Investigating the basis of substrate recognition in the pC221 relaxosome

The nicking of the origin of transfer (oriT) is an essential initial step in the conjugative mobilization of plasmid DNA. In the case of staphylococcal plasmid pC221, nicking by the plasmid-specific MobA relaxase is facilitated by the DNA-binding accessory protein MobC; however, the role of MobC in this process is currently unknown. In this study, the site of MobC binding was determined by DNase I footprinting. MobC interacts with oriT DNA at two directly repeated 9 bp sequences, mcb1 and mcb2, upstream of the oriT nic site, and additionally at a third, degenerate repeat within the mobC gene, mcb3. The binding activity of the conserved sequences was confirmed indirectly by competitive electrophoretic mobility shift assays and directly by Surface Plasmon Resonance studies. Mutation at mcb2 abolished detectable nicking activity, suggesting that binding of this site by MobC is a prerequisite for nicking by MobA. Sequential site-directed mutagenesis of each binding site in pC221 has demonstrated that all three are required for mobilization. The MobA relaxase, while unable to bind to oriT DNA alone, was found to associate with a MobC–oriT complex and alter the MobC binding profile in a region between mcb2 and the nic site. Mutagenesis of oriT in this region defines a 7 bp sequence, sra, which was essential for nicking by MobA. Exchange of four divergent bases between the sra of pC221 and the related plasmid pC223 was sufficient to swap their substrate identity in a MobA-specific nicking assay. Based on these observations we propose a model of layered specificity in the assembly of pC221-family relaxosomes, whereby a common MobC:mcb complex presents the oriT substrate, which is then nicked only by the cognate MobA

Antibacterial drug discovery: is it all downhill from here?

ABSTRACTThere has been a marked decline in the industrial research aimed at discovering novel antibacterial agents, including new drugs that target resistant organisms. While this decline may reflect past cyclical changes that often affect resource allocation at pharmaceutical companies, this decline is occurring at a time of increasing levels of antibacterial drug resistance and meagre pipelines of new agents that are active against them. There are multiple reasons for this decline, although few are unique to antibacterial drug discovery research. These include: lack of industry productivity, increasing size of clinical trials, increased generic competition and other pressures on drug pricing, a crowded and confused marketplace and industry consolidation. And while many (if not most) large companies and biotechs have exited the field or severely curtailed their research, others have made it a point to continue their efforts, citing both the unmet medical need and a large and apparently growing market. Despite the fact that some companies have remained engaged, the view here is that the current level of industrial effort is insufficient to sustain a healthy flow of new and better agents that are needed to counter the imminent threat of bacterial drug resistance. Therefore, a clear and urgent need for finding ways to improve the level and quality of industrial research in this area is apparent