlacZ occurred when cells were cultured microaerobically in unamended Sistroms medium (Fig. 3). When the strain was cultured in nitrate-amended medium, expression of the ppaZlacZ fusion was reduced by about 15%. Expression of the fusion under aerobic conditions was about 25-fold lower than when cells were incubated under limiting oxygen (Fig. 3
). This expression pattern is consistent with FnrL functioning as the primary regulator of ppaZ. To test this, expression of ppaZlacZ was monitored in R213, an FnrL-deficient strain of 2.4.3 (Fig. 3
). Expression in this strain was about 22-fold lower than in the wild-type strain. Under identical conditions, expression in R125, an NnrR-deficient strain of 2.4.3, was only about 2-fold less than wild-type (Fig. 3
). When the FnrL-deficient strain was grown in medium containing nitrate, expression of the fusion increased about threefold relative to nitrate-unamended medium. This suggests that NnrR may regulate ppaZ expression if FnrL is not active. Expression in strain R214, a 2.4.3 strain that lacks both FnrL and NnrR, was about 38-fold lower than expression in the wild-type strain in nitrate-unamended medium and did not increase when nitrate was present in the medium (Fig. 3
).
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Since nirV has only been found in strains encoding copper-containing nitrite reductases it is possible that NirV assists in assembly of an active nitrite reductase. To test if NirV might be required as a copper chaperone, wild-type and NirV-deficient cells were grown in medium containing the chelator diethyldithiocarbamate (DDC). DDC has been shown to effectively inhibit the activity of copper-containing nitrite reductases by removal of copper from the assembled protein (Shapleigh & Payne, 1985 ). If NirV is a copper chaperone, it might be expected that loss of this protein might more severely affect assembly of nitrite reductase in cells grown in medium in which copper insertion into the protein has been negatively affected. The effect on nitrite reductase activity was assessed by monitoring expression of the nirKlacZ fusion. Expression of the fusion was monitored in 2.4.3 and R351 cultured in media containing 0 to 50 µM DDC. While the expression of the fusion decreased as the DDC concentrations increased there was no significant difference in expression between the two strains (not shown). DDC concentrations higher than 50 µM decreased the growth rates and final cell yield of the two strains to such an extent that ß-galactosidase assays could not be run.
The ppaZ ORF was disrupted by insertion of an antibiotic-resistance cassette near the 5' end of the gene. The effect of ppaZ inactivation was assessed as done with the nirV mutant strain. There was no obvious effect of ppaZ inactivation on growth or on the taxis response to either nitrite or nitrate (not shown). Disruption also did not affect photosynthetic growth. The expression of nirKlacZ was similar in both the ppaZ mutant and the wild-type (not shown). Nitrite reductase activity in the ppaZ mutant was about 80% of the wild-type strain (data not shown). This slight decrease in activity did not result in the transient accumulation of nitrite during microaerobic growth in the presence of nitrate.
Expression of MBPPpaZ and MBPNirV fusions
To further test the affinity of both PpaZ and NirV for copper, a heterologous expression system for maltose-binding protein (MBP) fusions for each protein was developed. MBP fusions were constructed because it has been shown that a copper-containing nitrite reductaseMBP fusion (MBPNirK) can be purified from E. coli grown in copper-containing medium (Olesen et al., 1998 ). MBP fusions have also been used to assess the copper-binding capacities of other proteins (Hainaut et al., 1995
; Lutsenko et al., 1997
). Both fusions were expressed as cytoplasmic proteins and could be readily purified from induced E. coli strains (Fig. 4
). However, neither fusion was found to bind copper even when purified from cells grown in medium high in copper. The MBPPpaZ clone was colourless and a colorimetric copper assay showed no evidence of bound copper. Similar results were obtained with the MBPNirV fusion. Incubation of the MBPPpaZ fusion with extracts of 2.4.3 cells grown in nitrate-amended medium did not result in copper binding to the fusion.
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DISCUSSION |
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The role of the ppaZ product is obscure, particularly given its inability to bind copper. The regulation of ppaZ suggests that the protein is expressed whenever oxygen concentrations are low (Table 1). This, as well as the persistence of a copy of ppaZ in 2.4.1, suggests that the function of PpaZ is not limited to when nitrite reduction is occurring. In this context it is significant that many of the positively charged residues shown to be involved in pseudoazurin binding with nitrite reductase are present in PpaZ (Kukimoto et al., 1995
). It is possible then that PpaZ could bind to a number of electron-transport proteins. However, since PpaZ is unlikely to be redox active, the role of PpaZ in modulating electron flow is difficult to predict.
DNA sequence analysis suggests that the sequence changes that resulted in the loss of the ability of the ppaZ product to bind copper occurred before the divergence of 2.4.1 and 2.4.3. This is because the sequence changes leading to the loss of copper-binding residues are identical in both strains. Interestingly, the high identity of the 2.4.3 and 2.4.1 ppaZ products shows that there have been only minor sequence changes in PpaZ since the divergence of the two strains. The level of similarity of PpaZ from the two strains is similar to the levels of similarity found in other electron-transport proteins. This suggests that the primary sequence of PpaZ is constrained because it is functionally active in both strains and playing a similar physiological role.
Genome sequencing of the related bacterium Rhodobacter capsulatus has revealed that it also encodes a pseudoazurin, but unlike R. sphaeroides it retains all four of the copper-binding residues (not shown). This indicates that the modifications found in PpaZ are not widespread among photosynthetic bacteria. The sequence differences between PpaZ and other pseudoazurins are also not related to R. sphaeroides 2.4.3 having a copper nitrite reductase, since both Alcaligenes faecalis and Achromobacter cycloclastes have copper-containing nitrite reductases and copper-binding pseudoazurins (Godden et al., 1991 ; Inoue et al., 1999
; Kakutani et al., 1981a
, b
).
The complex regulation of ppaZ is also consistent with its encoding a functional protein with a role in the physiology of R. sphaeroides under low-oxygen conditions. At least three different regulatory proteins are involved in the activation of ppaZ expression. The related proteins FnrL and NnrR likely bind at the 5'-TTGATGCAGCGCAA-3' sequence. There is no other sequence upstream of ppaZ with significant identity to the Fnr consensus binding site 5'-TTGATNNNNATCAA-3', with N representing any base (Spiro, 1994 ). The change in the downstream half site in the ppaZ region to a modified consensus does not apparently affect the binding of FnrL to the DNA. This region also appears to be the target of NnrR since there is no sequence upstream of ppaZ with similarity to the NnrR consensus, which is 5'-TTG(C/T)GNNNNC(G/A)CAA-3'. However, the affinity of NnrR for this site does not appear to be as great as for its consensus, given the relatively low levels of expression of ppaZlacZ in an FnrL-deficient strain (Fig. 3
).
Expression of ppaZ also requires the two-component PrrB/A system (Table 2). The expression of a hemNlacZ fusion, which, like ppaZ, is regulated by both FnrL and NnrR, and has an identical binding site sequence in its promoter region, is not affected by prrB inactivation (R. Jain & J. P. Shapleigh, unpublished). The observation that a gene with many regulatory similarities to ppaZ is unaffected by PrrB inactivation suggests that the PrrB-deficient changes in expression of ppaZ are not likely to be a result of the physiological changes in the cell resulting from inactivation of this global regulatory protein. Rather, this suggests that phosphorylated PrrA likely binds to the ppaZ promoter region in concert with either FnrL or NnrR to activate gene transcription. Since there is no consensus sequence for the binding site of this family of proteins, it is not possible to determine if a PrrA-P binding site exists by sequence examination (Du et al., 1998
). This type of dependence on multiple transcription factors has been found in other genes regulated by PrrB/A (Oh et al., 2000
).
As with PpaZ, the role of NirV is unclear. However, given that 2.4.1 has lost both nirK and nirV it seems reasonable that NirV is required for some role related to nitrite reduction. The proteins with the highest similarity to NirV are only found in bacteria containing copper-containing nitrite reductases. This further supports a role for NirV during nitrite reduction. Given these observations it might be expected that NirV would be some type of copper chaperone. Alignment of closely related NirV sequences from bacteria with nitrite reductases reveals that there are several cysteine, histidine and acidic residues that are completely conserved and could potentially be copper-binding residues (Fig. 2a). However, these residues do not conform to known copper-binding motifs and could be involved in other functions (Koch et al., 1997
). The lack of effect of nirV inactivation on growth in copper-limited medium is also inconsistent with a copper-binding function for nirV. However, it is possible that other proteins compensate for the loss of NirV under these conditions.
There are at least two bacteria with copper-containing nitrite reductases that do not appear to encode an accompanying nirV. One of these is Rps. palustris. In this bacterium one of its two copper-containing nitrite reductases lacks an accompanying nirV. While it is possible that the single nirV is sufficient, preliminary experiments indicate that the regulation of the two nitrite reductases is different, suggesting that the nirK lacking a downstream nirV can be expressed independently of the single copy of nirV (D. Y. Lee & J. P. Shapleigh, unpublished). Nitrosomonas europaea encodes a nitrite reductase similar to those found in Neisseria spp., but unlike the situation in the neisseriae there is no nirV orthologue downstream of the nitrite reductase structural gene (http://wit.integratedgenomics.com/IGwit/). That there are bacteria that encode copper-containing nitrite reductases but lack nirV is further indication that the function of NirV is not essential for nitrite reductase activity.
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ACKNOWLEDGEMENTS |
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Received 16 May 2001;
accepted 22 May 2001.