Analysis of genes involved in nitrate reduction in Clostridium perfringens

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 d egenerate primer designed from its N-terminal amino acid sequence, a 9.5 kb fragment containing seven ORFs was cloned. The molecular mass and N-termin al amino acid sequence predicted from the nucleotide sequence of ORF 4 coin cided with those determined for the purified NarA, indicating that ORF 4 co rresponds to a narA gene. ORFs 5 and 6 encode a 15.4 kDa ferredoxin-like pr otein containing four iron-sulfur clusters and a 45 kDa protein homologous to NADH oxidase, respectively. Analyses involving primer extension and Nort hern blotting revealed that these three ORFs are transcribed as a polycistr onic message. The ORF 5- and ORF 6-encoded proteins were shown by immunoblo tting to be synthesized by cells grown in the presence of nitrate. Thus, th ese two proteins are likely to function as electron-transfer components in nitrate reduction in C. perfringens. The 9.5 kb fragment and a downstream r egion of 6.1 kb do not contain any genes involved in nitrate uptake or nitr ite reduction. Instead, all 5 ORFs downstream of ORF 6 are homologous to ge nes reported for molybdopterin biosynthesis, unlike the genomic organizatio n already determined for the respiratory and assimilatory nitrate-reduction systems. The evolutionary relationships between these two nitrate-reductio n systems and the fermentative one based on the results of comparative gene tic analysis are discussed.