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. 1986 Mar;165(3):831–836. doi: 10.1128/jb.165.3.831-836.1986

Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector.

R Wirth, F Y An, D B Clewell
PMCID: PMC214503  PMID: 3005240

Abstract

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.

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Selected References

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

  1. Behnke D., Gilmore M. S. Location of antibiotic resistance determinants, copy control, and replication functions on the double-selective streptococcal cloning vector pGB301. Mol Gen Genet. 1981;184(1):115–120. doi: 10.1007/BF00271206. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
  4. Clewell D. B. Plasmids, drug resistance, and gene transfer in the genus Streptococcus. Microbiol Rev. 1981 Sep;45(3):409–436. doi: 10.1128/mr.45.3.409-436.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clewell D. B., Tomich P. K., Gawron-Burke M. C., Franke A. E., Yagi Y., An F. Y. Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917. J Bacteriol. 1982 Dec;152(3):1220–1230. doi: 10.1128/jb.152.3.1220-1230.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dao M. L., Ferretti J. J. Streptococcus-Escherichia coli shuttle vector pSA3 and its use in the cloning of streptococcal genes. Appl Environ Microbiol. 1985 Jan;49(1):115–119. doi: 10.1128/aem.49.1.115-119.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunny G. M., Craig R. A., Carron R. L., Clewell D. B. Plasmid transfer in Streptococcus faecalis: production of multiple sex pheromones by recipients. Plasmid. 1979 Jul;2(3):454–465. doi: 10.1016/0147-619x(79)90029-5. [DOI] [PubMed] [Google Scholar]
  8. Engel H. W., Soedirman N., Rost J. A., van Leeuwen W. J., van Embden J. D. Transferability of macrolide, lincomycin, and streptogramin resistances between group A, B, and D streptococci, Streptococcus pneumoniae, and Staphylococcus aureus. J Bacteriol. 1980 May;142(2):407–413. doi: 10.1128/jb.142.2.407-413.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fitzgerald G. F., Clewell D. B. A conjugative transposon (Tn919) in Streptococcus sanguis. Infect Immun. 1985 Feb;47(2):415–420. doi: 10.1128/iai.47.2.415-420.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gawron-Burke C., Clewell D. B. Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria. J Bacteriol. 1984 Jul;159(1):214–221. doi: 10.1128/jb.159.1.214-221.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibson E. M., Chace N. M., London S. B., London J. Transfer of plasmid-mediated antibiotic resistance from streptococci to lactobacilli. J Bacteriol. 1979 Jan;137(1):614–619. doi: 10.1128/jb.137.1.614-619.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gonzalez C. F., Kunka B. S. Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501. Appl Environ Microbiol. 1983 Jul;46(1):81–89. doi: 10.1128/aem.46.1.81-89.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hershfield V. Plasmids mediating multiple drug resistance in group B streptococcus: transferability and molecular properties. Plasmid. 1979 Jan;2(1):137–149. doi: 10.1016/0147-619x(79)90012-x. [DOI] [PubMed] [Google Scholar]
  14. Horodniceanu T., Bouanchaud D. H., Bieth G., Chabbert Y. A. R plasmids in Streptococcus agalactiae (group B). Antimicrob Agents Chemother. 1976 Nov;10(5):795–801. doi: 10.1128/aac.10.5.795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ike Y., Craig R. A., White B. A., Yagi Y., Clewell D. B. Modification of Streptococcus faecalis sex pheromones after acquisition of plasmid DNA. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5369–5373. doi: 10.1073/pnas.80.17.5369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jacob A. E., Hobbs S. J. Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J Bacteriol. 1974 Feb;117(2):360–372. doi: 10.1128/jb.117.2.360-372.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Katsumata R., Ozaki A., Oka T., Furuya A. Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J Bacteriol. 1984 Jul;159(1):306–311. doi: 10.1128/jb.159.1.306-311.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. King J. R., Gooder H. Reversion to the streptococcal state of enterococcal protoplasts, spheroplasts, and L-forms. J Bacteriol. 1970 Sep;103(3):692–696. doi: 10.1128/jb.103.3.692-696.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Macrina F. L., Tobian J. A., Jones K. R., Evans R. P., Clewell D. B. A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis. Gene. 1982 Oct;19(3):345–353. doi: 10.1016/0378-1119(82)90025-7. [DOI] [PubMed] [Google Scholar]
  21. Marraro R. V., Pfister R. M., Rheins M. S. Growth inhibition of bacterial variants. Can J Microbiol. 1970 Jul;16(7):640–642. doi: 10.1139/m70-109. [DOI] [PubMed] [Google Scholar]
  22. Schaberg D. R., Clewell D. B., Glatzer L. Conjugative transfer of R-plasmids from Streptococcus faecalis to Staphylococcus aureus. Antimicrob Agents Chemother. 1982 Aug;22(2):204–207. doi: 10.1128/aac.22.2.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smith M. D. Transformation and fusion of Streptococcus faecalis protoplasts. J Bacteriol. 1985 Apr;162(1):92–97. doi: 10.1128/jb.162.1.92-97.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yagi Y., Clewell D. B. Recombination-deficient mutant of Streptococcus faecalis. J Bacteriol. 1980 Aug;143(2):966–970. doi: 10.1128/jb.143.2.966-970.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]

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