Identification and cloning of the conjugative transfer region of Staphylococcus aureus plasmid pGO1
Open Access
- 1 February 1989
- journal article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 171 (2), 684-691
- https://doi.org/10.1128/jb.171.2.684-691.1989
Abstract
The conjugative transfer (tra) genes of a 52-kilobase (kb) staphylococcal plasmid, pGO1, were localized by deletion analysis and transposon insertional inactivation. All transfer-defective (Tra-) deletions and Tn551 or Tn917 transposon insertions occurred within a 14.5-kb BglII fragment. Deletions and insertions outside this fragment all left the plasmid transfer proficient (Tra+). The tra region was found to be flanked by directly repeated DNA sequences, approximately 900 base pairs in length, at either end. Clones containing the 14.5-kb BglII fragment (pGO200) and subclones from this fragment were constructed in Escherichia coli on shuttle plasmids and introduced into Staphylococcus aureus protoplasts. Protoplasts could not be transformed with pGO200E (pGO200 on the staphylococcal replicon, pE194) or subclones containing DNA at one end of the tra fragment unless pGO1 or specific cloned tra DNA fragments were present in the recipient cell. However, once stabilized by sequences present on a second replicon, each tra fragment could be successfully introduced alone into other plasmid-free S. aureus recipients by conjugative mobilization or transduction. In this manner, two clones containing overlapping fragments comprising the entire 14.5-kb BglII fragment were shown to complement each other. The low-frequency transfer resulted in transconjugants containing one clone intact, deletions of that clone, and recombinants of the two clones. The resulting recombinant plasmid (pGO220), which regenerated the tra region intact on a single replicon, transferred at frequencies comparable to those of pGO1. Thus, all the genes necessary and sufficient for conjugative transfer of pGO1 are contained within a 14.5-kb region of DNA.Keywords
This publication has 24 references indexed in Scilit:
- IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgarisGene, 1987
- Homologous direct repeat sequences associated with mercury, methicillin, tetracycline and trimethoprim resistance determinants inStaphylococcus aureusFEMS Microbiology Letters, 1987
- THE CONJUGATION SYSTEM OF F, THE FERTILITY FACTOR OF ESCHERICHIA COLIAnnual Review of Genetics, 1986
- Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functionsPlasmid, 1986
- The use of cetyltrimethylammonium bromide for the rapid isolation from Staphylococcus aureus of relaxable and non-relaxable plasmid DNA suitable for in vitro manipulationLetters in Applied Microbiology, 1985
- Molecular Epidemiology of Transmissible Gentamicin Resistance Among Coagulase-Negative Staphylococci in a Cardiac Surgery UnitThe Journal of Infectious Diseases, 1985
- The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophageNature, 1983
- Overproduction of bacteriophage Qβ maturation (A2) protein leads to cell lysisCell, 1983
- Structural and phenotypic varieties of gentamicin resistance plasmids in hospital strains of Staphylococcus aureus and coagulase-negative staphylococciAntimicrobial Agents and Chemotherapy, 1982
- A complementation analysis of the restriction and modification of DNA in Escherichia coliJournal of Molecular Biology, 1969