Analysis of genes involved in nitrate reduction in Clostridium perfringens.

Abstract

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.

• Authors: Katsuyo Fujinaga, Yuki Taniguchi, Yezhou Sun, Seiichi Katayama, Junzaburo Minami, Osamu Matsushita, Akinobu Okabe
• Journal: Microbiology (Reading, England) (Microbiology (Reading)) Vol. 145 ( Pt 12) Pg. 3377-3387 (Dec 1999) ISSN: 1350-0872 [Print] England
• PMID: 10627036 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Ferredoxins
• Guanine Nucleotides
• Multienzyme Complexes
• Nitrates
• Pterins
• narA protein, Bacillus subtilis
• molybdopterin guanine dinucleotide
• NADH oxidase
• NADH, NADPH Oxidoreductases
• Nitrate Reductases
• Nitrate Reductase

Topics

• Amino Acid Sequence
• Bacterial Proteins (chemistry, genetics, metabolism)
• Cloning, Molecular
• Clostridium perfringens (enzymology, genetics)
• Ferredoxins (genetics, metabolism)
• Guanine Nucleotides (genetics, metabolism)
• Immunoblotting
• Molecular Sequence Data
• Multienzyme Complexes (chemistry, genetics, metabolism)
• Multigene Family
• NADH, NADPH Oxidoreductases (chemistry, genetics, metabolism)
• Nitrate Reductase
• Nitrate Reductases (chemistry, genetics, metabolism)
• Nitrates (metabolism)
• Operon
• Oxidation-Reduction
• Pterins (metabolism)
• Sequence Analysis, DNA