Stability of penicillinase plasmids in staphylococcus aureus

The stability of the penicillinase plasmids of Staphylococcus aureus has been investigated both by the effects of various agents on the elimination of the plasmids and by the characterisation of mutations influencing the stability.
Growth or plasmid-carrying strains in the presence of ethidium bromide enhanced the spontaneous rate of loss of plasmids in all seven strains of S.aureus tested. In one of these strains, growth in media containing acridine orange had a similar effect on plasmid loss. One of the strains grew in the presence of high concentrations of ethidium bromide. This resistance to ethidium bromide is determined by a gene located on the penicillinase plasmid and appears to be due, at least in part, to a permeability effect.
Two types of mutant affected in the stability of the penicillinase plasmids have been isolated from S.aureus PS80 after treatment with ethyl methane sulphonate. In one, the mutation is present on the penicillinase plasmid and results in an inability to replicate at 42andnbsp;C; replication at 30andnbsp;C being unaffected. The second type of mutation is not located on the plasmid and causes instability of penicillinase plasmids of both compatibility groups but does not affect a plasmid conferring resistance to tetracycline.
Reversion of the temperature-sensitive plasmid to temperature stability has been studied. Evidence is presented to show that this reversion is the result of integration of the plasmid into another replicon, probably the bacterial chromosome. Deletions encompassing a particular region or the plasmid reduce the frequency of this reversion. It is suggested that this region includes a gene specifying a protein involved in the integration process.
A penicillinase plasmid that has a gene conferring resistance to erythromycin (ero) has been used to integrate a fragment of this plasmid into the chromosome of S.aureus PS80. The fragment includes only the ero gene of the known plasmid genes. After transduction into this constructed strain, the temperature-sensitive plasmid integrated at a considerably increased rate. Such an integration can be effected at sites distant from the ero. since linkage between the integrated plasmid and the chromosomal ero is not obligatory. It is suggested that the effect is due to the fragment including,in addition to the ero gene, a gene int that produces a protein involved in recombination between a specific site on the plasmid and sites on the chromosome.
An integrated plasmid can be excised by a superinfecting plasmid. This excision is apparently independent of any recombination between the integrated and superinfecting plasmid. A gene xis that specifies a protein responsible for this excision is proposed, xis activity functions independently or int product.
Deletion mapping involving irradiation of transducing phage normally gives the same linear sequence of plasmid genes. This implies that the circular DNA in a host is ruptured at a specific site. Deletion mapping of a plasmid without this putative site results in an altered linear sequence. It is proposed that this site be called end.
The location on the plasmid of the genes specifying these functions is discussed and a tentative plasmid map presented.
In view of the similarities between the integration and excision functions specified by the penicillinase plasmid and those specified by temperate phage, such as lambda, it is proposed that temperate phages are the precursors of staphylococcal penicillinase plasmids.
It has proved impossible to remove all plasmid-like DNA from a penicillinase plasmid negative S.aureus PS80. Nevertheless there is some indication that the temperature-sensitive plasmid DNA can be separated from other plasmid-like DNA by centrifugation in neutral sucrose gradients.

Advisor: Dyke, K