Plasmids that replicate by a rolling-circle (RC) mechanism are ubiquitous in Gram-positive bacteria and a vast majority of over 200 rolling-circle replicating (RCR) plasmids belong to four major families. Many RCR plasmids carry antibiotic resistance genes and evidence suggests that horizontal transfer of RCR plasmids is quite common. Many RCR plasmids also contain genes that are involved in plasmid mobilization and transfer. While some RCR plasmids are able to replicate stably only in their native hosts, many have a broad host range. RC replication involves synthesis of the leading strand that requires the PcrA helicase, single strand DNA binding protein and DNA polymerase III. Lagging strand replication requires primer RNA synthesis by the host RNA polymerase, and subsequently replication is carried out by DNA Pol I and DNA Pol III. PcrA helicase is required for plasmid RC replication as well as survival of Gram-positive organisms. The goal of this R21 application is to test the hypothesis that interaction between plasmid initiator proteins and the PcrA helicases is critical for efficient plasmid replication and may determine narrow versus broad host range replication ofRCR plasmids. No studies have been performed dealing with this issue and the exploratory/development nature of the current application is consistent with the R21 format. We plan to utilize a few plasmids with a relatively narrow host range and a few with broad host range in our study. We will study the replication of these plasmids in S. aureus, B. subtilis, B. cereus and S. pneumoniae. RCR plasmids defective in replication in a particular host will be complemented by a cloned copy of their cognate pcrA gene. We will purify the PcrA helicases from the above Gram-positive organisms and study their interactions with the plasmid initiator proteins. We will also study the in vitro replication of the above plasmids in cell free extracts made from S. aureus in the presence and absence of the various PcrA helicases. These studies should reveal whether PcrA is critical in determining the efficient replication and host range of RCR plasmids. These studies may facilitate the development of antimicrobial drugs targeting the PcrA helicase and/or the initiators of RCR plasmids.
| Khan, Saleem A (2005) Plasmid rolling-circle replication: highlights of two decades of research. Plasmid 53:126-36 |
| Anand, Syam P; Mitra, Poulami; Naqvi, Asma et al. (2004) Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus. J Bacteriol 186:2195-9 |
| Naqvi, Asma; Tinsley, Eowyn; Khan, Saleem A (2003) Purification and characterization of the PcrA helicase of Bacillus anthracis. J Bacteriol 185:6633-9 |
