The Structure of the Plasmid pCU1 TraI Relaxase and the Role of the pCU1 TraI Relaxase-Helicase during Conjugative Plasmid Transfer

Bacteria disseminate genetic material to neighboring cells using conjugative plasmid transfer (CPT). During CPT, a donor bacterium transfers one strand of a double-stranded DNA plasmid to a recipient. Each conjugative plasmid encodes a complex of proteins necessary for its transfer. One of these proteins, the relaxase, initiates plasmid transfer by severing the nic site of the transferred strand (T-strand). A DNA helicase then separates the T-strand from the parent strand, starting at the nic site. The relaxase acts a second time to terminate transfer by resealing the nicked T-strand. The resistance plasmid pCU1 encodes a multi-domain protein, TraI, that supplies both the relaxase and helicase activities required for its transfer. We analyzed the structure and function of pCU1 TraI in order to compare it to similar plasmid-encoded proteins and to identify TraI-mediated activities that could be targeted by inhibitors. Characterization of the pCU1 relaxase revealed unique structural and functional modifications that this enzyme has introduced into the traditional relaxase-mediated DNA nicking mechanism. First, while the overall fold of the pCU1 relaxase is similar to that of homologous relaxases, its conserved DNA nicking residues (Y18,19,26,27) are flipped up to 14 � out of the relaxase active site. Second, the pCU1 relaxase preferentially utilizes Y26 or a combination of Y18+Y19 when nicking DNA. In contrast, homologous relaxases use the first tyrosine in amino acid sequence for DNA nicking. Third, the pCU1 relaxase lacks the sequence-specific DNA binding characteristic of homologous relaxase enzymes. However, it maintains highly sequence specific and metal-dependent DNA nicking. Analysis of the pCU1 helicase established the extent of the minimal helicase domain, the location of the seven conserved helicase motifs, and the substrate requirements of the helicase ATPase activity. The pCU1 helicase harnesses the energy released during ATP hydrolysis to drive DNA strand separation. After optimizing an ATPase assay for use with pCU1 TraI, small molecule libraries were screened for their ability to inhibit pCU1 TraI, and several potential TraI ATPase inhibitors were identified. In summary, this structural and functional characterization of pCU1 TraI identified features unique to this enzyme and revealed particular activities could be targeted by TraI-specific inhibitors.Doctor of Philosoph

The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 Å crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 Å out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid’s origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1

Investigating the impact of bisphosphonates and structurally related compounds on bacteria containing conjugative plasmids

Bacterial plasmids propagate through microbial populations via the directed process of conjugative plasmid transfer (CPT). Because conjugative plasmids often encode antibiotic resistance genes and virulence factors, several approaches to inhibit CPT have been described. Bisphosphonates and structurally related compounds (BSRCs) were previously reported to disrupt conjugative transfer of the F (fertility) plasmid in E. coli. We have further investigated the effect of these compounds on the transfer of two additional conjugative plasmids, pCU1 and R100, between E. coli. The impact of BSRCs on E. coli survival and plasmid transfer was found to be dependent on the plasmid type, the length of time the E. coli were exposed to the compounds, and the ratio of plasmid donor to plasmid recipient cells. Therefore, these data indicate that BSRCs produce a range of effects on the conjugative transfer of bacterial plasmids in E. coli. Since their impact appears to be plasmid type-dependent, BSRCs are unlikely to be applicable as broad inhibitors of antibiotic resistance propagation