Under constant conditions bacterial plasmids are maintained in a characteristic number of copies per cell, indicating that plasmid replication is under the control of a specific, finely tuned regulatory mechanism. The isolation of mutant plasmids maintained in a higher copy number (Cop mutants) from many different plasmids demonstrated the role of plasmids in the control of their own replication. The host cell provides most of the functions required for the synthesis of plasmid DNA, and our recent results suggest that it is involved in control as well. The goal of the proposed research is to investigate the involvement of the host in plasmid replication control, which would seem to represent an as yet unknown type of plasmid-host interrelationship. The approach will be based on the analysis of two classes of Staphylococcus aureus mutants isolated previously: (1) host mutants (plaC) in which Inc3 plasmids are specifically amplified, and (ii) host mutants (pcr) in which the copy number of Inc3 plasmids is reduced. The mechanisms responsible for these alterations in the plasmid copy numbers due to host mutations will be studied in the context of the present knowledge on the replication control of Inc3 plasmids. This will involve the quantitative estimation of the synthesis and activity in the host mutants of the plasmid-encoded small RNA species that act as negative effectors in replication control, and of RepC, a protein essential for plasmid replication. The level of Inc3 plasmid single-stranded DNA per plasmid copy in the pcr host mutants will be also determined. Other aspects of the study will include the behavior in the host mutants of Cop and temperature-sensitive replication (Tsr) mutants isolated from pT181, the Inc3 prototype. The chromosomal mutations identified will be mapped to determine the number of different host genes involved in plasmid copy control, and the functional relationships between different mutations will be determined. Cloning of the host genes identified by these mutations will be performed, and an attempt to identify the gene products and their function will be made.
