Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer

Meyer, Richard J.

doi:10.1128/jb.171.2.799-806.1989

Cited by 39 publications

(51 citation statements)

References 21 publications

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“…Two simultaneous C-to-T transitions at bp 30 and 32 ( Fig. 1, line 2) affect the initiation of transfer but not the termination step modeled by the recombination of oniT copies in M13 (10,14). Deletion of bp 1 to 8, which make up the outer arm of the inverted repeat in oriT ( Fig.…”

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confidence: 99%

“…RSF1010 DNA has been isolated as a relaxosome (16); more recently, this complex has been reconstructed from plasmid DNA and purified proteins (18). The recircularization of plasmid DNA after transfer is modeled by the recombination of directly repeated oriT copies on replicating M13 phage DNA (1,14). In this system of recombination, it is likely that the singlestranded oriT copies are treated as intermediates in transfer and are therefore cleaved and ligated at the nic site.…”

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confidence: 99%

“…5). The standard conditions for the assay are described in detail elsewhere (14). The oriT copies interrupt the lacZa-coding sequence, and phage plaques are therefore white on lawns of JM103 in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-13-D-galactopyranoside) and IPTG (isopropyl-3-D-thiogalactopyranoside).…”

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confidence: 99%

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Role of the origin of transfer in termination of strand transfer during bacterial conjugation

1992

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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 oniTs, with transfer initiated at one and terminated at the other, the inverted repeat within oniT 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.The basic steps in DNA processing during conjugal transfer are probably similar for many different plasmids in gram-negative bacteria. One of the DNA strands is first nicked within the origin of transfer (oriT), which is required in cis. The nicked strand is then linearly transferred into a recipient cell in the 5'-to-3' direction and subsequently recircularized. In both donor and recipient cells, the complementary strand of DNA is resynthesized to restore the duplex plasmid molecule. Many different plasmids can be isolated in the form of a relaxosome, a complex of proteins and supercoiled plasmid DNA, which can be induced to undergo site-and strand-specific nicking within oriT (for example, see references 5, 6, 12, and 16). This nicking models the initial DNA-processing step in plasmid transfer.R1162 (nearly identical to RSF1010) is a broad-host-range, IncQ plasmid that is efficiently mobilized during the conjugal transfer of IncPl group plasmids (15,20). The R1162 oriT consists of no more than 38 bp of DNA (Fig.

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Role of the origin of transfer in termination of strand transfer during bacterial conjugation

1992

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“…Recombination between oriTs on single-stranded M13 phage is a measure of the single-strand cleavage and rejoining activity of MobA (23). We found that a MobA amino-terminal fragment extending to residue 179 in MobA was inactive in recombination, whereas a fragment extending to residue 204 was fully active (pUT208 and pUT209 in Fig.…”

Section: Discussionmentioning

confidence: 81%

MobA, the DNA Strand Transferase of Plasmid R1162

Becker¹,

Meyer

2002

Journal of Biological Chemistry

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MobA is a DNA strand transferase encoded by the plasmid R1162 and required for plasmid DNA processing during conjugal transfer. The smallest active fragment was identified using phage display and partial enzymatic digestion of the purified protein. This fragment, consisting of approximately the first 184 amino acids, is able to bind and cleave its normal DNA substrate, the origin of transfer (oriT). Smaller fragments having one of these activities were not obtained. An active intermediate consisting of MobA linked to DNA was isolated and used to show that a single molecule of MobA is sufficient to carry out all of the DNA processing steps during transfer. These results, along with those obtained earlier, point to a single large, active site in MobA that makes several different contacts along the oriT DNA strand.Prior to conjugal transfer of the broad-host-range plasmid R1162, three plasmid-encoded proteins assemble at a unique site, the origin of transfer (oriT), to form the relaxosome (1). One component of the relaxosome is MobA, a large (708-amino acid) protein that consists of two domains. The carboxyl-terminal region is a primase and is not required for the interaction of the protein with oriT (2, 3). The primase is also translated separately (2), and the gene for this protein probably became fused to mobA as a secondary adaptation that increased the frequency of transfer (4). In agreement with this, pSC101 as well as other plasmids contain MobA homologs lacking the primase domain (5).The principal DNA processing reactions carried out by MobA at initiation and termination of transfer are shown in Fig. 1. The amino-terminal region of MobA, a strand transferase consisting of about 250 amino acids, cleaves one of the DNA strands at oriT and forms a tyrosyl phosphodiester bond with the 5Ј end (1, 6). The cleavage reaction is reversible and does not result in the loss of plasmid superhelicity (1). The two ends of the cleaved strand are probably held together by MobA, which prevents relaxation of the plasmid DNA. In the cell, cleavage and rejoining of the oriT DNA strand might occur as an idling reaction, awaiting a hypothetical signal to direct the complex into a productive round of transfer.Actual DNA transfer involves the unwinding of the cleaved strand and its passage, in the 5Ј to 3Ј direction (7), through an intercellular pore by a transporting machine assembled at this site. At the end of this process, the two ends of the strand are rejoined, presumably by a second trans-esterification carried out by the covalently linked MobA. This reaction has not been demonstrated to occur in the cell, but in vitro MobA can both cleave and rejoin single-stranded oriT DNA (8). Moreover, the putative intermediate, MobA covalently linked to singlestranded DNA, is stable and able to rejoin the strands (this work).During both initial strand cleavage within the relaxosome and subsequent strand rejoining after a round of transfer, MobA probably interacts with an oriT DNA structure made up of both double-and single-stranded doma...

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“…An oriT mutation which inhibits nicking in the relaxosome has no effect on the termination of transfer at a second oriT (Kim and Meyer, 1989). In addition, oriTs cloned into single-stranded M13 phage DNA can undergo site-specific recombination at the normal cleavage site (Meyer, 1989). This reaction requires the relaxase, but not the other components of the relaxosome.…”

Section: Electroporation and Matingmentioning

confidence: 99%

DNA Replication During Conjugal Transfer of R1162

Meyer¹

2002

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Public Reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comment regarding this burden estimates or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for information Operations and Reports, 1215 Conjugative DNA transfer is an important mechanism for the acquisition of multiple drug resistance by bacteria. The role of DNA synthesis in this process was examined under conditions where vegetative replication was strictly prevented. There was more than one round of transfer from a donor cell containing a single plasmid molecule, indicating replacement strand synthesis by a conjugation-specific mechanism. A significant time lag between rounds of transfer was detected, arguing against the spooling of DNA into recipients by a rolling-circle replication. The plasmid-e needed DNA primase, synthesized as a fusion product with one of the proteins required for conjugative mobilization, was required in the donor for transfer, but the canonical priming sites for vegetative replication could be deleted. The endogenous priming system of the host could not substitute for the plasmid-encoded system in the absence of vegetaive replication. Overall, the results suggest a plasmid-dependent, cryptic priming system active during conjugative transfer.14. SUBJECT FINAL REPORT DAAD190110467 Forward:Bacterial conjugation involves the transfer of DNA from donor to recipient cell by a process requiring cell-to-cell contact. A pore is generated between the mating pair, and a single strand of DNA is passed from one cell to the other. Conjugation has been the subject of intense concern because it is the molecular mechanism most often responsible for the acquisition of multiple drug resistance by microorganisms. The rapid spread of these resistant bacteria dramatically decreases the useful lifetime of antibiotics and increases the mortality due to formerly treatable bacterial infections. Adding significantly to the problem, genes for resistance are often encoded by promiscuous plasmids able to cross species barriers and maintain themselves in a large number of different hosts. The intentional or inadvertent introduction of these plasmids into pathogenic organisms intended for biowarfare or terrorism can in general be easily accomplished, and their presence would obviously complicate both treatment and containment. Statement of Problem Studied:The long-term objective of this project has been to develop strategies for the containment of plasmids by interfering with the spread of these elements by conjugation. To this end, we have been investigating the mechanism of conjugation-specific DNA replication. This replication is distinct from the vegetative replication of plasmids, and understanding its molecular requirem...

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Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer

Cited by 39 publications

References 21 publications

Role of the origin of transfer in termination of strand transfer during bacterial conjugation

Role of the origin of transfer in termination of strand transfer during bacterial conjugation

MobA, the DNA Strand Transferase of Plasmid R1162

DNA Replication During Conjugal Transfer of R1162

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