We have conducted the genetic analysis of fermentative nitrate reduction in Clostridium perfringens, a strict anaerobic bacterium. Nitrate reductase (NarA) was purified from the cytoplasmic fraction of the organism. Using a degenerate primer designed from its N- terminal amino acid sequence, a 9·5 kb fragment containing seven ORFs was cloned. The molecular mass and N-terminal amino acid sequence predicted from the nucleotide sequence of ORF 4 coincided with those determined for the purified NarA, indicating that ORF 4 corresponds to a narA gene. ORFs 5 and 6 encode a 15·4 kDa ferredoxin-like protein containing four iron–sulfur clusters and a 45 kDa protein homologous to NADH oxidase, respectively. Analyses involving primer extension and Northern blotting revealed that these three ORFs are transcribed as a polycistronic message. The ORF 5- and ORF 6-encoded proteins were shown by immunoblotting to be synthesized by cells grown in the presence of nitrate. Thus, these two proteins are likely to function as electron-transfer components in nitrate reduction in C. perfringens. The 9·5 kb fragment and a downstream region of 6·1 kb do not contain any genes involved in nitrate uptake or nitrite reduction. Instead, all 5 ORFs downstream of ORF 6 are homologous to genes reported for molybdopterin biosynthesis, unlike the genomic organization already determined for the respiratory and assimilatory nitrate-reduction systems. The evolutionary relationships between these two nitrate- reduction systems and the fermentative one based on the results of comparative genetic analysis are discussed.
AltschulS. F., MaddenT. L., SchafferA. A., ZhangJ., ZhangZ., MillerW., LipmanD. J.1997; Gapped blast and psi- blast: a new generation of protein database search programs. . Nucleic Acids Res 25:3389–3402[CrossRef]
Ba-TheinW., LyristisM., OhtaniK., NisbetI. T., HayashiH., RoodJ. I., ShimizuT.1996; The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens. J Bacteriol 178:2514–2520
BerksB. C., FergusonS. J., MoirJ. W., RichardsonD. J.1995; Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim Biophys Acta 1232:97–173[CrossRef]
BlascoF., Dos Santos, J. P., Magalon, A., Frixon, C., GuigliarelliB., SantiniC. L., GiordanoG.1998; NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli. Mol Microbiol 28:435–447[CrossRef]
CalmelsS., OhshimaH., HenryY., BartschH.1996; Characterization of bacterial cytochrome cd(1)-nitrite reductase as one enzyme responsible for catalysis of nitrosation of secondary amines. Carcinogenesis 17:533–536[CrossRef]
EavesD. J., PalmerT., BoxerD. H.1997; The product of the molybdenum cofactor gene mobB of Escherichia coli is a GTP-binding protein. Eur J Biochem 246:690–697[CrossRef]
GennisR. B., StewartV.1996; Respiration. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn. pp. 217–261Edited byNeidhardtF. C.others Washington, DC: American Society for Microbiology;
HoffmannT., TroupB., SzaboA., HungererC., JahnD.1995; The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system. FEMS Microbiol Lett 131:219–225[CrossRef]
IshimotoM., UmeyamaM., ChibaS.1974; Alteration of fermentation products from butyrate to acetate by nitrate reduction in Clostridium perfringens. Z Allg Mikrobiol 14:115–121[CrossRef]
MatsushitaO., YoshiharaK., KatayamaS.-I., MinamiJ., OkabeA.1994; Purification and characterization of a Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene. J Bacteriol 176:149–156
OgawaK., AkagawaE., YamaneK., SunZ. W., LaCelleM., ZuberP., NakanoM. M.1995; The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis. J Bacteriol 177:1409–1413
SekiS., HagiwaraM., KudoK., IshimotoM.1979; Studies on nitrate reductase of Clostridium perfringens. II. Purification and some properties of ferredoxin. J Biochem 85:833–838
SekiS., HattoriY., HasegawaT., HaraguchiH., IshimotoM.1987; Studies on nitrate reductase of Clostridium perfringens. IV. Identification of metals, molybdenum cofactor, and iron–sulfur cluster. J Biochem 101:503–509
SekiS., IkedaA., IshimotoM.1988; Rubredoxin as an intermediary electron carrier for nitrate reduction by NAD(P)H in Clostridium perfringens. J Biochem 103:583–584
Seki-ChibaS., IshimotoM.1977; Studies on nitrate reductase of Clostridium perfringens. I. Purification, some properties, and effect of tungstate on its formation. J Biochem 82:1663–1671
SodaS., YamamotoA., ItoA., MurataR.1968; On the maintenance of toxigenicity of seed cultures of Clostridium perfringens PB6K with special reference to alpha toxin. Jpn J Med Sci Biol 21:91–94[CrossRef]
TakahashiH., TaniguchiS., EgamiF.1963; Inorganic nitrogen compounds: distribution and metabolism. In Comparative Biochemistry pp. 91–202Edited byFlorkinM., MasonH. S. New York: Academic Press;
VermeerI. T., PachenD. M., DallingaJ. W., KleinjansJ. C., van MaanenJ. M. S.1998; Volatile N-nitrosamine formation after intake of nitrate at the ADI level in combination with an amine-rich diet. Environ Health Perspect 106:459–463[CrossRef]
Analysis of genes involved in nitrate reduction in Clostridium perfringensThe GenBank accession number for the sequence reported in this paper is AB017192.