NSF Award
9004169
The present proposal is centered on the study of two classes vof Staphylococcus aureus host mutants which affect the replication of plasmid pT181. The studies conducted so far on the characterization of these two classes of mutants have shown that both illustrate new types of host-plasmid interactions. The first class of mutations, exemplified by pcrA3, maintain plasmid pT181 at a reduced copy number, without dramatically affecting its hereditary stability. The pcrA mutations does not act by interfering with the plasmid control mechanism, and its effect can not be explained simply by a host factor limiting for plasmid replication. It was shown that in the pcrA host there is an accumulation of the plasmid pT181 Rep-origin replication initiation complex. These results suggest that the pcrA gene product enables the plasmid replication initiation complex to make efficient use of the host enzymatic machinery for replication. Several classes of plasmid mutations that affect the response to the pcrA3 mutation have been isolated and characterized. A more detailed study of the pcrA3 mutations is proposed which will include in vivo and in vitro replication experiments as well as genetical studies. Work is in progress for the cloning of the pcrA gene. This will permit us to evaluate its role in the cell and will contribute to the understanding of the way in which it affects plasmid pT181 replication. The mutation placC1, representative of the second class which leads to the maintenance of plasmid pT181 at much increased copy number, was found to specifically affect the control mechanism of this plasmid, by depressing the synthesis of the countertranscripts ( ctRNA), which act as negative effectors. The placC gene has been cloned and its sequencing is in progress. The cloned wild type allele was found to complement the placC1 allele present on the chromosome preventing the amplification of plasmid pT181. The isolation of the plaC gene makes possible the study of its role in host metabolism and of the way in which its expression is regulated. The results to be obtained will help to determine what is the biological significance of the dependence of the pT181 ctRNA promoter upon the function of the plaC gene.
