Sequencing of the region flanking nirK, the gene encoding the copper-containing nitrite reductase in Rhodobacter sphaeroides 2.4.3, has identified two genes whose products could potentially be involved in nitrite reductase expression and activity. One of the genes has been designated nirV. Putative nirV orthologues are found in other denitrifiers, where they are also located downstream of the structural gene for nitrite reductase. The nirV in 2.4.3 is apparently cotranscribed with nirK. Inactivation of nirV had no effect on cell growth, or on nitrite reductase expression or activity. Downstream of nirV and divergently transcribed is a gene, designated ppaZ, encoding a protein with significant similarity to pseudoazurins from other denitrifiers. However, three of the four residues required for binding of the type I copper centre are not conserved in the deduced sequence of the protein in 2.4.3. ppaZ is expressed only when oxygen becomes limiting. ppaZ expression is dependent on both FnrL and NnrR, and a putative binding site for these proteins has been identified. Expression of ppaZ is also dependent on the two-component PrrB/PrrA system. Inactivation of ppaZ had no significant effect on cell growth or on nitrite reductase expression or activity. Expression of a maltose-binding protein–PpaZ fusion indicated that the protein could not bind copper. Examination of the genome of the related bacterium R. sphaeroides 2.4.1 revealed that it encodes ppaZ but not nirV and evidence is presented suggesting that a common ancestor of 2.4.3 and 2.4.1 had both nitrite and nitric oxide reductase activity but as the strains diverged 2.4.1 lost nirK and nirV, making it incapable of nitrite reduction.
BedzykL., WangT.,
YeR. W.1999; The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step in denitrification. J Bacteriol 181:2802–2806
ChungC. T., NiemelaS. L.,
MillerR. H.1989; One-step transformation of competent Escherichia coli : transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA 86:2172–2175[CrossRef]
DuS., BirdT. H.,
BauerC. E.1998; DNA binding characteristics of RegA*. A constitutively active anaerobic activator of photosynthesis gene expression in Rhodobacter capsulatus . J Biol Chem 273:18509–18513[CrossRef]
ErasoJ. M.,
KaplanS.1994; prrA , a putative response regulator involved in oxygen regulation of photosynthetic gene expression in Rhodobacter sphaeroides . J Bacteriol 176:32–43
FulopV., MoirJ. W. B., FergusonS. J.,
HajduJ.1995; The anatomy of a bifunctional enzyme: structural basis for reduction of oxygen to water and synthesis of nitric oxide by cytochrome cd 1. Cell 81:369–377[CrossRef]
HainautP., RolleyN., DaviesM.,
MilnerJ.1995; Modulation by copper of p53 conformation and sequence-specific DNA binding: role for Cu(II)/Cu(I) redox mechanism. Oncogene 10:27–32
HormelS., AdmanE., WalshK. A., BeppuT.,
TitaniK.1986; The amino acid sequence of the blue copper protein of Alcaligenes faecalis . FEBS Lett 197:301–304[CrossRef]
InoueT., NishioN., SuzukiS., KataokaK., KohzumaT.,
KaiY.1999; Crystal structure determinations of oxidized and reduced pseudoazurins from Achromobacter cycloclastes . Concerted movement of copper site in redox forms with the rearrangement of hydrogen bond at a remote histidine. J Biol Chem 274:17845–17852[CrossRef]
KakutaniT., WatanabeH., ArimaK.,
BeppuT.1981a; Purification and properties of a copper-containing nitrite reductase from a denitrifying bacterium Alcaligenes faecalis S-6. J Biochem 89:453–461
KakutaniT., WatanabeH., ArimaK.,
BeppuT.1981b; A blue protein as an activating factor for nitrite reductase from Alcaligenes faecalis strain S-6. J Biochem 89:463–472
KokotekW.,
LotzW.1989; Construction of a lacZ -kanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe. Gene 84:467–471[CrossRef]
KoutnyM., KuceraI., TesarikR., TuranekJ.,
Van SpanningR. J.1999; Pseudoazurin mediates periplasmic electron flow in a mutant strain of Paracoccus denitrificans lacking cytochrome c 550. FEBS Lett 448:157–159[CrossRef]
KukimotoM., NishiyamaM., OhnukiT., TurleyS., AdmanE. T., HorinouchiS.,
BeppuT.1995; Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6. Protein Eng 8:153–158[CrossRef]
KwiatkowskiA. V., LarattaW. P., ToffaninA.,
ShapleighJ. P.1997; Analysis of the role of the nnrR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide. J Bacteriol 179:5618–5620
LutsenkoS., PetrukhinK., CooperM. J., GilliamC. T.,
KaplanJ. H.1997; N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson’s and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat. J Biol Chem 272:18939–18944[CrossRef]
McGowanS. J., SebaihiaM., O’LearyS., HardieK. R., WilliamsP., StewartG. S., BycroftB. W.,
SalmondG. P.1997; Analysis of the carbapenem gene cluster of Erwinia carotovora : definition of the antibiotic biosynthetic genes and evidence for a novel beta-lactam resistance mechanism. Mol Microbiol 26:545–556[CrossRef]
OhJ. I., ErasoJ. M.,
KaplanS.2000; Interacting regulatory circuits involved in orderly control of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. J Bacteriol 182:3081–3087[CrossRef]
OlesenK. O., VeselovA., ZhaoY., WangY., DannerB., ScholesC. P.,
ShapleighJ. P.1998; Spectroscopic, kinetic and electrochemical characterization of heterologously expressed wild type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3. Biochemistry 37:6086–6094[CrossRef]
PetratosK., BannerD. W., BeppuT., WilsonK. S.,
TsernoglouD.1987; The crystal structure of pseudoazurin from Alcaligenes faecalis S-6 determined at 2·9 Å resolution. FEBS Lett 218:209–214[CrossRef]
ShapleighJ. P.,
PayneW. J.1985; Differentiation of c,d 1 cytochrome and copper nitrite reductase production in denitrifiers. FEMS Microbiol Lett 26:275–279
SimonR., PrieferU., PühlerA.1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1:784–791[CrossRef]
StewartV., ParalesJ.Jr.1988; Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12. J Bacteriol 170:1589–1597
ToffaninA., WuQ., MaskusM., CasellaS., ShapleighJ. P., AbruñaH. D.1996; Characterization of the gene encoding nitrite reductase and the physiological consequences of its expression in the nondenitrifying Rhizobium ′hedysari ′ strain HCNT1. Appl Environ Microbiol 62:4019–4025
TosquesI. E., ShiJ.,
ShapleighJ. P.1996; Cloning and characterization of nnrR , whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. J Bacteriol 178:4958–4964
TosquesI. E., KwiatkowskiA. V., ShiJ.,
ShapleighJ. P.1997; Characterization and regulation of the gene encoding nitrite reductase in Rhodobacter sphaeroides 2.4.3. J Bacteriol 179:1090–1095
WilliamsP. A., FulopV., LeungY. C., ChanC., MoirJ. W., HowlettG., FergusonS. J., RadfordS. E.,
HajduJ.1995; Pseudospecific docking surfaces on electron transfer proteins as illustrated by pseudoazurin, cytochrome c 550 and cytochrome cd 1 nitrite reductase. Nat Struct Biol 2:975–982[CrossRef]
YamamotoK., UozumiT.,
BeppuT.1987; 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
Zeilstra-RyallsJ. H.,
KaplanS.1995; Aerobic and anaerobic regulation in Rhodobacter sphaeroides 2.4.1: the role of the fnrL gene. J Bacteriol 177:6422–6431
Characterization of nirV and a gene encoding a novel pseudoazurin in Rhodobacter sphaeroides 2.4.3The GenBank accession number for the sequence determined in this work is AF339883.