Molecular Analysis of a Mobilizable Theta-Mode Trimethoprim Resistance Plasmid from Coagulase-Negative Staphylococci

Apisiridej, Sumalee; Leelaporn, Amornrut; Scaramuzzi, Carol D.; Skurray, Ronald A.; Firth, Neville

doi:10.1006/plas.1997.1292

Cited by 36 publications

(27 citation statements)

References 36 publications

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“…These contain the conserved motif suggested by Apisiridej et al (2) (L/FxxxG/SxNxNQxAxxxN), although we have not been able to assign a DNA-binding domain comparable to the functionally equivalent VirD1 (63) nor reveal significant sequence homology with the essential TraJ protein of RP4 (18). pSK639, pIP1629, and pIP1630 also encode an additional overlapping reading frame of ϳ64 aa between MobC and MobA with unknown function: this may serve to provide translational coupling from MobC to MobA within a single transcript.…”

Section: Discussionmentioning

confidence: 95%

An Accessory Protein Is Required for Relaxosome Formation by Small Staphylococcal Plasmids

Smith

Thomas

2004

J Bacteriol

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Mobilization of the staphylococcal plasmid pC221 requires at least one plasmid-encoded protein, MobA, in order to form a relaxosome. pC221 and closely related plasmids also possess an overlapping reading frame encoding a protein of 15 kDa, termed MobC. By completing the nucleotide sequence of plasmid pC223, we have found a further example of this small protein, and gene knockouts have shown that MobC is essential for relaxosome formation and plasmid mobilization in both pC221 and pC223. Primer extension analysis has been used to identify the nic site in both of these plasmids, located upstream of the mobC gene in the sense strand. Although the sequence surrounding the nic site is highly conserved between pC221 and pC223, exchange of the oriT sequence between plasmids significantly reduces the extent of relaxation complex formation, suggesting that the Mob proteins are selective for their cognate plasmids in vivo.The horizontal transfer of plasmid DNA by conjugative transfer requires two distinct processes: the formation of a mating pair between donor and recipient bacteria, and a series of DNA processing reactions to prepare the plasmid for transfer (45,65,66). The latter requires the formation of a protein-DNA complex at the origin of transfer (oriT), termed the relaxosome (10, 31), requiring a plasmid-encoded tyrosine transesterase (also referred to as a relaxase, or Mob protein) capable of introducing a single-stranded nick within oriT. This nick permits the transfer of single-stranded DNA into the recipient and may be captured by sodium dodecyl sulfate (SDS), alkali, heat, or protease treatment of the complex in vitro.Relaxases typically carry three conserved amino acid sequence motifs (23, 27) related to those required for rolling circle replication initiator (Rep) proteins: these contain (motif I) the sequence surrounding the active site tyrosine, which nicks the DNA and typically forms a phosphodiester linkage with the nucleotide at the 5Ј side of the nick; (motif II) a broadly hydrophobic region implicated in maintaining the stability of the trapped nick by the relaxosome (46); and (motif III) the histidine-hydrophobic-histidine motif responsible for binding the divalent metal cation essential for the cleavage process (22). Relaxases may be further classified into families based on both the amino acid sequence within these motifs and the nucleotide sequence surrounding the nick site: these include those related to the IncP plasmids such as TraI of RP4 (68) and VirD2 of the agrobacterial Ti plasmids (67); the IncQ plasmids such as MobA of RSF1010 and R1162 (54) and the Nes protein of staphylococcal pGO1 (13); the IncF/IncW family, including TraI of R1 and F (17) and TrwC of R388 (32); and relatives of the MobM protein of streptococcal plasmid pMV158 (48). Further characteristics are associated within these family groupings: for example, the IncQ-family MobA polypeptides include a primase activity which may be separated from the relaxase activity (7); and the IncF/IncW family TraI/ TrwC polypeptides includ...

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Section: Discussionmentioning

confidence: 95%

An Accessory Protein Is Required for Relaxosome Formation by Small Staphylococcal Plasmids

Smith

Thomas

2004

J Bacteriol

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“…A relaxase is responsible for initiation and completion of the transfer process, as it cleaves one strand of the double-stranded plasmid to begin transfer and then ligates the strands back together to complete transfer (8,(12)(13)(14)(15). There are two classes of relaxases: multityrosine relaxases, which use a "thumb" motif to position the plasmid DNA for processing, and single-tyrosine relaxases, which lack this thumb motif (9,16).…”

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confidence: 99%

“…pSK41-like conjugative plasmids have also been shown to mobilize several smaller coresident plasmids, such as pC221 and pSK639, which carry their own mob genes (9,12,13,15,16). Recently, O'Brien et al showed that another staphylococcal conjugative plasmid, pWBG749, which is unrelated to pSK41, can facilitate the mobilization of other plasmids that lack mob genes (17).…”

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Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

et al. 2016

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Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at the oriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, pSK156 and pCA347 are nonconjugative Staphylococcus aureus plasmids that contain sequences similar to the oriT region of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate the oriT sequences of these nonconjugative plasmids in vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognate oriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variant oriT mimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-like oriT. These data indicate that the conjugative relaxase in trans mechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids. IMPORTANCEUnderstanding the mechanism of antimicrobial resistance transfer in bacteria such as Staphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of the Staphylococcus aureus multiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variant oriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase in trans mechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.

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“…In contrast to relaxase alignments, the various accessory proteins align poorly between the plasmid groups and yet are often found encoded adjacent to the relaxase in plasmid mobilization regions in both gram-positive and gram-negative bacteria. A potential conserved motif within a number of MobC homologues from gram-positive and gram-negative bacteria has been identified (L/FxxxG/SxNxNQxAxxxN), although no function has yet been assigned (1).…”

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Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro

2004

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The isolation of plasmid-protein relaxation complexes from bacteria is indicative of the plasmid nickingclosing 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 Mg 2؉ or Mn 2؉ , 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 Mg 2؉ or Mn 2؉ 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.The transfer of plasmids between bacterial cells by conjugation is the result of two processes: mating-pair formation between the donor and recipient, and DNA processing reactions, which prepare the plasmid for transfer (38). In keeping with the generalized models of DNA processing based on gramnegative systems, conjugation can proceed only after a siteand strand-specific cleavage at a unique nick site (nic) within an origin of transfer (oriT). The transesterification reaction is catalyzed by a relaxase (transesterase) within a nucleoprotein complex, the relaxosome, in the presence or absence of accessory proteins. A single strand is unwound by a host or plasmidencoded helicase and transferred with a 5Ј-to-3Ј polarity to the recipient cell (15).The DNA processing components of several gram-negative conjugative and mobilizable plasmids, e.g., RP4, pTiC58, F, R388, and R1162/RSF1010 have been genetically and biochemically characterized in vitro. This facilitates the determination of the minimal components and confirms roles and specificities of factors otherwise identified in vivo (15,38). In contrast, conjugative and mobilizable plasmids of gram-positive bacteria are comparatively under-represented (8).Plasmids pC221 and pC223 are small (4.6 kb), low-copynumber chloramphenicol resistance plasmids and are mobilizable members of the rolling-circle replicating small staphylococcal plasmid family. They contain four genetic loci involved in their mobilization: the cis-acting locus origin of transfer (oriT) and the overlapping trans-acting loci mobC, mobA, and mobB ( Fig. 1) (33). An oriT region, functional in mobilization assays, has been broadly defined on an intergenic 692-bp AluI fragment (28, 36).Site-and strand-specific nicking of pC221 and pC223 in vivo requires mobA, mobC, and superhelical oriT, the positions of which have been determined (33). The nic regions share significant similarity with relaxase family 1 consensus nic region...

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Molecular Analysis of a Mobilizable Theta-Mode Trimethoprim Resistance Plasmid from Coagulase-Negative Staphylococci

Cited by 36 publications

References 36 publications

An Accessory Protein Is Required for Relaxosome Formation by Small Staphylococcal Plasmids

An Accessory Protein Is Required for Relaxosome Formation by Small Staphylococcal Plasmids

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro

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