Staphylococcus aureus is a major human pathogen that causes a variety of diseases. Strains of S. aureus have increasingly become resistant to a number of commonly used antibiotics. Rolling-circle replicating (RCR) plasmids are ubiquitous in S. aureus and many other Gram-positive bacteria. These plasmids encode drug resistance and play a major role in the horizontal spread of these genes in nature. We will continue our studies on the replication of one of the best studied RCR plasmid, pT181 of S. aureus. We will generate mutants of the pT181-encoded initiator protein, RepC, and identify its domains involved in the termination of pT181 replication using biochemical approaches. A chromosome-encoded, essential helicase, PcrA, is present in S. aureus and other Gram-positive bacteria. PcrA is required for cell growth and viability, and plasmid rolling-circle (RC) replication. We have shown that it promotes RepC-dependent pT181 DNA unwinding and in vitro replication. We will use both in vitro and in vivo approaches to identify domains of RepC and PcrA that are involved in their interaction. We have shown that the S. aureus PcrA is unusual in that it has equally robust bipolar 5'->3'and 3'->5'helicase activities. We plan to study the structure-function relationship of PcrA to identify its domains and enzymatic activities (ssDNA translocation, 5'->3'and 3'->5'helicase, and DNA unwinding) that are important in plasmid replication. Site-directed and random mutants of PcrA will be generated, overexpressed, purified and analyzed for their biochemical activities. PcrA mutants expressed from constitutive promoters will be introduced into strains containing an inducible, wild-type pcrA gene and tested for their ability to support plasmid RC replication under non-inducing conditions. The PcrAS mutant is defective in supporting pT181 replication, but is competent in the replication of plasmids belonging to other RCR families such as pC194, pE194 and pSN2. We will test various pcrA mutants for their ability to support replication of the above plasmids. These studies should provide information on domains of PcrA that are critical for the replication of particular RCR plasmid families and may identify PcrA domains that interact with the initiator proteins of various plasmids. Our studies could provide new avenues for the development of novel drugs targeting RCR plasmids, as well as targeting an essential helicase in S. aureus and other Gram-positive bacteria.
NIH ExPorter
NIH ExPorter