1887

Abstract

Pneumococci that have developed the competent state kill and lyse non-competent sister cells and members of closely related species during co-cultivation . The key component in this process, called fratricide, is the product of the late competence gene . In addition, the peptidoglycan hydrolases LytA and LytC are required for efficient lysis of target cells. Here, we have investigated the relative contribution and possible role of each of the proteins mentioned above. Previous studies have shown that CbpD is produced exclusively by competent cells, whereas LytA and LytC can be provided by the competent attackers as well as the non-competent target cells. By using an improved assay to compare the effect of - versus -acting LytA and LytC, we were able to show that target cells are lysed much more efficiently when LytA and LytC are provided , i.e. by the target cells themselves. Western analysis demonstrated that considerable amounts of LytC are present in the growth medium. In contrast, we were not able to detect any extracellular LytA. This finding indicates that LytA- and LytC-mediated fratricide represent different processes. In the absence of LytA and LytC, only a tiny fraction of the target cells were lysed, demonstrating that CbpD does not function efficiently on its own. However, in the presence of 1 mM EDTA, the fraction of target cells lysed directly by CbpD increased dramatically, indicating that divalent cations are involved in the regulation of fratricide under natural conditions.

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2009-07-01
2019-10-22
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References

  1. Bateman, A. & Rawlings, N. D. ( 2003; ). The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases. Trends Biochem Sci 28, 234–237.[CrossRef]
    [Google Scholar]
  2. Briese, T. & Hakenbeck, R. ( 1985; ). Interaction of the pneumococcal amidase with lipoteichoic acid and choline. Eur J Biochem 146, 417–427.[CrossRef]
    [Google Scholar]
  3. Chi, F., Nolte, O., Bergmann, C., Ip, M. & Hakenbeck, R. ( 2007; ). Crossing the barrier: evolution and spread of a major class of mosaic pbp2x in Streptococcus pneumoniae, S. mitis, and S. oralis. Int J Med Microbiol 297, 503–512.[CrossRef]
    [Google Scholar]
  4. Claverys, J. P. & Håvarstein, L. S. ( 2007; ). Cannibalism and fratricide: mechanisms and raison d'être. Nat Rev Microbiol 5, 219–229.[CrossRef]
    [Google Scholar]
  5. Claverys, J. P., Dintilhac, A., Pestova, E. V., Martin, B. & Morrison, D. A. ( 1995; ). Construction and evaluation of new drug-resistance cassettes for gene disruption mutagenesis in Streptococcus pneumoniae, using an ami test platform. Gene 164, 123–128.[CrossRef]
    [Google Scholar]
  6. Dagkessamanskaia, A., Moscoso, M., Hénard, V., Guiral, S., Overweg, K., Reuter, M., Martin, B., Wells, J. & Claverys, J. P. ( 2004; ). Interconnection of competence, stress and CiaR regulons in Streptococcus pneumoniae: competence triggers stationary phase autolysis of ciaR mutant cells. Mol Microbiol 51, 1071–1086.[CrossRef]
    [Google Scholar]
  7. Díaz, E., López, R. & García, J. L. ( 1990; ). Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes. Proc Natl Acad Sci U S A 87, 8125–8129.[CrossRef]
    [Google Scholar]
  8. Dubos, R. J. ( 1937; ). Mechanism of the lysis of pneumococci by freezing and thawing, bile, and other agents. J Exp Med 66, 101–112.[CrossRef]
    [Google Scholar]
  9. Fiser, A., Sergio, R. F. & Tomasz, A. ( 2003; ). Cell wall branches, penicillin resistance and the secrets of the MurM protein. Trends Microbiol 11, 547–553.[CrossRef]
    [Google Scholar]
  10. Giudicelli, S. & Tomasz, A. ( 1984; ). Attachment of pneumococcal autolysin to wall teichoic acids, an essential step in enzymatic wall degradation. J Bacteriol 158, 1188–1190.
    [Google Scholar]
  11. Guiral, S., Mitchell, T. J., Martin, B. & Claverys, J. P. ( 2005; ). Competence-programmed predation of noncompetent cells in the human pathogen Streptococcus pneumoniae: genetic requirements. Proc Natl Acad Sci U S A 102, 8710–8715.[CrossRef]
    [Google Scholar]
  12. Hakenbeck, R. ( 1995; ). Target mediated resistance to β-lactam antibiotics. Biochem Pharmacol 50, 1121–1127.[CrossRef]
    [Google Scholar]
  13. Håvarstein, L. S., Coomaraswami, G. & Morrison, D. A. ( 1995; ). An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc Natl Acad Sci U S A 92, 11140–11144.[CrossRef]
    [Google Scholar]
  14. Håvarstein, L. S., Gaustad, P., Nes, I. F. & Morrison, D. A. ( 1996; ). Identification of the streptococcal competence-pheromone receptor. Mol Microbiol 21, 863–869.[CrossRef]
    [Google Scholar]
  15. Håvarstein, L. S., Martin, B., Johnsborg, O., Granadel, C. & Claverys, J. P. ( 2006; ). New insights into the pneumococcal fratricide: relationship to clumping and identification of a novel immunity factor. Mol Microbiol 59, 1297–1307.[CrossRef]
    [Google Scholar]
  16. Hiller, N. L., Janto, B., Hogg, J. S., Boissy, R., Yu, S., Powell, E., Keefe, R., Ehrlich, N. E., Shen, K. & other authors ( 2007; ). Comparative genomic analyses of seventeen Streptococcus pneumoniae strains: insights into the pneumococcal supragenome. J Bacteriol 189, 8186–8195.[CrossRef]
    [Google Scholar]
  17. Hui, F. M. & Morrison, D. A. ( 1991; ). Genetic transformation in Streptococcus pneumoniae: nucleotide sequence analysis shows comA, a gene required for competence induction, to be a member of the bacterial ATP-dependent transport protein family. J Bacteriol 173, 372–381.
    [Google Scholar]
  18. Jenkins, S. G., Brown, S. D. & Farrell, D. J. ( 2008; ). Trends in antibacterial resistance among Streptococcus pneumoniae isolated in the USA: update from PROTEKT US Years 1–4. Ann Clin Microbiol Antimicrob 7, 1–11.[CrossRef]
    [Google Scholar]
  19. Johnsborg, O., Eldholm, V. & Håvarstein, L. S. ( 2007; ). Natural genetic transformation: prevalence, mechanisms and function. Res Microbiol 158, 767–778.[CrossRef]
    [Google Scholar]
  20. Johnsborg, O., Eldholm, V., Bjørnstad, M. L. & Håvarstein, L. S. ( 2008; ). A predatory mechanism dramatically increases the efficiency of lateral gene transfer in Streptococcus pneumoniae and related commensal species. Mol Microbiol 69, 245–253.[CrossRef]
    [Google Scholar]
  21. Kausmally, L., Johnsborg, O., Lunde, M., Knutsen, E. & Håvarstein, L. S. ( 2005; ). Choline-binding protein D (CbpD) in Streptococcus pneumoniae is essential for competence-induced cell lysis. J Bacteriol 187, 4338–4345.[CrossRef]
    [Google Scholar]
  22. Lacks, S. A. ( 1970; ). Mutants of Diplococcus pneumoniae that lack deoxy-ribonucleases and other activities possibly pertinent to genetic transformation. J Bacteriol 101, 373–383.
    [Google Scholar]
  23. Lacks, S. A. & Hotchkiss, R. D. ( 1960; ). A study of the genetic material determining an enzyme activity in pneumococcus. Biochim Biophys Acta 39, 508–517.[CrossRef]
    [Google Scholar]
  24. Lacks, S. A., Ayalew, S., de la Campa, A. G. & Greenberg, B. ( 2000; ). Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system. Mol Microbiol 35, 1089–1098.[CrossRef]
    [Google Scholar]
  25. Lee, M. S. & Morrison, D. A. ( 1999; ). Identification of a new regulator in Streptococcus pneumoniae linking quorum sensing to competence for genetic transformation. J Bacteriol 181, 5004–5016.
    [Google Scholar]
  26. López, R. & García, E. ( 2004; ). Recent trends in the molecular biology of pneumococcal capsules, lytic enzymes, and bacteriophage. FEMS Microbiol Rev 28, 553–580.[CrossRef]
    [Google Scholar]
  27. Mascher, T., Heintz, M., Zähner, D., Merai, M. & Hakenbeck, R. ( 2006; ). The CiaRH system of Streptococcus pneumoniae prevents lysis during stress induced by treatment with cell wall inhibitors and by mutations in pbp2x involved in β-lactam resistance. J Bacteriol 188, 1959–1968.[CrossRef]
    [Google Scholar]
  28. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  29. Mortier-Barrière, I., de Saizieu, A., Claverys, J. P. & Martin, B. ( 1998; ). Competence-specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae. Mol Microbiol 27, 159–170.[CrossRef]
    [Google Scholar]
  30. Neuhaus, F. C. & Baddiley, J. ( 2003; ). A continuum of anionic charge: structures and functions of d-alanyl-teichoic acids in gram-positive bacteria. Microbiol Mol Biol Rev 67, 686–723.[CrossRef]
    [Google Scholar]
  31. Obaro, S. & Adegbola, R. ( 2002; ). The pneumococcus: carriage, disease, and conjugate vaccines. J Med Microbiol 51, 98–104.
    [Google Scholar]
  32. Pestova, E. V., Håvarstein, L. S. & Morrison, D. A. ( 1996; ). Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system. Mol Microbiol 21, 853–862.[CrossRef]
    [Google Scholar]
  33. Peterson, S. N., Sung, C. K., Cline, R., Desai, B. V., Snesrud, E. C., Luo, P., Walling, J., Li, H., Mintz, M. & other authors ( 2004; ). Identification of competence pheromone responsive genes in Streptococcus pneumoniae by use of DNA microarrays. Mol Microbiol 51, 1051–1070.[CrossRef]
    [Google Scholar]
  34. Regev-Yochay, G., Trzcinski, K., Thompson, C. M., Lipsitch, M. & Malley, R. ( 2007; ). SpxB is a suicide gene of Streptococcus pneumoniae and confers a selective advantage in an in vivo competitive colonization model. J Bacteriol 189, 6532–6539.[CrossRef]
    [Google Scholar]
  35. Rigden, D. J., Jedrzejas, M. J. & Galperin, M. Y. ( 2003; ). Amidase domains from bacterial and phage autolysins define a family of γ-d,l-glutamate-specific amidohydrolases. Trends Biochem Sci 28, 230–234.[CrossRef]
    [Google Scholar]
  36. Shoemaker, N. B. & Guild, W. R. ( 1974; ). Destruction of low efficiency markers is a slow process occuring at a heteroduplex stage of transformation. Mol Gen Genet 128, 283–290.
    [Google Scholar]
  37. Steinmoen, H., Knutsen, E. & Håvarstein, L. S. ( 2002; ). Induction of natural competence in Streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population. Proc Natl Acad Sci U S A 99, 7681–7686.[CrossRef]
    [Google Scholar]
  38. Steinmoen, H., Teigen, A. & Håvarstein, L. S. ( 2003; ). Competence induced cells of Streptococcus pneumoniae lyse competence-deficient cells of the same strain during co-cultivation. J Bacteriol 185, 7176–7183.[CrossRef]
    [Google Scholar]
  39. Sung, C. K., Li, H., Claverys, J. P. & Morrison, D. A. ( 2001; ). An rpsL cassette, Janus, for gene replacement through negative selection in Streptococcus pneumoniae. Appl Environ Microbiol 67, 5190–5196.[CrossRef]
    [Google Scholar]
  40. Tomasz, A. & Waks, S. ( 1975a; ). Mechanism of action of penicillin: triggering of the pneumococcal autolytic enzyme by inhibitors of cell wall synthesis. Proc Natl Acad Sci U S A 72, 4162–4166.[CrossRef]
    [Google Scholar]
  41. Tomasz, A. & Waks, S. ( 1975b; ). Enzyme replacement in a bacterium: phenotypic correction by the experimental introduction of the wild type enzyme into a live enzyme defective mutant pneumococcus. Biochem Biophys Res Commun 65, 1311–1319.[CrossRef]
    [Google Scholar]
  42. Tomasz, A. & Westphal, M. ( 1971; ). Abnormal autolytic enzyme in a pneumococcus with altered teichoic acid composition. Proc Natl Acad Sci U S A 68, 2627–2630.[CrossRef]
    [Google Scholar]
  43. Ween, O., Gaustad, P. & Håvarstein, L. S. ( 1999; ). Identification of DNA binding sites for ComE, a key regulator of natural competence in Streptococcus pneumoniae. Mol Microbiol 33, 817–827.[CrossRef]
    [Google Scholar]
  44. Yother, J. & White, J. M. ( 1994; ). Novel surface attachment mechanism of the Streptococcus pneumoniae protein PspA. J Bacteriol 176, 2976–2985.
    [Google Scholar]
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vol. , part 7, pp. 2223 - 2234

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