Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Feb;170(2):630–637. doi: 10.1128/jb.170.2.630-637.1988

Genetic transformation in Streptococcus pneumoniae: molecular cloning and characterization of recP, a gene required for genetic recombination.

D K Rhee 1, D A Morrison 1
PMCID: PMC210701  PMID: 2828317

Abstract

A 225-base-pair fragment of a recombination gene was identified by insertion-duplication mutagenesis and used as a radioactive probe to clone the corresponding rec locus from Streptococcus pneumoniae in Escherichia coli plasmid vectors. Attempts to clone large pieces of this locus were unsuccessful, but small pieces of DNA from this region were cloned in the E. coli transcriptional terminator vectors pKK232-8 and pJDC9. The extent of the rec region, 2.1 to 2.2 kilobases, was defined by determining the competence phenotype of insertion mutations constructed in vitro. A deletion of the rec locus showed it to be necessary for chromosomal integration but not for plasmid establishment. A plasmid carrying the entire locus encoded a 72-kilodalton polypeptide in a cell-free E. coli transcription-translation system.

Full text

PDF
634

Images in this article

635Image
on p.635

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brehm J., Salmond G., Minton N. Sequence of the adenine methylase gene of the Streptococcus faecalis plasmid pAM beta 1. Nucleic Acids Res. 1987 Apr 10;15(7):3177–3177. doi: 10.1093/nar/15.7.3177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  4. Burdett V. Identification of tetracycline-resistant R-plasmids in Streptococcus agalactiae (group B). Antimicrob Agents Chemother. 1980 Nov;18(5):753–760. doi: 10.1128/aac.18.5.753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chandler M. S., Morrison D. A. Competence for genetic transformation in Streptococcus pneumoniae: molecular cloning of com, a competence control locus. J Bacteriol. 1987 May;169(5):2005–2011. doi: 10.1128/jb.169.5.2005-2011.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dreyfuss G., Adam S. A., Choi Y. D. Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription. Mol Cell Biol. 1984 Mar;4(3):415–423. doi: 10.1128/mcb.4.3.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. FOX M. S., ALLEN M. K. ON THE MECHANISM OF DEOXYRIBONUCLEATE INTEGRATION IN PNEUMOCOCCAL TRANSFORMATION. Proc Natl Acad Sci U S A. 1964 Aug;52:412–419. doi: 10.1073/pnas.52.2.412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gasc A. M., Sicard N., Claverys J. P., Sicard A. M. Lack of SOS repair in Streptococcus pneumoniae. Mutat Res. 1980 Apr;70(2):157–165. doi: 10.1016/0027-5107(80)90155-4. [DOI] [PubMed] [Google Scholar]
  10. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gurney T., Jr, Fox M. S. Physical and genetic hybrids formed in bacterial transformation. J Mol Biol. 1968 Feb 28;32(1):83–100. doi: 10.1016/0022-2836(68)90147-2. [DOI] [PubMed] [Google Scholar]
  12. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  13. Irino N., Nakayama K., Nakayama H. The recQ gene of Escherichia coli K12: primary structure and evidence for SOS regulation. Mol Gen Genet. 1986 Nov;205(2):298–304. doi: 10.1007/BF00430442. [DOI] [PubMed] [Google Scholar]
  14. LACKS S. Molecular fate of DNA in genetic transformation of Pneumococcus. J Mol Biol. 1962 Jul;5:119–131. doi: 10.1016/s0022-2836(62)80067-9. [DOI] [PubMed] [Google Scholar]
  15. Lacks S. A., 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. doi: 10.1016/0022-2836(86)90026-4. [DOI] [PubMed] [Google Scholar]
  16. Lacks S., Greenberg B. Competence for deoxyribonucleic acid uptake and deoxyribonuclease action external to cells in the genetic transformation of Diplococcus pneumoniae. J Bacteriol. 1973 Apr;114(1):152–163. doi: 10.1128/jb.114.1.152-163.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lacks S., Greenberg B. Single-strand breakage on binding of DNA to cells in the genetic transformation of Diplococcus pneumoniae. J Mol Biol. 1976 Feb 25;101(2):255–275. doi: 10.1016/0022-2836(76)90376-4. [DOI] [PubMed] [Google Scholar]
  18. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  19. Low B. Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K12. Proc Natl Acad Sci U S A. 1968 May;60(1):160–167. doi: 10.1073/pnas.60.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marko M. A., Chipperfield R., Birnboim H. C. A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem. 1982 Apr;121(2):382–387. doi: 10.1016/0003-2697(82)90497-3. [DOI] [PubMed] [Google Scholar]
  21. Martin B., Prats H., Claverys J. P. Cloning of the hexA mismatch-repair gene of Streptococcus pneumoniae and identification of the product. Gene. 1985;34(2-3):293–303. doi: 10.1016/0378-1119(85)90138-6. [DOI] [PubMed] [Google Scholar]
  22. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morrison D. A., Baker M. F. Competence for genetic transformation in pneumococcus depends on synthesis of a small set of proteins. Nature. 1979 Nov 8;282(5735):215–217. doi: 10.1038/282215a0. [DOI] [PubMed] [Google Scholar]
  24. Morrison D. A., Guild W. R. Breakage prior to entry of donor DNA in Pneumococcus transformation. Biochim Biophys Acta. 1973 Apr 11;299(4):545–556. doi: 10.1016/0005-2787(73)90226-8. [DOI] [PubMed] [Google Scholar]
  25. Morrison D. A., Guild W. R. Structure of deoxyribonucleic acid on the cell surface during uptake by pneumococcus. J Bacteriol. 1973 Sep;115(3):1055–1062. doi: 10.1128/jb.115.3.1055-1062.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morrison D. A., Lacks S. A., Guild W. R., Hageman J. M. Isolation and characterization of three new classes of transformation-deficient mutants of Streptococcus pneumoniae that are defective in DNA transport and genetic recombination. J Bacteriol. 1983 Oct;156(1):281–290. doi: 10.1128/jb.156.1.281-290.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morrison D. A., Mannarelli B. Transformation in pneumococcus: nuclease resistance of deoxyribonucleic acid in the eclipse complex. J Bacteriol. 1979 Nov;140(2):655–665. doi: 10.1128/jb.140.2.655-665.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [DOI] [PubMed] [Google Scholar]
  29. Morrison D. A. Transformation in pneumococcus: existence and properties of a complex involving donor deoxyribonucleate single strands in eclipse. J Bacteriol. 1977 Nov;132(2):576–583. doi: 10.1128/jb.132.2.576-583.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Morrison D. A. Transformation in pneumococcus: protein content of eclipse complex. J Bacteriol. 1978 Nov;136(2):548–557. doi: 10.1128/jb.136.2.548-557.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morrison D. A., Trombe M. C., Hayden M. K., Waszak G. A., Chen J. D. Isolation of transformation-deficient Streptococcus pneumoniae mutants defective in control of competence, using insertion-duplication mutagenesis with the erythromycin resistance determinant of pAM beta 1. J Bacteriol. 1984 Sep;159(3):870–876. doi: 10.1128/jb.159.3.870-876.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Méjean V., Claverys J. P., Vasseghi H., Sicard A. M. Rapid cloning of specific DNA fragments of Streptococcus pneumoniae by vector integration into the chromosome followed by endonucleolytic excision. Gene. 1981 Nov;15(2-3):289–293. doi: 10.1016/0378-1119(81)90139-6. [DOI] [PubMed] [Google Scholar]
  33. Nakayama K., Irino N., Nakayama H. The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants. Mol Gen Genet. 1985;200(2):266–271. doi: 10.1007/BF00425434. [DOI] [PubMed] [Google Scholar]
  34. Porter R. D., Guild W. R. Transfection in pneumococcus: single-strand intermediates in the formation of infective centers. J Virol. 1978 Jan;25(1):60–72. doi: 10.1128/jvi.25.1.60-72.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Prats H., Martin B., Claverys J. P. The hexB mismatch repair gene of Streptococcus pneumoniae: characterisation, cloning and identification of the product. Mol Gen Genet. 1985;200(3):482–489. doi: 10.1007/BF00425735. [DOI] [PubMed] [Google Scholar]
  36. Prats H., Martin B., Pognonec P., Burger A. C., Claverys J. P. A plasmid vector allowing positive selection of recombinant plasmids in Streptococcus pneumoniae. Gene. 1985;39(1):41–48. doi: 10.1016/0378-1119(85)90105-2. [DOI] [PubMed] [Google Scholar]
  37. Raina J. L., Macrina F. L. A competence specific inducible protein promotes in vivo recombination in Streptococcus sanguis. Mol Gen Genet. 1982;185(1):21–29. doi: 10.1007/BF00333785. [DOI] [PubMed] [Google Scholar]
  38. Saunders C. W., Guild W. R. Monomer plasmid DNA transforms Streptococcus pneumoniae. Mol Gen Genet. 1981;181(1):57–62. doi: 10.1007/BF00339005. [DOI] [PubMed] [Google Scholar]
  39. Saunders C. W., Guild W. R. Pathway of plasmid transformation in Pneumococcus: open circular and linear molecules are active. J Bacteriol. 1981 May;146(2):517–526. doi: 10.1128/jb.146.2.517-526.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sicard N. Possible correlation between transformability and deficiency in error-prone repair. J Bacteriol. 1983 May;154(2):995–997. doi: 10.1128/jb.154.2.995-997.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stassi D. L., Lacks S. A. Effect of strong promoters on the cloning in Escherichia coli of DNA fragments from Streptococcus pneumoniae. Gene. 1982 Jun;18(3):319–328. doi: 10.1016/0378-1119(82)90170-6. [DOI] [PubMed] [Google Scholar]
  42. Tomasz A. Control of the competent state in Pneumococcus by a hormone-like cell product: an example for a new type of regulatory mechanism in bacteria. Nature. 1965 Oct 9;208(5006):155–159. doi: 10.1038/208155a0. [DOI] [PubMed] [Google Scholar]
  43. Tomasz A., Mosser J. L. On the nature of the pneumococcal activator substance. Proc Natl Acad Sci U S A. 1966 Jan;55(1):58–66. doi: 10.1073/pnas.55.1.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vijayakumar M. N., Morrison D. A. Fate of DNA in eclipse complex during genetic transformation in Streptococcus pneumoniae. J Bacteriol. 1983 Nov;156(2):644–648. doi: 10.1128/jb.156.2.644-648.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vijayakumar M. N., Morrison D. A. Localization of competence-induced proteins in Streptococcus pneumoniae. J Bacteriol. 1986 Mar;165(3):689–695. doi: 10.1128/jb.165.3.689-695.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Witkin E. M. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev. 1976 Dec;40(4):869–907. doi: 10.1128/br.40.4.869-907.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES