1887

Abstract

In this paper, the construction and evaluation of a chromosomal expression platform (CEP), which allows controlled gene expression following ectopic integration into the chromosome of , is described. CEP is based on the well-studied maltosaccharide-inducible system. To facilitate integration at CEP, a plasmid, pCEP, capable of replication in , but not in , was assembled. This plasmid contains an expression/selection cassette flanked on each side by more than 2 kb of pneumococcal DNA. The cassette comprises a maltose-inducible promoter, P, separated from a kanamycin-resistance gene by I and HI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of P can be obtained through direct transformation of an recipient with ligation products between that gene and I/HI-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.

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2006-02-01
2019-10-15
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References

  1. Alloing, G., Trombe, M. C. & Claverys, J. P. ( 1990; ). The ami locus of the Gram-positive bacterium Streptococcus pneumoniae is similar to binding protein-dependent transport operons of Gram-negative bacteria. Mol Microbiol 4, 633–644.[CrossRef]
    [Google Scholar]
  2. Balganesh, T. S. & Lacks, S. A. ( 1985; ). Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: cloning and expression of the hexA gene. J Bacteriol 162, 979–984.
    [Google Scholar]
  3. Bergé, M., Moscoso, M., Prudhomme, M., Martin, B. & Claverys, J. P. ( 2002; ). Uptake of transforming DNA in Gram-positive bacteria: a view from Streptococcus pneumoniae. Mol Microbiol 45, 411–421.[CrossRef]
    [Google Scholar]
  4. Chan, P. F., O'Dwyer, K. M., Palmer, L. M. & 8 other authors ( 2003; ). Characterization of a novel fucose-regulated promoter (PfcsK) suitable for gene essentiality and antibacterial mode-of-action studies in Streptococcus pneumoniae. J Bacteriol 185, 2051–2058.[CrossRef]
    [Google Scholar]
  5. 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]
  6. de Saizieu, A., Gardes, C., Flint, N., Wagner, C., Kamber, M., Mitchell, T. J., Keck, W., Amrein, K. E. & Lange, R. ( 2000; ). Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide. J Bacteriol 182, 4696–4703.[CrossRef]
    [Google Scholar]
  7. Díaz, A., Lacks, S. A. & López, P. ( 1992; ). The 5′ to 3′ exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae. Mol Microbiol 6, 3009–3019.[CrossRef]
    [Google Scholar]
  8. Guiral, S., Hénard, V., Granadel, C., Martin, B. & Claverys, J. P. ( 2006; ). Inhibition of competence development in Streptococcus pneumoniae by increased basal-level expression of the ComDE two-component regulatory system. Microbiology, 152, 323–331.[CrossRef]
    [Google Scholar]
  9. Hoskins, J., Alborn, W. E., Jr, Arnold, J. & 40 other authors ( 2001; ). Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol 183, 5709–5717.[CrossRef]
    [Google Scholar]
  10. 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]
  11. Kong, L., Siranosian, K. J., Grossman, A. D. & Dubnau, D. ( 1993; ). Sequence and properties of mecA, a negative regulator of genetic competence in Bacillus subtilis. Mol Microbiol 9, 365–373.[CrossRef]
    [Google Scholar]
  12. Lacks, S. A. ( 1968; ). Genetic regulation of maltosaccharide utilization in pneumococcus. Genetics 60, 685–706.
    [Google Scholar]
  13. Luo, P., Li, H. & Morrison, D. A. ( 2003; ). ComX is a unique link between multiple quorum sensing outputs and competence in Streptococcus pneumoniae. Mol Microbiol 50, 623–633.[CrossRef]
    [Google Scholar]
  14. Mannarelli, B. M. & Lacks, S. A. ( 1984; ). Ectopic integration of chromosomal genes in Streptococcus pneumoniae. J Bacteriol 160, 867–873.
    [Google Scholar]
  15. Martin, B., Prats, H. & Claverys, J. P. ( 1985; ). Cloning of the hexA mismatch repair of Streptococcus pneumoniae and identification of the product. Gene 34, 293–303.[CrossRef]
    [Google Scholar]
  16. Martin, B., Alloing, G., Boucraut, C. & Claverys, J. P. ( 1989; ). The difficulty of cloning Streptococcus pneumoniae mal and ami loci in Escherichia coli: toxicity of malX and amiA gene products. Gene 80, 227–237.[CrossRef]
    [Google Scholar]
  17. Martin, B., Prudhomme, M., Alloing, G., Granadel, C. & Claverys, J. P. ( 2000; ). Cross-regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae. Mol Microbiol 38, 867–878.[CrossRef]
    [Google Scholar]
  18. Méjean, V., Claverys, J. P., Vasseghi, H. & Sicard, A. M. ( 1981; ). Rapid cloning of specific DNA fragments of Streptococcus pneumoniae by vector integration into the chromosome followed by endonucleolytic excision. Gene 15, 289–293.[CrossRef]
    [Google Scholar]
  19. 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]
  20. Nieto, C., Espinosa, M. & Puyet, A. ( 1997; ). The maltose/maltodextrin regulon of Streptococcus pneumoniae. J Biol Chem 272, 30860–30865.[CrossRef]
    [Google Scholar]
  21. Nieto, C., Fernández de Palencia, P., López, P. & Espinosa, M. ( 2000; ). Construction of a tightly regulated plasmid vector for Streptococcus pneumoniae: controlled expression of the green fluorescent protein. Plasmid 43, 205–213.[CrossRef]
    [Google Scholar]
  22. Peterson, S., Sung, C. K., Cline, R. & 13 other authors ( 2004; ). Identification of competence pheromone responsive genes in Streptococcus pneumoniae. Mol Microbiol 51, 1051–1070.[CrossRef]
    [Google Scholar]
  23. Pichoff, S., Vollrath, B., Touriol, C. & Bouché, J. P. ( 1995; ). Deletion analysis of gene minE which encodes the topological specificity factor of cell division in Escherichia coli. Mol Microbiol 18, 321–329.[CrossRef]
    [Google Scholar]
  24. Pozzi, G. & Guild, W. R. ( 1985; ). Modes of integration of heterologous plasmid DNA into the chromosome of Streptococcus pneumoniae. J Bacteriol 161, 909–912.
    [Google Scholar]
  25. Prats, H., Martin, B. & Claverys, J. P. ( 1985; ). The hexB mismatch repair gene of Streptococcus pneumoniae: characterization, cloning and identification of the product. Mol Gen Genet 200, 482–489.[CrossRef]
    [Google Scholar]
  26. Prudhomme, M. & Claverys, J. P. ( 2006; ). There will be a light: the use of luc transcriptional fusions in living pneumococcal cells. In The Molecular Biology of Streptococci. Edited by R. Hakenbeck & G. S. Chhatwal. Norwich, UK: Horizon Scientific Press.
  27. Puyet, A., Ibáñez, A. M. & Espinosa, M. ( 1993; ). Characterization of the Streptococcus pneumoniae maltosaccharide regulator MalR, a member of the LacI-GalR family of repressors displaying distinctive genetic features. J Biol Chem 268, 25402–25408.
    [Google Scholar]
  28. Robertson, G. T., Ng, W. L., Foley, J., Gilmour, R. & Winkler, M. E. ( 2002; ). Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and virulence. J Bacteriol 184, 3508–3520.[CrossRef]
    [Google Scholar]
  29. Robertson, G. T., Ng, W. L., Gilmour, R. & Winkler, M. E. ( 2003; ). Essentiality of clpX, but not clpP, clpL, clpC, or clpE, in Streptococcus pneumoniae R6. J Bacteriol 185, 2961–2966.[CrossRef]
    [Google Scholar]
  30. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  31. Stassi, D. L., Dunn, J. J. & Lacks, S. A. ( 1982; ). Nucleotide sequence of DNA controlling expression of genes for maltosaccharide utilization in Streptococcus pneumoniae. Gene 20, 359–366.[CrossRef]
    [Google Scholar]
  32. Stieger, M., Wohlgensinger, B., Kamber, M., Lutz, R. & Keck, W. ( 1999; ). Integrational plasmids for the tetracycline-regulated expression of genes in Streptococcus pneumoniae. Gene 226, 243–251.[CrossRef]
    [Google Scholar]
  33. 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]
  34. Vasseghi, H. & Claverys, J. P. ( 1983; ). Amplification of a chimeric plasmid carrying an erythromycin-resistance determinant introduced into the genome of Streptococcus pneumoniae. Gene 21, 285–292.[CrossRef]
    [Google Scholar]
  35. Vasseghi, H., Claverys, J. P. & Sicard, A. M. ( 1981; ). Mechanism of integrating foreign DNA during transformation of Streptococcus pneumoniae. In Transformation-1980, pp. 137–154. Edited by M. Polsinelli & G. Mazza. Oxford: Cotswold Press.
  36. Zähner, D. & Hakenbeck, R. ( 2000; ). The Streptococcus pneumoniae beta-galactosidase is a surface protein. J Bacteriol 182, 5919–5921.[CrossRef]
    [Google Scholar]
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