The primase of broad-host-range plasmid R1162 is active in conjugal transfer

Henderson, Dorian; Meyer, Richard J.

doi:10.1128/jb.178.23.6888-6894.1996

Cited by 21 publications

(23 citation statements)

References 30 publications

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“…MobA is a multifunctional protein consisting of an N-terminal relaxase domain and a C-terminal DNA primase domain. Both domains are involved in vegetative plasmid replication (21) as well as in transfer replication (13). The RSF1010 relaxase family shows the conserved active-site tyrosine (Tyr-26 for TraA of pIP501) that is located at the very N terminus of the respective proteins, similar to the case for the other relaxase types.…”

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confidence: 90%

The tra Region of the Conjugative Plasmid pIP501 Is Organized in an Operon with the First Gene Encoding the Relaxase

et al. 2002

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The tra genes orf1 to orf11 of pIP501 were shown to be transcribed as a single operon of 11.3 kb in Enterococcus faecalis by reverse transcription-PCR. The transcriptional start site of the tra mRNA was mapped at 110 bp upstream from the predicted TTG start codon of the first gene of the operon, the traA relaxase. The TraA protein (660 amino acids) and a C-terminally truncated version of the TraA protein (293 amino acids) were purified as fusions with glutathione S-transferase. oriT cleavage activity of both TraA proteins was demonstrated in vitro on supercoiled plasmid pVA2241 DNA containing oriT pIP501 . The activity of the DNA relaxase TraA is strictly dependent on the presence of Mg 2؉ or Mn 2؉ and is highest at temperatures of between 42 and 45°C.pIP501 is a 30.2-kb Inc18 broad-host-range conjugative plasmid that was originally isolated from Streptococcus agalactiae. It confers resistance to macrolide, lincosamide, and streptogramin B antibiotics and to chloramphenicol. In addition to self-transfer to a wide variety of gram-positive genera, pIP501 is able to mobilize non-self-transmissible plasmids such as pMV158, the prototype of family 2 of rolling-circle-replicating plasmids (6). Krah and Macrina (16) postulated that the determinants conferring pIP501-mediated conjugation are encoded by two separate regions, a so-called region A and another region, region B, which has not been further characterized. Wang and Macrina (25) reported the nucleotide sequence of the complete 8,121-bp region A. Analysis of this DNA sequence revealed six contiguous open reading frames (orf1 to orf6) and an oriT site with high sequence identity to oriT regions of plasmids from gram-negative bacteria (Fig. 1). The oriT of pIP501 belongs to the RSF1010 family (27). An alignment of the nick regions of the RSF1010 family (modified from reference 27), including two more plasmids of grampositive origin, i.e., pGO1 from Staphylococcus aureus (5) and a new member, pRE25 from Enterococcus faecalis (GenBank accession no. X92945), is presented in Fig. 1. pRE25 is a 50.237-kb conjugative multiresistance plasmid whose putative tra region shows high overall identity with the tra region of pIP501 (B. Kurenbach and E. Grohmann, unpublished data).The precise nick site within oriT pIP501 was mapped by primer extension analysis on relaxed (open circular) pVA1857 DNA containing the entire tra region (26). To only one of the genes encoded by the pIP501 tra region A was a putative function ascribed. Wang and Macrina (26) demonstrated nicking of the oriT pIP501 site with plasmid pVA1860 carrying only oriT pIP501 and the traA gene, which gave rise to amounts of open circular plasmid DNA similar to those with pVA1857 containing the entire tra region.The six proteins encoded by tra region A of pIP501 show high overall similarity (between 24 and 36% identity over the whole amino acid sequence) with the gene products of the tra regions of the staphylococcal plasmids pGO1 (5) and pSK41 (3) and of the lactococcal conjugative plasmid pMRC01 (7). The TraA prote...

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The tra Region of the Conjugative Plasmid pIP501 Is Organized in an Operon with the First Gene Encoding the Relaxase

et al. 2002

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“…1) (27). This arrangement increases the frequency of mobilization, probably because the MobA-linked primase is delivered more efficiently to the replicative origin, where it initiates synthesis of the complement to the transferred strand (17).…”

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Identification of the mob Genes of Plasmid pSC101 and Characterization of a Hybrid pSC101-R1162 System for Conjugal Mobilization

Meyer

2000

J Bacteriol

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“…The larger form is a fusion product with one of the proteins for moblization, and we had shown earlier that the fusion increases the frequency of transfer under conditions where this transfer is inefficient (11). In the electroporation/transfer assay used in this project, only the larger form is active for transfer in the donor.…”

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confidence: 87%

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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The primase of broad-host-range plasmid R1162 is active in conjugal transfer

Cited by 21 publications

References 30 publications

The tra Region of the Conjugative Plasmid pIP501 Is Organized in an Operon with the First Gene Encoding the Relaxase

The tra Region of the Conjugative Plasmid pIP501 Is Organized in an Operon with the First Gene Encoding the Relaxase

Identification of the mob Genes of Plasmid pSC101 and Characterization of a Hybrid pSC101-R1162 System for Conjugal Mobilization

DNA Replication During Conjugal Transfer of R1162

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