1887

Abstract

We have conducted the genetic analysis of fermentative nitrate reduction in , 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 95 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 gene. ORFs 5 and 6 encode a 154 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 . The 95 kb fragment and a downstream region of 61 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.

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1999-12-01
2020-01-25
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