1887

Abstract

A set of shuttle plasmids containing four different constitutive promoters was generated to facilitate overexpression of foreign and native genes in streptococci, such as . The four promoters that were chosen were: P, P, P and P. These promoters are active in many Gram-positive bacteria, and allow various levels of gene expression depending on the host bacterium. Shuttle plasmids were constructed based on two types of broad-host-range replication origins: a rolling-circle replicon (pSH71) and a theta replicon (pAM1). Shuttle plasmids derived from the pAM1 replicon were generated to avoid the structural and segregational stability problems associated with rolling-circle replication, since these problems may be encountered during large gene cloning. In a complementation assay, we used one such plasmid to express a gene to show the utility of these plasmids. In addition, a series of plasmids was generated for the expression of recombinant proteins with an N-terminal 6×His tag or a C-terminal Strep-tag fusion, and, using a gene derived from , we showed a high level of recombinant protein expression in and . Since these plasmids contain broad-host-range replication origins, and because the selected promoters are functional in many bacteria, they can be used for gene expression studies, such as complementation and recombinant protein expression.

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2008-08-01
2024-03-28
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References

  1. Ajdic D., McShan W. M., McLaughlin R. E., Savic G., Chang J., Carson M. B., Primeaux C., Tian R., Kenton S. other authors 2002; Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A 99:14434–14439
    [Google Scholar]
  2. Apfel C. M., Locher H., Evers S., Takacs B., Hubschwerlen C., Pirson W., Page M. G., Keck W. 2001; Peptide deformylase as an antibacterial drug target: target validation and resistance development. Antimicrob Agents Chemother 45:1058–1064
    [Google Scholar]
  3. Barnett T. C., Bugrysheva J. V., Scott J. R. 2007; Role of mRNA stability in growth phase regulation of gene expression in the group A streptococcus. J Bacteriol 189:1866–1873
    [Google Scholar]
  4. Beard S. J., Salisbury V., Lewis R. J., Sharpe J. A., MacGowan A. P. 2002; Expression of lux genes in a clinical isolate of Streptococcus pneumoniae: using bioluminescence to monitor gemifloxacin activity. Antimicrob Agents Chemother 46:538–542
    [Google Scholar]
  5. Biswas S., Biswas I. 2006; Regulation of the glucosyltransferase (gtfBC) operon by CovR in Streptococcus mutans . J Bacteriol 188:988–998
    [Google Scholar]
  6. Biswas I., Maguin E., Ehrlich S. D., Gruss A. 1995; A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis . Proc Natl Acad Sci U S A 92:2244–2248
    [Google Scholar]
  7. Biswas I., Drake L., Biswas S. 2007a; Regulation of gbpC expression in Streptococcus mutans . J Bacteriol 189:6521–6531
    [Google Scholar]
  8. Biswas I., Drake L., Johnson S., Thielen D. 2007b; Unmarked gene modification in Streptococcus mutans by a cotransformation strategy with a thermosensitive plasmid. Biotechniques 42:487–490
    [Google Scholar]
  9. Biswas I., Drake L., Erkina D., Biswas S. 2008; Involvement of sensor kinases in the stress tolerance response of Streptococcus mutans . J Bacteriol 190:68–77
    [Google Scholar]
  10. Brockmeier U., Wendorff M., Eggert T. 2006; Versatile expression and secretion vectors for Bacillus subtilis . Curr Microbiol 52:143–148
    [Google Scholar]
  11. Cutting S. M., Vander-Horn P. B. 1990; Genetic Analysis. . In Molecular Biological Methods for Bacillus pp 27–74 Edited by Harwood C. R., Cutting S. M. Chichester: John Wiley;
    [Google Scholar]
  12. Dabert P., Ehrlich S. D., Gruss A. 1992; High-molecular-weight linear multimer formation by single-stranded DNA plasmids in Escherichia coli . J Bacteriol 174:173–178
    [Google Scholar]
  13. de Vos W. M. 1987; Gene cloning and expression in lactic streptococci. FEMS Microbiol Rev 46:281–295
    [Google Scholar]
  14. Eichenbaum Z., Federle M. J., Marra D., de Vos W. M., Kuipers O. P., Kleerebezem M., Scott J. R. 1998; Use of the lactococcal nisA promoter to regulate gene expression in Gram-positive bacteria: comparison of induction level and promoter strength. Appl Environ Microbiol 64:2763–2769
    [Google Scholar]
  15. Facklam R. 2002; What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin Microbiol Rev 15:613–630
    [Google Scholar]
  16. Fujimoto S., Ike Y. 2001; pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis . Appl Environ Microbiol 67:1262–1267
    [Google Scholar]
  17. Goodman S. D., Gao Q. 2000; Characterization of the gtfB and gtfC promoters from Streptococcus mutans GS-5. Plasmid 43:85–98
    [Google Scholar]
  18. Gruss A., Ehrlich S. D. 1988; Insertion of foreign DNA into plasmids from Gram-positive bacteria induces formation of high-molecular-weight plasmid multimers. J Bacteriol 170:1183–1190
    [Google Scholar]
  19. Gruss A., Ehrlich S. D. 1989; The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev 53:231–241
    [Google Scholar]
  20. Halfmann A., Hakenbeck R., Bruckner R. 2007; A new integrative reporter plasmid for Streptococcus pneumoniae . FEMS Microbiol Lett 268:217–224
    [Google Scholar]
  21. Kiewiet R., Kok J., Seegers J. F., Venema G., Bron S. 1993; The mode of replication is a major factor in segregational plasmid instability in Lactococcus lactis . Appl Environ Microbiol 59:358–364
    [Google Scholar]
  22. Li Y., Burne R. A. 2001; Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate. Microbiology 147:2841–2848
    [Google Scholar]
  23. Loesche W. J. 1986; Role of Streptococcus mutans in human dental decay. Microbiol Rev 50:353–380
    [Google Scholar]
  24. Luchansky J. B., Muriana P. M., Klaenhammer T. R. 1988; Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium . Mol Microbiol 2:637–646
    [Google Scholar]
  25. Michel B., Ehrlich S. D. 1986; Illegitimate recombination occurs between the replication origin of the plasmid pC194 and a progressing replication fork. EMBO J 5:3691–3696
    [Google Scholar]
  26. Moran C. P. Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. 1982; Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis . Mol Gen Genet 186:339–346
    [Google Scholar]
  27. O'Sullivan T. F., Fitzgerald G. F. 1999; Electrotransformation of industrial strains of Streptococcus thermophilus . J Appl Microbiol 86:275–283
    [Google Scholar]
  28. Opdyke J. A., Scott J. R., Moran C. P. Jr 2003; Expression of the secondary sigma factor sigmaX in Streptococcus pyogenes is restricted at two levels. J Bacteriol 185:4291–4297
    [Google Scholar]
  29. Perez-Arellano I., Zuniga M., Perez-Martinez G. 2001; Construction of compatible wide-host-range shuttle vectors for lactic acid bacteria and Escherichia coli . Plasmid 46:106–116
    [Google Scholar]
  30. Perez-Casal J., Caparon M. G., Scott J. R. 1991; Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins of two-component regulatory systems. J Bacteriol 173:2617–2624
    [Google Scholar]
  31. Que Y. A., Haefliger J. A., Francioli P., Moreillon P. 2000; Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector. Infect Immun 68:3516–3522
    [Google Scholar]
  32. Sato Y., Yamamoto Y., Kizaki H. 2000; Xylitol-induced elevated expression of the gbpC gene in a population of Streptococcus mutans cells. Eur J Oral Sci 108:538–545
    [Google Scholar]
  33. Simon D., Chopin A. 1988; Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis . Biochimie 70:559–566
    [Google Scholar]
  34. Takahashi Y., Konishi K., Cisar J. O., Yoshikawa M. 2002; Identification and characterization of hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1. Infect Immun 70:1209–1218
    [Google Scholar]
  35. Turgeon N., Laflamme C., Ho J., Duchaine C. 2006; Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579. J Microbiol Methods 67:543–548
    [Google Scholar]
  36. Yoshida A., Kuramitsu H. K. 2002; Streptococcus mutans biofilm formation: utilization of a gtfB promoter–green fluorescent protein (PgtfB : :  gfp) construct to monitor development. Microbiology 148:3385–3394
    [Google Scholar]
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