Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly.

Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase ß- and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the ß-clamp contributes to the formation of the ß-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the ß-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the ß-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and ß-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined.

[Active efflux as the multidrug resistance mechanism]. / Czynne usuwanie leku z komórki jako jeden z mechanizmów opornosci bakterii na srodki przeciwdrobnoustrojowe i metody jego zwalczania.

Active efflux is a common resistance mechanism in a wide range of bacterial pathogens. It is responsible for the transport of such toxic compounds as drugs, toxins, and detergents. Pumps with broad substrate profiles promote the emergence of multidrug-resistant bacterial pathogens that are a particular threat to contemporary medicine, such as Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Salmonella enterica sv Typhimurium. One can distinguish five major class of transport systems on the basis of their structure and function: MFS (major facilitator superfamily), SMR (small multidrug-resistance family), MATE (multidrug and toxic compound extrusion family), ABC (ATP binding cassette superfamily), and RND (resistance nodulation cell division family). Substrate transport may include hydrogen proton exchange or energy generated via ATP hydrolysis. The transport is effectively regulated by local regulatory proteins.such as BmrR from Bacillus subtilis, or by the global bacterial regulatory system. Investigations into efflux pumps and their substrate profiles and regulatory mechanisms have led to the discovery of new therapeutics and pump inhibitors that could potentially become alternative and effective antimicrobial drugs. Additionally, some alternative therapies such as photodynamic inactivation could be more effective if the synergistic action of efflux pump inhibitors are used.Thus research into efflux transport systems seems to be an important element of contemporary medicine .