Conjugative transfer of plasmid DNA via close cell-cell junctions is the main route by which antibiotic resistance genes spread between bacterial strains. Relaxases are essential for conjugative transfer and act by cleaving DNA strands and forming covalent phosphotyrosine linkages. Based on data indicating that multityrosine relaxase enzymes can accommodate two phosphotyrosine intermediates within their divalent metal-containing active sites, we hypothesized that bisphosphonates would inhibit relaxase activity and conjugative DNA transfer. We identified bisphosphonates that are nanomolar inhibitors of the F plasmid conjugative relaxase in vitro. Furthermore, we used cell-based assays to demonstrate that these compounds are highly effective at preventing DNA transfer and at selectively killing cells harboring conjugative plasmids. Two potent inhibitors, clodronate and etidronate, are already clinically approved to treat bone loss. Thus, the inhibition of conjugative relaxases is a potentially novel antimicrobial approach, one that selectively targets bacteria capable of transferring antibiotic resistance and generating multidrug resistant strains.antimicrobial ͉ bacterial conjugation ͉ bisphosphonates ͉ F plasmid TraI ͉ relaxase inhibition C onjugative elements are responsible for the majority of horizontal gene transfers within and between bacterial strains (reviewed in ref. 1), as first described for the Escherichia coli F plasmid by Lederberg and Tatum in 1946 (2). Conjugative DNA transfer is also the central mechanism by which antibiotic resistance and virulence factors are propagated in bacterial populations (reviewed in ref.3). Indeed, it is well established that antibiotic resistance can be rapidly acquired in clinical settings and that such acquisition is critically dependent on conjugative DNA transfer (reviewed in ref. 4). Small-molecule inhibition of conjugation could prove to be a powerful method for curbing the generation and spread of multidrug-resistant strains. Past studies suggested that various antibiotics, polycyclic chemicals, and crude extracts inhibit conjugation at concentrations less than the antibacterial minimum inhibitory concentration (5-11); however, most of these effects have been attributed to nonconjugation-specific inhibition of bacterial growth or DNA synthesis (12)(13)(14)(15). This study describes a bottom-up approach used to identify the first small molecule inhibitors of conjugative DNA transfer that target an enzyme of the conjugative system.The DNA relaxase is a central enzyme in each conjugative system (16-18) and thus is a prime target for inhibition. The conjugative relaxase initiates DNA transfer with a site-and strand-specific ssDNA nick in the transferred strand (T-strand) at the origin of transfer (oriT), forming a covalent 5Ј-phosphotyrosine intermediate (16,(19)(20)(21)(22)(23). The nicked T-strand moves from the donor cell (plasmid ϩ ) to the recipient cell (plasmid Ϫ ) via an intercellular junction mediated by a type IV secretion system (reviewed in refs. 19, 24,...

Alfalfa mosaic virus genomic RNAs are infectious only when the viral coat protein binds to the RNA 3´ termini. The crystal structure of an alfalfa mosaic virus RNA-peptide complex reveals that conserved AUGC repeats and Pro-Thr-x-Arg-Ser-x-x-Tyr coat protein amino acids cofold upon interacting. Alternating AUGC residues have opposite orientation, and they base pair in different adjacent duplexes. Localized RNA backbone reversals stabilized by arginine-guanine interactions place the adenosines and guanines in reverse order in the duplex. The results suggest that a uniform, organized 3´ conformation, similar to that found on viral RNAs with transfer RNA-like ends, may be essential for replication.A general problem in positive-strand RNA virology is understanding how viral RNA replication is initiated by the RNA-dependent RNA polymerase (replicase) on the correct template and nucleotide in an infected cell. Alfalfa mosaic virus (AMV) and ilarviruses are unusual positive-sense viruses, the genomic RNAs of which are replicated only in the presence of the viral coat protein (CP) (1,2). These viruses are distinguished from many other members of the virus family Bromoviridae because they lack canonical features of the tRNA-like structure (TLS) common at the 3´ termini of the viral RNA genomes. The TLS is a necessary and sufficient feature for recruitment of the bromovirus replicase (3,4). CP-induced structural organization of the AMV RNA 3´ terminus may create a functional homolog of the tRNA tail and thereby permit recognition by the RNA-dependent RNA polymerase.CP binds specifically to the 3´ untranslated regions (3´UTRs) found on all four RNAs of the segmented AMV genome (5). The 180-nucleotide 3´UTR secondary structure likely consists of six hairpins, most of which are separated by single-stranded tetranucleotide AUGC repeats (5-8). These repeats are characteristic of AMV and ilarvirus RNA sequences and are important for CP binding (8-11). We previously identified a 39-nucleotide minimal high affinity AMV CP-binding site, consisting of the two terminal hairpins and their flanking AUGC nucleotides (nucleotides 843 to 881 in RNA4; i.e., AMV 843-881 ) (8,12,13) (fig. S1A). This fragment is competent to bind either full-length CP or a 26-amino acid peptide (CP26, fig. S1B) (13) representing the N-terminal RNA binding domain (14). The CP N terminus contains a ProThr-x-Arg-Ser-x-x-Tyr (PTxRSxxY) RNA binding domain conserved among AMV and ilarvirus CPs (14).

SummaryThe TraI relaxase-helicase is the central catalytic component of the multi-protein relaxosome complex responsible for conjugative DNA transfer (CDT) between bacterial cells. CDT is a primary mechanism for the lateral propagation of microbial genetic material, including the spread of antibiotic resistance genes. The 2.4 Å resolution crystal structure of the C-terminal domain of the multifunctional Escherichia coli F plasmid TraI protein (TraI-CT) is presented, and specific structural regions essential for CDT are identified. The crystal structure reveals a novel fold composed of a 28-residue N-terminal α-Domain connected by a proline-rich loop to a compact α/β-Domain. Both the globular nature of the α/β-Domain and the presence and rigidity of the proline-rich loop are required for DNA transfer and single-stranded DNA binding. Taken together, these data establish the specific structural features of this non-catalytic domain that are essential to DNA conjugation.

Alfalfa mosaic virus (AMV) and ilarvirusRNA-protein interactions play important roles in a variety of biological processes and are of particular importance in the life cycle of positive-strand RNA viruses, where the genomic RNA serves as the template for protein synthesis and replication. Alfalfa mosaic virus (AMV) and ilarviruses provide an interesting model system in which to study RNA-protein interactions because the viral coat protein (CP) has multiple roles in the viral life cycle, not all of which are completely understood. In addition to virion assembly (21, 44) the viral CP has roles in cell-to-cell movement (12, 43), infectivity (6, 23), and translation (28,29).Most bromoviruses have a tRNA-like structure (TLS) on the 3Ј end of the RNA genome. The TLS has been found to be important for recruiting the viral replicase (8, 13). Conversely, AMV and ilarviruses lack a canonical CCA 3Ј end common to the TLS and further require the presence of their own viral CP to initiate replication (6). It has been shown that CP binds specifically to the 3Ј untranslated region found on all three AMV genomic RNAs as well as the subgenomic RNA4 (5, 22, 24). Genomic RNA1 and -2 encode proteins with replicase functions (33). RNA3 is dicistronic, encoding the viral movement protein (MP) and CP. Only the upstream open reading frame, encoding the viral MP, is translated from genomic RNA3. The viral CP is translated from a subgenomic RNA4, which is generated by the internal initiation of transcription from minus-strand RNA3. In vitro-transcribed RNA4 (CP mRNA) can substitute for CP in the activation of replication when inoculated with the genomic RNAs into tobacco protoplasts (46).It was first proposed by Houwing and Jaspars that an RNA conformational change accompanies CP binding to the 3Ј ends of the genomic RNAs and thereby presents a recognition signal for replicase binding (20). Results from circular dichroism analyses and native polyacrylamide gel electrophoresis experiments support the idea that the 3Ј untranslated region (UTR) of AMV RNA3 and -4 undergoes a conformational change upon binding amino-terminal CP peptides (3). Further investigation of this RNA-protein interaction revealed a 26-aminoacid RNA binding motif in the N terminus of the CP that is sufficient to activate replication in mesophyll protoplasts (3,39). A minimal CP binding site was also identified at the terminal 39 nucleotides of the AMV 3Ј UTR (1, 17). While it is known that the genomic RNAs are not infectious in the absence of CP (6, 47), it is unclear how CP initiates infection or how it influences ongoing replication.Several models have been proposed to explain the genome activation phenomenon. The interaction of CP with the genomic RNA 3Ј UTR may serve to protect the RNAs from degradation (30); however, AMV genomic RNAs have been shown to be stable for several hours when inoculated into protoplasts in the absence of CP or polymerase (19). Olsthoorn et al. (31) described the conformational switch model, wherein the protein-free 3Ј UTR assumes a pseu...