• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Sep 1992; 174(17): 5633–5638.
PMCID: PMC206509

New thermosensitive plasmid for gram-positive bacteria.


We isolated a replication-thermosensitive mutant of the broad-host-range replicon pWV01. The mutant pVE6002 is fully thermosensitive above 35 degrees C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon Tn10 derivative in Bacillus subtilis, we observed transposition frequencies of about 1%.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bates EE, Gilbert HJ. Characterization of a cryptic plasmid from Lactobacillus plantarum. Gene. 1989 Dec 21;85(1):253–258. [PubMed]
  • Bergemann AD, Whitley JC, Finch LR. Homology of mycoplasma plasmid pADB201 and staphylococcal plasmid pE194. J Bacteriol. 1989 Jan;171(1):593–595. [PMC free article] [PubMed]
  • Bravo A, Alonso JC. The generation of concatemeric plasmid DNA in Bacillus subtilis as a consequence of bacteriophage SPP1 infection. Nucleic Acids Res. 1990 Aug 25;18(16):4651–4657. [PMC free article] [PubMed]
  • Byeon WH, Weisblum B. Replication genes of plasmid pE194-cop and repF: transcripts and encoded proteins. J Bacteriol. 1990 Oct;172(10):5892–5900. [PMC free article] [PubMed]
  • Chopin A, Chopin MC, Moillo-Batt A, Langella P. Two plasmid-determined restriction and modification systems in Streptococcus lactis. Plasmid. 1984 May;11(3):260–263. [PubMed]
  • Chopin MC, Chopin A, Rouault A, Galleron N. Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome. Appl Environ Microbiol. 1989 Jul;55(7):1769–1774. [PMC free article] [PubMed]
  • Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988 Nov 25;16(22):10881–10890. [PMC free article] [PubMed]
  • Gasson MJ. In vivo genetic systems in lactic acid bacteria. FEMS Microbiol Rev. 1990 Sep;7(1-2):43–60. [PubMed]
  • Gasson MJ. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol. 1983 Apr;154(1):1–9. [PMC free article] [PubMed]
  • Gruss A, Ehrlich SD. The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev. 1989 Jun;53(2):231–241. [PMC free article] [PubMed]
  • Hamilton CM, Aldea M, Washburn BK, Babitzke P, Kushner SR. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. [PMC free article] [PubMed]
  • Holo H, Nes IF. High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media. Appl Environ Microbiol. 1989 Dec;55(12):3119–3123. [PMC free article] [PubMed]
  • Horinouchi S, Weisblum B. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J Bacteriol. 1982 May;150(2):804–814. [PMC free article] [PubMed]
  • Kleanthous H, Clayton CL, Tabaqchali S. Characterization of a plasmid from Helicobacter pylori encoding a replication protein common to plasmids in gram-positive bacteria. Mol Microbiol. 1991 Oct;5(10):2377–2389. [PubMed]
  • Kok J, van der Vossen JM, Venema G. Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol. 1984 Oct;48(4):726–731. [PMC free article] [PubMed]
  • Krah ER, 3rd, Macrina FL. Identification of a region that influences host range of the streptococcal conjugative plasmid pIP501. Plasmid. 1991 Jan;25(1):64–69. [PubMed]
  • Lacks SA, Lopez P, Greenberg B, Espinosa M. Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. J Mol Biol. 1986 Dec 20;192(4):753–765. [PubMed]
  • Langella P, Chopin A. Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus helveticus. FEMS Microbiol Lett. 1989 Jul 15;51(1):149–152. [PubMed]
  • Le Bourgeois P, Mata M, Ritzenthaler P. Genome comparison of Lactococcus strains by pulsed-field gel electrophoresis. FEMS Microbiol Lett. 1989 May;50(1-2):65–69. [PubMed]
  • Leenhouts KJ, Kok J, Venema G. Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis. Appl Environ Microbiol. 1989 Feb;55(2):394–400. [PMC free article] [PubMed]
  • Leenhouts KJ, Tolner B, Bron S, Kok J, Venema G, Seegers JF. Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1. Plasmid. 1991 Jul;26(1):55–66. [PubMed]
  • Luchansky JB, Muriana PM, Klaenhammer TR. Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium. Mol Microbiol. 1988 Sep;2(5):637–646. [PubMed]
  • McKay LL, Baldwin KA. Applications for biotechnology: present and future improvements in lactic acid bacteria. FEMS Microbiol Rev. 1990 Sep;7(1-2):3–14. [PubMed]
  • Mercenier A, Chassy BM. Strategies for the development of bacterial transformation systems. Biochimie. 1988 Apr;70(4):503–517. [PubMed]
  • Minton NP, Oultram JD, Brehm JK, Atkinson T. The replication proteins of plasmids pE194 and pLS1 have N-terminal homology. Nucleic Acids Res. 1988 Apr 11;16(7):3101–3101. [PMC free article] [PubMed]
  • Niaudet B, Ehrlich SD. In vitro genetic labeling of Bacillus subtilis cryptic plasmid pHV400. Plasmid. 1979 Jan;2(1):48–58. [PubMed]
  • Noirot P, Petit MA, Ehrlich SD. Plasmid replication stimulates DNA recombination in Bacillus subtilis. J Mol Biol. 1987 Jul 5;196(1):39–48. [PubMed]
  • Novick RP. Penicillinase plasmids of Staphylococcus aureus. Fed Proc. 1967 Jan-Feb;26(1):29–38. [PubMed]
  • Novick RP, Edelman I, Lofdahl S. Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes. J Mol Biol. 1986 Nov 20;192(2):209–220. [PubMed]
  • Otto R, de Vos WM, Gavrieli J. Plasmid DNA in Streptococcus cremoris Wg2: Influence of pH on Selection in Chemostats of a Variant Lacking a Protease Plasmid. Appl Environ Microbiol. 1982 Jun;43(6):1272–1277. [PMC free article] [PubMed]
  • Petit MA, Bruand C, Jannière L, Ehrlich SD. Tn10-derived transposons active in Bacillus subtilis. J Bacteriol. 1990 Dec;172(12):6736–6740. [PMC free article] [PubMed]
  • Petit MA, Mesas JM, Noirot P, Morel-Deville F, Ehrlich SD. Induction of DNA amplification in the Bacillus subtilis chromosome. EMBO J. 1992 Apr;11(4):1317–1326. [PMC free article] [PubMed]
  • Projan SJ, Carleton S, Novick RP. Determination of plasmid copy number by fluorescence densitometry. Plasmid. 1983 Mar;9(2):182–190. [PubMed]
  • Puyet A, del Solar GH, Espinosa M. Identification of the origin and direction of replication of the broad-host-range plasmid pLS1. Nucleic Acids Res. 1988 Jan 11;16(1):115–133. [PMC free article] [PubMed]
  • Raya RR, Klaenhammer TR. High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage phiadh. Appl Environ Microbiol. 1992 Jan;58(1):187–193. [PMC free article] [PubMed]
  • Romero DA, Slos P, Robert C, Castellino I, Mercenier A. Conjugative mobilization as an alternative vector delivery system for lactic streptococci. Appl Environ Microbiol. 1987 Oct;53(10):2405–2413. [PMC free article] [PubMed]
  • Scheer-Abramowitz J, Gryczan TJ, Dubnau D. Origin and mode of replication of plasmids pE194 and pUB110. Plasmid. 1981 Jul;6(1):67–77. [PubMed]
  • te Riele H, Michel B, Ehrlich SD. Single-stranded plasmid DNA in Bacillus subtilis and Staphylococcus aureus. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2541–2545. [PMC free article] [PubMed]
  • Terzaghi BE, Sandine WE. Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol. 1975 Jun;29(6):807–813. [PMC free article] [PubMed]
  • van der Vossen JM, van der Lelie D, Venema G. Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol. 1987 Oct;53(10):2452–2457. [PMC free article] [PubMed]
  • Villafane R, Bechhofer DH, Narayanan CS, Dubnau D. Replication control genes of plasmid pE194. J Bacteriol. 1987 Oct;169(10):4822–4829. [PMC free article] [PubMed]
  • Xu FF, Pearce LE, Yu PL. Genetic analysis of a lactococcal plasmid replicon. Mol Gen Genet. 1991 May;227(1):33–39. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)