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 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.
Fujinaga
Katsuyo
K
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan 1.
Taniguchi
Yuki
Y
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan 1.
Sun
Yezhou
Y
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan 1.
Katayama
Seiichi
S
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan 1.
Minami
Junzaburo
J
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan 1.
Matsushita
Osamu
O
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan 1.
Okabe
Akinobu
A
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, Miki-cho, Kita-gun, Kagawa 761-0793, Japan 1.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics*; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism; D006150:Guanine Nucleotides / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D009876:Operon; D010084:Oxidation-Reduction; D011622:Pterins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D005288:Ferredoxins; D006150:Guanine Nucleotides; D009097:Multienzyme Complexes; D009566:Nitrates; D011622:Pterins; C092057:narA protein, Bacillus subtilis; C063761:molybdopterin guanine dinucleotide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D009565:Nitrate Reductases; D050901:Nitrate Reductase