Richard J. Meyer
Although most plasmids in Gram-negative bacteria have a restricted host-range, a few groups are widely distributed among unrelated species. These plasmids are disseminated primarily by conjugation, a process that can result in the transfer of plasmids not only between bacterial species, but also into eucaryotic cells. In our laboratory, we are focussing on how plasmid DNA is processed for conjugal transfer. This problem has practical medical significance, since broad host-range plasmids generally encode resistance to one or more antibiotics, and are probably contributing to the increasing frequency of multiple drug-resistance in bacteria by providing a mechanism for interspecific gene transfer and maintenance. We have been investigating the conjugal transfer of the broad host-range plasmid R1162. Our objectives are to map the genes involved and to characterize their regulation, and also to identify the gene products and the sites at which they act. Ultimately, we wish to describe in detail the molecular steps in transfer, and to identify those properties which free the plasmid from the constraints imposed by different cytoplasms. We have identified the R1162 genes required for transfer, and have mapped oriT, the origin of transfer, to a 38 base-pair segment of the plasmid. We are in the process of determining how the R1162-encoded proteins interact at oriT to carry out the DNA processing steps required for the initiation and termination of transfer. To this end, several specific questions now are being investigated in our laboratory:
Elements in the co-evolution of relaxases and their origins of transfer. Mechanisms of Strand Replacement Synthesis for Plasmid DNA Transferred by Conjugation.
Relaxed specificity of the R1162 relaxase: a model for evolution of a system for conjugative mobilization of plasmids. Molecular handcuffing of the relaxosome at the origin of conjugative transfer of the plasmid R1162.
MobA, the DNA strand transferase of plasmid R1162. The minimal domain required for DNA processing at the origin of transfer.
Identification of the mob genes of plasmid pSC101 and characterization of a hybrid pSC101/R1162 system for conjugal mobilization. Recognition of oriT for DNA processing at termination of a round of conjugal transfer. Becker, E.C. and R.J. Meyer Journal of Molecular Biology 300 (2000):1069-79 ABSTRACT: Conjugal transfer of plasmid DNA is terminated when the transferred strand, linearized at the 38 base-pair origin of transfer (oriT), is recircularized. For the plasmid R1162, it is the protein MobA, covalently linked to the linear strand, that rejoins the ends by a reversible transesterification reaction. We have identified from those oligonucleotides with a partially degenerate oriT base sequence, subpopulations bound by MobA that undergo transesterification, and support efficient termination of conjugal transfer. Two domains of oriT, a ten base-pair inverted repeat and an adjacent TAA, are required for tight binding by the protein, whereas the location of the dinucleotide YG determines the site of strand cleavage. The results indicate that capture of MobA by oriT, and subsequent processing of the DNA for termination, are determined by different sequence motifs within this locus.
Perwez, T. and R.J. Meyer The Journal of Bacteriology 181 (1999): 2124-2131. ABSTRACT: MobB is a small protein encoded by the broad-host-range plasmid R1162 and required for efficient mobilization of its DNA during conjugation. The protein was shown previously to stabilize the relaxosome, the complex of plasmid DNA and mobilization proteins at the origin of transfer (oriT). We have generated in-frame mobB deletions that specifically inactivate the stabilizing effect of MobB while still allowing a high rate of transfer. Thus, MobB has two genetically distinct functions in transfer. The effect of another deletion, extending into mobA, indicates that both functions require a specific region of MobA protein that is distinct from the nicking-ligating domain. The mobB mutations that specifically affected stability also resulted in poor growth of cells, due to increased transcription from the promoters adjacent to oriT. The effects of the mutations could be suppressed not only by full-length MobB provided in trans, as expected, but also by additional copies of oriT, cloned in pBR322. In addition, in the presence of MobA both the full-length and truncated forms of MobB stimulated recombination between oriT-containing plasmids. We propose a model in which MobB regulates expression of plasmid genes by altering the stability of the relaxosome, in a manner that involves the coupling of plasmid molecules.
The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer Becker, E. C. and R. J. Meyer The Journal of Bacteriology 179 (1997): 5947-5950. ABSTRACT: R1162 is a representative member of the broad-host-range IncQ group of multicopy plasmids. Lower-copy-number derivatives of R1162 were constructed in vitro and shown to be unstable, indicating that partitioning of plasmid copies at cell division is due to random distribution and not to an active partitioning mechanism. However, the normal copy number of R1162 reduces cell fitness during growth in broth and favors the emergence of unstable, lower-copy-number variants. As a result, plasmid-borne antibiotic resistance genes active at a low copy number eventually result in plasmid loss during periods of no selection. We argue that the maintenance of R1162 in a population requires a gene that is selected only at high levels. This reduces the potential for acquiring genes from other R factors and could explain the limited variety of antibiotic resistance genes contained by naturally occurring IncQ plasmids.
Becker, E. C., H. Zhou, and R.J. Meyer The Journal of Bacteriology 78 (1996): 4870-4876. ABSTRACT: The origin of replication of the plasmid R1162 contains an initiation site for the synthesis of each DNA strand. When one of these sites (oriL) is deleted, synthesis on the corresponding strand is no longer initiated efficiently in vitro by the R1162-encoded replication proteins, and the plasmid is no longer stably maintained in the cell. However, in vivo the two strands of the plasmid duplex molecule are active at a similar level as templates for DNA synthesis, and newly synthesized copies of each strand are incorporated into daughter molecules at a similar rate. No secondary, strong initiation sites on the delta oriL strand were detected in the region of the origin. The delta oriL plasmid induces the SOS response, and this is important for plasmid maintenance even in a recombination-proficient strain. Our results indicate that an SOS-induced host system can maintain an R1162 derivative lacking one of its initiation sites.MobB protein stimulates nicking at the R1162 origin of transfer by increasing the proportion of complexed plasmid DNA Perwez, T., and R.J. Meyer The Journal of Bacteriology 178 (1996): 5762-5767. ABSTRACT: An essential early step in conjugal mobilization of R1162, nicking of the DNA strand that is subsequently transferred, is carried out in the relaxosome, a complex of two plasmid-encoded proteins and DNA at the origin of transfer (oriT). A third protein, MobB, is also required for efficient mobilization. We show that in the cell this protein increases the proportion of molecules specifically nicked at oriT, resulting in lower yields of covalently closed molecules after alkaline extraction. These nicked molecules largely remain supercoiled, with unwinding presumably constrained by the relaxosome. MobB enhances the sensitivity of the oriT DNA to oxidation by permanganate, indicating that the protein acts by increasing the fraction of complexed molecules. Mutations that significantly reduce the amount of complexed DNA in the cell were isolated. However, plasmids with these mutations were mobilized at nearly the normal frequency, were nicked at a commensurate level, and still required MobB. Our results indicate that the frequency of transfer is determined both by the amount of time each molecule is in the nicked form and by the proportion of complexed molecules in the total population.The primase of broad host-range plasmid R1162 is active in conjugal transfer Henderson, D. and R.J. Meyer The Journal of Bacteriology 178 (1996): 6888-6894. ABSTRACT: The broad-host-range plasmid R1162 is conjugally mobilized at high frequency by the IncP-1 plasmid R751 but is poorly mobilized by pOX38, a derivative of the F factor. In both cases, the origin of transfer (oriT) and the Mob proteins of R1162 are required, indicating that these plasmids are mobilized by similar mechanisms. R1162 encodes a primase, essential for vegetative replication of the plasmid, that is made both as a separate protein and as the carboxy-terminal domain of MobA, one of the R1162 mobilization proteins (P. Scholz, V. Haring, B. Wittman-Liebold, K. Ashman, M. Bagdasarian, and E. Scherzinger, Gene 75:271-288, 1989). When R751 is the mobilizing vector, the primase is not required for mobilization of plasmids containing cloned mob-oriT R1162 DNA. However, detectable mobilization of such plasmids by pOX38 requires both the primase and its cognate initiation site, oriented for synthesis of the complement to the transferred strand. The long form of the primase is required for optimal transfer: R1162 replicons lacking this form also are not transferred detectably by pOX38 and are less well mobilized by R751. The distance between oriT and the primase initiation site affects the frequency of mobilization, and this effect is polar in the direction of transfer. Our results indicate that the R1162 primase is active in mobilization of R1162 and suggest that the MobA-linked form is an adaptation increasing its effectiveness during transfer.
Localized denaturation of oriT DNA within relaxosomes of the broad-host-range plasmid R1162
Rao, X.-M. and R.J. Meyer The Journal of Bacteriology 176 (1994): 5958-5961. ABSTRACT: Conjugal transfer of plasmid R1162 is initiated and terminated at a 38-bp origin of transfer (oriT). Plasmids containing two directly repeated copies of oriT were used to determine the actual frequency of termination at this site. This frequency was calculated both for oriTnic, a mutated origin that cannot act as the initiation site of transfer, and for an unmutated oriT where transfer had been initiated. In both cases, the termination frequency decreased as the distance between the initiation and termination sites became greater and was significantly less than one for plasmids the size of R1162. A substantial proportion of recipient cells received more than one plasmid copy during transfer. Our results indicate that termination is inefficient but that this is partly compensated for by the transmission of multiple plasmid copies.
Bhattacharjee, M. and R.J. Meyer Nucleic Acids Research 21 (1993): 4563-4568. ABSTRACT: MobA protein, encoded by the broad host-range plasmid R1162, is required for conjugal mobilization of this plasmid. The protein is an essential part of the relaxosome, and is also necessary for the termination of strand transfer. In vitro, MobA is a nuclease specific for one of the two DNA strands of the origin of transfer (oriT). The protein can cleave this strand at the same site that is nicked in the relaxosome, and can also ligate the DNA. We show here that purified MobA protein forms a complex that is specific for this single oriT strand. The complex is unusually stable, with a half-life of approximately 95 min, is not disrupted by hybridization with the complementary strand, and reforms rapidly after boiling. Both the inverted repeat within oriT, and the eight bases between this repeat and the site cleaved by MobA, are required for binding by the protein. Mutations reducing base complementarity between the arms of the inverted repeat also decrease binding. This effect is partially suppressed by second-site mutations restoring complementarity. These results parallel the effects of these mutations on termination. Footprinting experiments with P1 nuclease indicate that the DNA between the inverted repeat and the nick site is protected by MobA, but that pairing between the arms of the repeat, which occurs in the absence of protein, is partially disrupted. Our results suggest that termination of strand transfer during conjugation involves tight binding of the MobA protein to the inverted repeat and adjacent oriT DNA. This complex positions the protein for ligation of the ends of the transferred strand, to reform a circular plasmid molecule.Erickson, M.J. and R.J. Meyer The origin of greater-than-unit-length plasmids generated during bacterial conjugation. Molecular Microbiology 7 (1993): 289-298. ABSTRACT: In Gram-negative bacteria, the general mechanism of conjugal plasmid transfer, which is probably similar for many different groups of plasmids, involves the transfer of a single plasmid DNA strand with 5' to 3' polarity. Transfer is initiated by nicking of the duplex DNA at a particular site, i.e. the origin of transfer (oriT). We constructed plasmids containing two directly repeated copies of oriT, derived from the broad-host-range plasmid R1162 and flanking the lac operator. The number of lacO copies in the plasmid after transfer could be determined from the colour of transconjugant colonies on medium containing X-Gal. When the oriTs were mutated to prevent initiation and termination of transfer at the same oriT, almost all of the transconjugant cells contained greater-than-unit-length plasmids with two copies of lacO and three copies of oriT. We show that these molecules were generated by an intramolecular, conjugation-dependent mechanism unlikely to depend solely on a pre-existing population of circular or linear multimers in donor cells. We propose that the greater-than-unit-length molecules were instead generated by a rolling-circle mechanism of DNA replication.
Bhattacharjee, M., X.-M. Rao, and R. J. Meyer The Journal of Bacteriology 174 (1992): 6659-6665. ABSTRACT: Conjugal transfer of the broad-host-range plasmid R1162 is initiated and terminated at the nic site within the 38-bp origin of transfer (oriT). Termination involves ligation of the transferred single strand by the plasmid-encoded MobA protein. Several different assays were used to identify the oriT DNA required for termination. For plasmids containing two oriTs, with transfer initiated at one and terminated at the other, the inverted repeat within oriT is important for termination. Deletion of the outer arm reduces the termination frequency; those terminations that do occur probably depend upon nicking at this oriT prior to transfer. The locations of second-site suppressor mutations indicate that base pairing between the arms of the inverted repeat is important for termination. In vitro, the inverted repeat is not required for specific cleavage of single-stranded DNA at nic, but competition experiments indicate that oriTs with the inverted repeat are preferentially cleaved. We propose that the function of the oriT inverted repeat is to trap the plasmid-encoded MobA protein at the end of a round of strand transfer, thus ensuring that the protein is available for the ligation step.
Kim, Y. J., and R.J. Meyer. The Journal of Bacteriology 173 (1991): 5539-5545. ABSTRACT: The R1162-encoded protein RepIB is essential for replication of the plasmid and binds specifically to iterons within the replicative origin. The protein causes the localized melting of DNA (determined by sensitivity to P1 nuclease) at a site within the AT-rich region of the origin, about 60 bp from the iteron binding sites and separated from them by a GC-rich tract. Point mutations have been isolated in the AT-rich DNA. These mutations interfere with origin activity and also prevent the protein-induced sensitivity to P1. A second-site suppressor of one of these mutations maps in the repIb gene and restores both origin function and sensitivity to P1. The results suggest a specific interaction between RepIB and origin DNA at a position distant from its primary binding site.Probing the activation of the replicative origin of broad host-range plasmid R1162 with Tus, the E. coli anti-helicase protein. Zhou, H., C. Byrd, and R.J. Meyer Nucleic Acids Research 19 (1991): 5379-5383. ABSTRACT: The E.coli Tus protein is an anti-helicase involved in the termination of chromosome replication. The binding site for this protein, ter, was cloned into derivatives of the broad host-range plasmid R1162. The ter site caused the orientation-specific termination of plasmid replication fork movement in cell extracts containing Tus. Plasmids were constructed so that two sites for initiation of R1162 replication flanked the iteron-containing domain of the origin. In these plasmids, the site next to the AT-rich region within the iteron-containing domain was more active. In addition, when ter was placed between the more active site and the iterons, initiation of replication from this site was specifically inhibited. The data support a model for entry of the essential, plasmid-encoded helicase at one side of the direct repeats, and for its movement primarily in one direction away from these repeats to activate the initiation sites for DNA replication. (a new browser window will open) |
||||||||||||||||||||||||||||||||||
|
