1887

Abstract

, 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 . 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 and were 3.08 nmol ml min and 2.84 mM, respectively, with -nitrophenyl phosphate (PNPP) as substrate. A CPE0201 mutant did not grow in a minimal medium containing PNPP, while it showed normal growth when NaHPO 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 in the host tissue.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.030395-0
2010-01-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/1/167.html?itemId=/content/journal/micro/10.1099/mic.0.030395-0&mimeType=html&fmt=ahah

References

  1. Aiba, H., Adhya, S. & de Crombrugghe, B. ( 1981; ). Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem 256, 11905–11910.
    [Google Scholar]
  2. Ba-Thein, W., Lyristis, M., Ohtani, K., Nisbet, I. T., Hayashi, H., Rood, J. I. & Shimizu, T. ( 1996; ). The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens. J Bacteriol 178, 2514–2520.
    [Google Scholar]
  3. Beacham, I. R. ( 1979; ). Periplasmic enzymes in gram-negative bacteria. Int J Biochem 10, 877–883.[CrossRef]
    [Google Scholar]
  4. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  5. Carmany, D. O., Hollingsworth, K. & McCleary, W. R. ( 2003; ). Genetic and biochemical studies of phosphatase activity of PhoR. J Bacteriol 185, 1112–1115.[CrossRef]
    [Google Scholar]
  6. Dissing, K. & Uerkvitz, W. ( 2006; ). Class B nonspecific acid phosphatase from Salmonella typhimurium LT2: phosphotransferase activity, stability and thiol group reactivity. Enzyme Microb Technol 38, 683–688.[CrossRef]
    [Google Scholar]
  7. Eisgruber, H., Geppert, P., Sperner, B. & Stolle, A. ( 2003; ). Evaluation of different methods for the detection of Clostridium perfringens phosphatases. Int J Food Microbiol 82, 81–86.[CrossRef]
    [Google Scholar]
  8. Felts, R. L., Reilly, T. J., Calcutt, M. J. & Tanner, J. J. ( 2006; ). Cloning, purification and crystallization of Bacillus anthracis class C acid phosphatase. Acta Crystallogr Sect F Struct Biol Cryst Commun 62, 705–708.[CrossRef]
    [Google Scholar]
  9. Green, B. A., Farley, J. E., Quinn-Dey, T., Deich, R. A. & Zlotnick, G. W. ( 1991; ). The e (P4) outer membrane protein of Haemophilus influenzae: biologic activity of anti-e serum and cloning and sequencing of the structural gene. Infect Immun 59, 3191–3198.
    [Google Scholar]
  10. Mahony, D. E. & Moore, T. I. ( 1976; ). Stable L-forms of Clostridium perfringens and their growth on glass surfaces. Can J Microbiol 22, 953–959.[CrossRef]
    [Google Scholar]
  11. Okabayashi, K., Futai, M. & Mizuno, D. ( 1974; ). Localization of acid and alkaline phosphatases in Staphylococcus aureus. Jpn J Microbiol 18, 287–294.[CrossRef]
    [Google Scholar]
  12. Poirier, T. P. & Holt, S. C. ( 1983; ). Acid and alkaline phosphatases of Capnocytophaga species. I. Production and cytological localization of the enzymes. Can J Microbiol 29, 1350–1360.[CrossRef]
    [Google Scholar]
  13. Riha, W. E., Jr & Solberg, M. ( 1971; ). Chemically defined medium for the growth of Clostridium perfringens. Appl Microbiol 22, 738–739.
    [Google Scholar]
  14. Rood, J. I. ( 1998; ). Virulence genes of Clostridium perfringens. Annu Rev Microbiol 52, 333–360.[CrossRef]
    [Google Scholar]
  15. Rossolini, G. M., Schippa, S., Riccio, M. L., Berlutti, F., Macaskie, L. E. & Thaller, M. C. ( 1998; ). Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology. Cell Mol Life Sci 54, 833–850.[CrossRef]
    [Google Scholar]
  16. Saitou, N. & Nei, M. ( 1987; ). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
    [Google Scholar]
  17. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  18. Shimizu, T., Ba-Thein, W., Tamaki, M. & Hayashi, H. ( 1994; ). The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens. J Bacteriol 176, 1616–1623.
    [Google Scholar]
  19. Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S. & Hayashi, H. ( 2002a; ). Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci U S A 99, 996–1001.[CrossRef]
    [Google Scholar]
  20. Shimizu, T., Yaguchi, H., Ohtani, K., Banu, S. & Hayashi, H. ( 2002b; ). Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins. Mol Microbiol 43, 257–265.[CrossRef]
    [Google Scholar]
  21. Tamura, K., Dudley, J., Nei, M. & Kumar, S. ( 2007; ). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596–1599.[CrossRef]
    [Google Scholar]
  22. Thaller, M. C., Berlutti, F., Schippa, S., Lombardi, G. & Rossolini, G. M. ( 1994; ). Characterization and sequence of PhoC, the principal phosphate-irrepressible acid phosphatase of Morganella morganii. Microbiology 140, 1341–1350.[CrossRef]
    [Google Scholar]
  23. Thaller, M. C., Lombardi, G., Berlutti, F., Schippa, S. & Rossolini, G. M. ( 1995; ). Cloning and characterization of the NapA acid phosphatase/phosphotransferase of Morganella morganii: identification of a new family of bacterial acid-phosphatase-encoding genes. Microbiology 141, 147–154.[CrossRef]
    [Google Scholar]
  24. Thaller, M. C., Schippa, S., Bonci, A., Cresti, S. & Rossolini, G. M. ( 1997; ). Identification of the gene (aphA) encoding the class B acid phosphatase/phosphotransferase of Escherichia coli MG1655 and characterization of its product. FEMS Microbiol Lett 146, 191–198.[CrossRef]
    [Google Scholar]
  25. Thaller, M. C., Berlutti, F., Schippa, S., Selan, L. & Rossolini, G. M. ( 1998; ). Bacterial acid phosphatase gene fusions useful as targets for cloning-dependent insertional inactivation. Biotechnol Prog 14, 241–247.[CrossRef]
    [Google Scholar]
  26. Ueno, K., Fujii, H., Marui, T., Takahashi, J. & Sugitani, T. ( 1970; ). Acid phosphatase in Clostridium perfringens. A new rapid and simple identification method. Jpn J Microbiol 14, 171–173.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.030395-0
Loading
/content/journal/micro/10.1099/mic.0.030395-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error