Genetic and biochemical analysis of a class C non-specific acid phosphatase (NSAP) of Clostridium perfringens.
Clostridium perfringens, a Gram-positive anaerobe, is a human pathogen that causes gas gangrene in muscle tissues. Its ability to grow and survive in the host is believed to be due to the production of numerous enzymes that enable the organism to obtain essential nutrients from the host. In this study, CPE0201, a putative acid phosphatase gene deduced by genome analysis, was shown to encode a non-specific acid phosphatase in C. perfringens. Multiple alignments of the amino acid sequence showed that CPE0201 shares two signature motifs that belong to a class C acid phosphatase family. Expression of CPE0201 was shown to be positively regulated by the global VirR/VirS-VR-RNA regulatory cascade at the transcriptional level. To determine the acid phosphatase activity of the CPE0201-encoded protein, cloning, expression, purification and several biochemical characterizations were carried out. The optimum pH for activity of the CPE0201 enzyme was 4.8, and its V(max) and K(m) were 3.08 nmol ml(-1) min(-1) and 2.84 mM, respectively, with p-nitrophenyl phosphate (PNPP) as substrate. A CPE0201 mutant did not grow in a minimal medium containing PNPP, while it showed normal growth when Na(2)HPO(4) was added to the medium. The enzyme appears to be associated with the surface of the cell, where it may function to acquire inorganic phosphate from organic phosphomonoesters in acidic conditions, which could play an important role in the survival and growth of C. perfringens in the host tissue.
Wang
Ruoyu
R
Department of Bacteriology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan.
Ohtani
Kaori
K
Department of Bacteriology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan.
Wang
Yun
Y
Department of Bacteriology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan.
Yuan
Yonghui
Y
Department of Bacteriology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan.
Hassan
Sufi
S
Department of Bacteriology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan.
Shimizu
Tohru
T
Department of Bacteriology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8640, Japan.
D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology / Q000235:genetics*; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D003470:Culture Media; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D000135:Acid Phosphatase