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. 1987 Jul;84(14):4964–4968. doi: 10.1073/pnas.84.14.4964

Transformation of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and Escherichia coli with the C. diphtheriae plasmid pNG2.

T M Serwold-Davis, N Groman, M Rabin
PMCID: PMC305227  PMID: 3110777

Abstract

The transfection and transformation of members of two species of pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, is described. Protoplasts were produced by treatment with lysozyme following growth in glycine, and a medium was defined on which a significant fraction of the osmotically sensitive cells were regenerated. Transfections were carried out with DNA from corynephage 782, a member of the beta family of converting phages, and transformations were performed with DNA of plasmid pNG2, a 9500-kDa plasmid that was isolated from an erythromycin-resistant strain of C. diphtheriae and carries the resistance gene. Strains of Corynebacterium glutamicum and Escherichia coli were also successfully transformed with pNG2 DNA. Transfection frequencies were in the range of 3-8 X 10(3) plaque-forming units/micrograms of phage DNA, and transformation frequencies were in the range of 0.2-150 colony-forming units/micrograms of plasmid DNA. Plasmid pNG2 replicated and was stably maintained in all transformants both in the presence or absence of erythromycin. Thus, it displayed the ability to replicate in strains of both Gram-positive and Gram-negative bacteria without the intervention of genetic engineering. pNG2 DNA isolated from any of the transformed strains was able to transform all parental strains. The host range of pNG2 suggests its possible utility in or as a shuttle vector for the study and manipulation of genes from corynebacterial strains of animal origin.

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Selected References

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

  1. Barany F., Boeke J. D., Tomasz A. Staphylococcal plasmids that replicate and express erythromycin resistance in both Streptococcus pneumoniae and Escherichia coli. Proc Natl Acad Sci U S A. 1982 May;79(9):2991–2995. doi: 10.1073/pnas.79.9.2991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buck G. A., Cross R. E., Wong T. P., Loera J., Groman N. DNA relationships among some tox-bearing corynebacteriophages. Infect Immun. 1985 Sep;49(3):679–684. doi: 10.1128/iai.49.3.679-684.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cianciotto N., Groman N. A beta-related corynebacteriophage which lacks a tox allele but can acquire it by recombination with converting phage. Infect Immun. 1985 Jul;49(1):32–35. doi: 10.1128/iai.49.1.32-35.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Goze A., Ehrlich S. D. Replication of plasmids from Staphylococcus aureus in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7333–7337. doi: 10.1073/pnas.77.12.7333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Groman N. B. Conversion by corynephages and its role in the natural history of diphtheria. J Hyg (Lond) 1984 Dec;93(3):405–417. doi: 10.1017/s0022172400065001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Groman N., Cianciotto N., Bjorn M., Rabin M. Detection and expression of DNA homologous to the tox gene in nontoxinogenic isolates of Corynebacterium diphtheriae. Infect Immun. 1983 Oct;42(1):48–56. doi: 10.1128/iai.42.1.48-56.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  8. Hirai K., Yanagawa R. Generalized transduction in Corynebacterium renale. J Bacteriol. 1970 Mar;101(3):1086–1087. doi: 10.1128/jb.101.3.1086-1087.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Kok J., van der Vossen J. M., 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. doi: 10.1128/aem.48.4.726-731.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laird W., Groman N. Prophage map of converting corynebacteriophage beta. J Virol. 1976 Jul;19(1):208–219. doi: 10.1128/jvi.19.1.208-219.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Miwa K., Matsui K., Terabe M., Ito K., Ishida M., Takagi H., Nakamori S., Sano K. Construction of novel shuttle vectors and a cosmid vector for the glutamic acid-producing bacteria Brevibacterium lactofermentum and Corynebacterium glutamicum. Gene. 1985;39(2-3):281–286. doi: 10.1016/0378-1119(85)90324-5. [DOI] [PubMed] [Google Scholar]
  13. Ozaki A., Katsumata R., Oka T., Furuya A. Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum. Mol Gen Genet. 1984;196(1):175–178. doi: 10.1007/BF00334113. [DOI] [PubMed] [Google Scholar]
  14. Santamaria R. I., Gil J. A., Martin J. F. High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA. J Bacteriol. 1985 Apr;162(1):463–467. doi: 10.1128/jb.162.1.463-467.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schiller J., Groman N., Coyle M. Plasmids in Corynebacterium diphtheriae and diphtheroids mediating erythromycin resistance. Antimicrob Agents Chemother. 1980 Nov;18(5):814–821. doi: 10.1128/aac.18.5.814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Schiller J., Strom M., Groman N., Coyle M. Relationship between pNG2, an Emr plasmid in Corynebacterium diphtheriae, and plasmids in aerobic skin coryneforms. Antimicrob Agents Chemother. 1983 Dec;24(6):892–901. doi: 10.1128/aac.24.6.892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Serwold-Davis T. M., Groman N. B. Mapping and cloning of Corynebacterium diphtheriae plasmid pNG2 and characterization of its relatedness to plasmids from skin coryneforms. Antimicrob Agents Chemother. 1986 Jul;30(1):69–72. doi: 10.1128/aac.30.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Trieu-Cuot P., Gerbaud G., Lambert T., Courvalin P. In vivo transfer of genetic information between gram-positive and gram-negative bacteria. EMBO J. 1985 Dec 16;4(13A):3583–3587. doi: 10.1002/j.1460-2075.1985.tb04120.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yoshihama M., Higashiro K., Rao E. A., Akedo M., Shanabruch W. G., Follettie M. T., Walker G. C., Sinskey A. J. Cloning vector system for Corynebacterium glutamicum. J Bacteriol. 1985 May;162(2):591–597. doi: 10.1128/jb.162.2.591-597.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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