Abstract
The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters.
Full text
PDF







Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Baltz R. H. Genetic recombination in Streptomyces fradiae by protoplast fusion and cell regeneration. J Gen Microbiol. 1978 Jul;107(1):93–102. doi: 10.1099/00221287-107-1-93. [DOI] [PubMed] [Google Scholar]
- 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]
- Coetzee J. N., Sirgel F. A., Lecatsas G. Genetic recombination in fused spheroplasts of Providence alcalifaciens. J Gen Microbiol. 1979 Oct;114(2):313–322. doi: 10.1099/00221287-114-2-313. [DOI] [PubMed] [Google Scholar]
- Fodor K., Alföldi L. Fusion of protoplasts of Bacillus megaterium. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2147–2150. doi: 10.1073/pnas.73.6.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frehel C., Lheritier A. M., Sanchez-Rivas C., Schaeffer P. Electron microscopic study of Bacillus subtilis protoplast fusion. J Bacteriol. 1979 Mar;137(3):1354–1361. doi: 10.1128/jb.137.3.1354-1361.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gabor M. H., Hotchkiss R. D. Parameters governing bacterial regeneration and genetic recombination after fusion of Bacillus subtilis protoplasts. J Bacteriol. 1979 Mar;137(3):1346–1353. doi: 10.1128/jb.137.3.1346-1353.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hopwood D. A., Wright H. M. Bacterial protoplast fusion: recombination in fused protoplasts of Streptomyces coelicolor. Mol Gen Genet. 1978 Jul 4;162(3):307–317. doi: 10.1007/BF00268856. [DOI] [PubMed] [Google Scholar]
- Iordanescu S., Surdeanu M. Two restriction and modification systems in Staphylococcus aureus NCTC8325. J Gen Microbiol. 1976 Oct;96(2):277–281. doi: 10.1099/00221287-96-2-277. [DOI] [PubMed] [Google Scholar]
- Johnston J. H., Richmond M. H. The increased rate of loss of penicillinase plasmids from Staphylococcus aureus in the presence of rifampicin. J Gen Microbiol. 1970 Jan;60(1):137–139. doi: 10.1099/00221287-60-1-137. [DOI] [PubMed] [Google Scholar]
- Kloos W. E., Schleifer K. H. Simplified scheme for routine identification of human Staphylococcus species. J Clin Microbiol. 1975 Jan;1(1):82–88. doi: 10.1128/jcm.1.1.82-88.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuhl S. A., Pattee P. A., Baldwin J. N. Chromosomal map location of the methicillin resistance determinant in Staphylococcus aureus. J Bacteriol. 1978 Aug;135(2):460–465. doi: 10.1128/jb.135.2.460-465.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lindberg M., Novick R. P. Plasmid-specific transformation in Staphylococcus aureus. J Bacteriol. 1973 Jul;115(1):139–145. doi: 10.1128/jb.115.1.139-145.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lindberg M., Rudin L. Thymineless bacteriophage induction in Staphylococcus aureus. II. Specific transduction of constitutive and inducible erythromycin resistance. J Virol. 1975 Dec;16(6):1367–1374. doi: 10.1128/jvi.16.6.1367-1374.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lindberg M., Sjöström J. E., Johansson T. Transformation of chromosomal and plasmid characters in Staphylococcus aureus. J Bacteriol. 1972 Feb;109(2):844–847. doi: 10.1128/jb.109.2.844-847.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- NOVICK R. P. ANALYSIS BY TRANSDUCTION OF MUTATIONS AFFECTING PENICILLINASE FORMATION IN STAPHYLOCOCCUS AUREUS. J Gen Microbiol. 1963 Oct;33:121–136. doi: 10.1099/00221287-33-1-121. [DOI] [PubMed] [Google Scholar]
- Novick R. P., Bouanchaud D. The problems of drug-resistant pathogenic bacteria. Extrachromosomal nature of drug resistance in Staphylococcus aureus. Ann N Y Acad Sci. 1971 Jun 11;182:279–294. doi: 10.1111/j.1749-6632.1971.tb30664.x. [DOI] [PubMed] [Google Scholar]
- Novick R. P., Edelman I., Schwesinger M. D., Gruss A. D., Swanson E. C., Pattee P. A. Genetic translocation in Staphylococcus aureus. Proc Natl Acad Sci U S A. 1979 Jan;76(1):400–404. doi: 10.1073/pnas.76.1.400. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Novick R. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology. 1967 Sep;33(1):155–166. doi: 10.1016/0042-6822(67)90105-5. [DOI] [PubMed] [Google Scholar]
- Ochi K., Hitchcock M. J., Katz E. High-frequency fusion of Streptomyces parvulus or Streptomyces antibioticus protoplasts induced by polyethylene glycol. J Bacteriol. 1979 Sep;139(3):984–992. doi: 10.1128/jb.139.3.984-992.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pattee P. A., Neveln D. S. Transformation analysis of three linkage groups in Staphylococcus aureus. J Bacteriol. 1975 Oct;124(1):201–211. doi: 10.1128/jb.124.1.201-211.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peberdy J. F. Fungal protoplasts: isolation, reversion, and fusion. Annu Rev Microbiol. 1979;33:21–39. doi: 10.1146/annurev.mi.33.100179.000321. [DOI] [PubMed] [Google Scholar]
- Pontecorvo G., Riddle P. N., Hales A. Time and mode of fusion of human fibroblasts treated with polyethylene glycol (PEG). Nature. 1977 Jan 20;265(5591):257–258. doi: 10.1038/265257a0. [DOI] [PubMed] [Google Scholar]
- Rosendorf L. L., Kayser F. H. Transduction and plasmid deoxyribonucleic acid analysis in a multiply antibiotic-resistant strain of Staphylococcus epidermidis. J Bacteriol. 1974 Nov;120(2):679–686. doi: 10.1128/jb.120.2.679-686.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schaeffer P., Cami B., Hotchkiss R. D. Fusion of bacterial protoplasts. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2151–2155. doi: 10.1073/pnas.73.6.2151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sjöström J. E., Löfdahl S., Philipson L. Transformation reveals a chromosomal locus of the gene(s) for methicillin resistance in Staphylococcus aureus. J Bacteriol. 1975 Sep;123(3):905–915. doi: 10.1128/jb.123.3.905-915.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wyman L., Goering R. V., Novick R. P. Genetic control of chromosomal and plasmid recombination in Staphylococcus aureus. Genetics. 1974 Apr;76(4):681–702. doi: 10.1093/genetics/76.4.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wyrick P. B., Rogers H. J. Isolation and characterization of cell wall-defective variants of Bacillus subtilis and Bacillus licheniformis. J Bacteriol. 1973 Oct;116(1):456–465. doi: 10.1128/jb.116.1.456-465.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]