Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Nov;78(11):7028–7032. doi: 10.1073/pnas.78.11.7028

Cloning of chromosomal genes in Streptococcus pneumoniae.

D L Stassi, P Lopez, M Espinosa, S A Lacks
PMCID: PMC349187  PMID: 6273903

Abstract

A system for molecular cloning in Streptococcus pneumoniae was developed. The multicopy plasmids pMV158 (5.4 kilobases) and pLS1 (4.3 kilobases), which confer tetracycline resistance, were used as vectors to clone chromosomal genes of S. pneumoniae in host cells of this species. A 3.3-kilobase restriction fragment containing the malM gene, which codes for amylomaltase, was cloned in a deletion mutant lacking chromosomal homology with the fragment. The recombinant plasmid pLS70, could transform over 50% of a recipient population to maltose utilization. Amylomaltase constituted up to 10% of the protein of cells containing pLS70. A derivative with a deletion, pLS69, appeared to gain a selective advantage by producing less enzyme. A 10-kilobase restriction fragment containing the sul-d gene for sulfonamide resistance was cloned in the presence of the homologous chromosomal gene. De novo establishment of a recombinant plasmid was just as frequent as transformation in an endogenous plasmid. Despite the processing of DNA during uptake in the transformation of S. pneumoniae, recombinant plasmids can be introduced. Models for the reconstruction of recombinant DNA in cells of S. pneumoniae and Bacillus subtilis are considered and compared.

Full text

PDF
7028

Images in this article

Selected References

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

  1. Barany F., Tomasz A. Genetic transformation of Streptococcus pneumoniae by heterologous plasmid deoxyribonucleic acid. J Bacteriol. 1980 Nov;144(2):698–709. doi: 10.1128/jb.144.2.698-709.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Canosi U., Iglesias A., Trautner T. A. Plasmid transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194. Mol Gen Genet. 1981;181(4):434–440. doi: 10.1007/BF00428732. [DOI] [PubMed] [Google Scholar]
  5. Canosi U., Morelli G., Trautner T. A. The relationship between molecular structure and transformation efficiency of some S. aureus plasmids isolated from B. subtilis. Mol Gen Genet. 1978 Nov 9;166(3):259–267. doi: 10.1007/BF00267617. [DOI] [PubMed] [Google Scholar]
  6. Claverys J. P., Lefevre J. C., Sicard A. M. Transformation of Streptococcus pneumoniae with S. pneumoniae-lambda phage hybrid DNA: induction of deletions. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3534–3538. doi: 10.1073/pnas.77.6.3534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Contente S., Dubnau D. Marker rescue transformation by linear plasmid DNA in Bacillus subtilis. Plasmid. 1979 Oct;2(4):555–571. doi: 10.1016/0147-619x(79)90054-4. [DOI] [PubMed] [Google Scholar]
  8. Ehrlich S. D. Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1680–1682. doi: 10.1073/pnas.74.4.1680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gryczan T., Contente S., Dubnau D. Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis. Mol Gen Genet. 1980 Feb;177(3):459–467. doi: 10.1007/BF00271485. [DOI] [PubMed] [Google Scholar]
  10. HOTCHKISS R. D., EVANS A. H. Analysis of the complex sulfonamide resistance locus of pneumococcus. Cold Spring Harb Symp Quant Biol. 1958;23:85–97. doi: 10.1101/sqb.1958.023.01.012. [DOI] [PubMed] [Google Scholar]
  11. Kapuściński J., Skoczylas B. Simple and rapid fluorimetric method for DNA microassay. Anal Biochem. 1977 Nov;83(1):252–257. doi: 10.1016/0003-2697(77)90533-4. [DOI] [PubMed] [Google Scholar]
  12. Keggins K. M., Lovett P. S., Duvall E. J. Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB110. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1423–1427. doi: 10.1073/pnas.75.3.1423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lacks S. Genetic regulation of maltosaccharide utilization in Pneumococcus. Genetics. 1968 Dec;60(4):685–706. doi: 10.1093/genetics/60.4.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lacks S., Greenberg B. A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA. J Biol Chem. 1975 Jun 10;250(11):4060–4066. [PubMed] [Google Scholar]
  15. Lacks S., Greenberg B. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J Mol Biol. 1977 Jul;114(1):153–168. doi: 10.1016/0022-2836(77)90289-3. [DOI] [PubMed] [Google Scholar]
  16. Lacks S. Integration efficiency and genetic recombination in pneumococcal transformation. Genetics. 1966 Jan;53(1):207–235. doi: 10.1093/genetics/53.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  18. Michel B., Palla E., Niaudet B., Ehrlich S. D. DNA cloning in Bacillus subtilis. III. Efficiency of random-segment cloning and insertional inactivation vectors. Gene. 1980 Dec;12(1-2):147–154. doi: 10.1016/0378-1119(80)90025-6. [DOI] [PubMed] [Google Scholar]
  19. Raibaud O., Schwartz M. Restriction map of the Escherichia coli malA region and identification of the malT product. J Bacteriol. 1980 Aug;143(2):761–771. doi: 10.1128/jb.143.2.761-771.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Saunders C. W., Guild W. R. Properties and transforming activities of two plasmids in Streptococcus pneumoniae. Mol Gen Genet. 1980;180(3):573–578. doi: 10.1007/BF00268062. [DOI] [PubMed] [Google Scholar]
  22. Smith M. D., Shoemaker N. B., Burdett V., Guild W. R. Transfer of plasmids by conjugation in Streptococcus pneumonias. Plasmid. 1980 Jan;3(1):70–79. doi: 10.1016/s0147-619x(80)90035-9. [DOI] [PubMed] [Google Scholar]
  23. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES