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Genetic and biochemical analysis of a class C nonspecific acid phosphatase (NSAP) of Clostridium perfringens

¹ Graduate School of Medical Science, Kanazawa University;
² Graduate School pf Medical Science, Kanazawa University

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 responsible for the production of numerous enzymes that enable the organism to obtain essential nutritional sources from the host. In this study, CPE0201, a putative acid phosphatase gene deduced by genome analysis, was shown to encode the nonspecific 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 CPE0201's acid phosphatase activity, cloning, expression, purification and several biochemical characterizations of CPE0201 were carried out to reveal related properties. The optimum pH for CPE0201 activity was 4.8, and its Vmax and Km were 3.08 nmol ml^-1 min^-1 and 2.84 mM, respectively, when p-nitrophenyl phosphate (PNPP) was used as a substrate. The CPE0201 mutant did not grow in a minimum medium containing PNPP, while it showed normal growth when Na2HPO4 was added to the medium. We presume that the enzyme is possibly associated with the surface of the cell to acquire inorganic phosphates derived from organic phosphomonoesters in acidic conditions, which would play an important role in the survival and growth of C. perfringens in the host tissue.

³ E-mail: tshimizu{at}med.kanazawa-u.ac.jp