Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro

Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Journal of Bacteriology (Impact Factor: 2.81). 07/2004; 186(11):3374-83. DOI: 10.1128/JB.186.11.3374-3383.2004
Source: PubMed


The isolation of plasmid-protein relaxation complexes from bacteria is indicative of the plasmid nicking-closing equilibrium
in vivo that serves to ready the plasmids for conjugal transfer. In pC221 and pC223, the components required for in vivo site-
and strand-specific nicking at oriT are MobC and MobA. In order to investigate the minimal requirements for nicking in the absence of host-encoded factors, the
reactions were reconstituted in vitro. Purified MobA and MobC, in the presence of Mg2+ or Mn2+, were found to nick at oriT with a concomitant phosphorylation-resistant modification at the 5′ end of nic. The position of nic is consistent with that determined in vivo. MobA, MobC, and Mg2+ or Mn2+ therefore represent the minimal requirements for nicking activity. Cross-complementation analyses showed that the MobC proteins
possess binding specificity for oriT DNA of either plasmid and are able to complement each other in the nicking reaction. Conversely, nicking by the MobA proteins
is plasmid specific. This suggests the MobA proteins may encode the nicking specificity determinant.

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    • "Thus, the conclusion that plasmid RCR is mechanistically similar to conjugative transfer was soon achieved (Waters and Guiney, 1993). It was interesting to learn that some of the RCR-plasmids encoded, in addition to the Rep topoisomerase-like initiator, another protein with the ability to relax DNA (Caryl et al., 2004; Grohmann et al., 2003; Guzmán and Espinosa, 1997; Smith and Thomas, 2004). These proteins were thought to be involved in inter-plasmidic recombination (plasmid recombination enzymes, Pre; (Projan and Novick, 1988)), although it was later shown that Pre proteins were required for plasmid mobilization (Priebe and Lacks, 1989). "
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    ABSTRACT: Rolling circle-replicating plasmids constitute a vast family that is particularly abundant in, but not exclusive of, Gram-positive bacteria. These plasmids are constructed as cassettes that harbor genes involved in replication and its control, mobilization, resistance determinants and one or two origins of lagging strand synthesis. Any given plasmid may contain all, some, or just only the replication cassette. We discuss here the family of the promiscuous streptococcal plasmid pMV158, with emphasis on its mobilization functions: the product of the mobM gene, prototype of the MOBV relaxase family, and its cognate origin of transfer, oriT. Amongst the subfamily of MOBV1 plasmids, three groups of oriT sequences, represented by plasmids pMV158, pT181, and p1414 were identified. In the same subfamily, we found four types of single-strand origins, namely ssoA, ssoU, ssoW, and ssoT. We found that plasmids of the rolling-circle Rep_2 family (to which pMV158 belongs) are more frequently found in Lactobacillales than in any other bacterial order, whereas Rep_1 initiators seemed to prefer hosts included in the Bacillales order. In parallel, MOBV1 relaxases associated with Rep_2 initiators tended to cluster separately from those linked to Rep_1 plasmids. The updated inventory of MOBV1 plasmids still contains exclusively mobilizable elements, since no genes associated with conjugative transfer (other than the relaxase) were detected. These plasmids proved to have a great plasticity at using a wide variety of conjugative apparatuses. The promiscuous recognition of non-cognate oriT sequences and the role of replication origins for lagging-strand origin in the host range of these plasmids are also discussed.
    Full-text · Article · Jun 2014 · Plasmid
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    • "These findings contrast with those observed with the highly identical oriT s plasmids pC221 and pC223, where relaxosome formation does not occur when their oriT regions are exchanged [Smith and Thomas, 2004]. Further studies with these two plasmids showed that the MobC protein encoded by each one is able to recognize and to bind to the oriT region of the other, but the MobA protein is not capable of doing the same, indicating that the binding specificity is determined by the relaxase [Caryl et al., 2004]. When comparing the sra sites of plasmids pC221 and pC223, three-base substitutions are found, which may account for the inability of the relaxase encoded by each one to recognize the oriT region of the other. "
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    ABSTRACT: Plasmid pRJ9 is a non-self-mobilizable bacteriocinogenic plasmid from Staphylococcus aureus. Despite this feature, DNA sequencing and RT-PCR experiments showed that it presents a Mob region with three genes (mobCAB), transcribed as an operon. In silico analysis of the Mob proteins encoded by pRJ9 showed that they present all the conserved functional features reported until present as being essential for plasmid mobilization. Moreover, they showed a high identity to Mob proteins encoded by mobilizable plasmids from Staphylococcus spp., especially to those encoded by plasmid pRJ6, which presents four mob genes (mobCDAB). A putative oriT region was also found upstream of the pRJ9 mob operon. pRJ9 could only be successfully mobilized by pGO1 when pRJ6 was present in the same strain. Further experiments showed that the pRJ9 oriT can be recognized by the pRJ6 Mob proteins, confirming its functionality. As pRJ9 does not possess a mobD gene while pRJ6 does, the absence of this gene was believed to be responsible for its lack of mobilization. However, conjugation experiments with a donor strain carrying also mobD cloned into an S. aureus vector showed that pRJ9 does not become mobilized even in the presence of the protein MobD encoded by pRJ6. Therefore, the reasons for pRJ9 failure to be mobilized are presently unknown.
    Full-text · Article · Jan 2011 · Journal of Molecular Microbiology and Biotechnology
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    • "As PcfF and LtrF only share 47% protein identity, amino acids involved in DNA recognition might be highly conserved in these two proteins. Interestingly, the two MobC proteins (73% identical) of pC221 and pC223 also are unable to distinguish between cognate and noncognate oriT DNA (Caryl et al., 2004). In contrast, the well-characterized RP4 accessory protein TraJ, which lacks the conserved (iGnNiNQiA) MobC motif, only interacts with its cognate oriT, and not with that of the closely related R751 (Furste et al., 1989). "
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    ABSTRACT: The DNA-processing region of the Enterococcus faecalis pheromone-responsive plasmid pCF10 is highly similar to that of the otherwise unrelated plasmid pRS01 from Lactococcus lactis. A transfer-proficient pRS01 derivative was unable to mobilize plasmids containing the pCF10 origin of transfer, oriT. In contrast, pRS01 oriT-containing plasmids could be mobilized by pCF10 at a low frequency. Relaxases PcfG and LtrB were both capable of binding to single-stranded oriT DNAs; LtrB was highly specific for its cognate oriT, whereas PcfG could recognize both pCF10 and pRS01 oriT. However, pcfG was unable to complement an ltrB insertion mutation. Genetic analysis showed that pcfF of pCF10 and ltrF of pRS01 are also essential for plasmid transfer. Purified PcfF and LtrF possess double-stranded DNA binding activities for the inverted repeat within either oriT sequence. PcfG and LtrB were recruited into their cognate F-oriT DNA complex through direct interactions with their cognate accessory protein. PcfG also could interact with LtrF when pCF10 oriT was present. In vivo cross-complementation analysis showed that ltrF partially restored the pCF10DeltapcfF mutant transfer ability when provided in trans, whereas pcfF failed to complement an ltrF mutation. Specificity of conjugative DNA processing in these plasmids involves both DNA-protein and protein-protein interactions.
    Preview · Article · Apr 2007 · Molecular Microbiology
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