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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/019265-0
2008-08-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/8/2275.html?itemId=/content/journal/micro/10.1099/mic.0.2008/019265-0&mimeType=html&fmt=ahah

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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [Google Scholar]
  5. Biswas, S. & Biswas, I. ( 2006; ). Regulation of the glucosyltransferase (gtfBC) operon by CovR in Streptococcus mutans. J Bacteriol 188, 988–998.[CrossRef]
    [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.[CrossRef]
    [Google Scholar]
  7. Biswas, I., Drake, L. & Biswas, S. ( 2007a; ). Regulation of gbpC expression in Streptococcus mutans. J Bacteriol 189, 6521–6531.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [Google Scholar]
  10. Brockmeier, U., Wendorff, M. & Eggert, T. ( 2006; ). Versatile expression and secretion vectors for Bacillus subtilis. Curr Microbiol 52, 143–148.[CrossRef]
    [Google Scholar]
  11. Cutting, S. M. & Vander-Horn, P. B. ( 1990; ). Genetic Analysis. In Molecular Biological Methods for Bacillus, pp. 27–74. Edited by C. R. Harwood & S. M. Cutting. Chichester: John Wiley.
  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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [Google Scholar]
  27. O'Sullivan, T. F. & Fitzgerald, G. F. ( 1999; ). Electrotransformation of industrial strains of Streptococcus thermophilus. J Appl Microbiol 86, 275–283.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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.[CrossRef]
    [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]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/019265-0
Loading
/content/journal/micro/10.1099/mic.0.2008/019265-0
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