Relaxosome function and conjugation regulation in F‐like plasmids – a structural biology perspective

Wong, Jillian W.; Lu, Jun; Glover, J. N. Mark

doi:10.1111/j.1365-2958.2012.08131.x

Cited by 80 publications

(63 citation statements)

References 138 publications

(225 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Structural studies have determined that the last 7 amino acids (V 712 EPGDDF 717 ) of TraD's CTD fit into a positively charged groove of TraM. Further mutational analyses of this partner interaction confirmed that the TraD CTD-TraM interaction is required for highly efficient F plasmid transfer and, conversely, is strongly inhibitory for F plasmid mobilization of MOBQ plasmids (68,69). These findings warrant further studies exploring the importance of the CTDs of PcfC pCF10 , VirD4 At , and other T4CPs that carry such domains for possible DNA or effector protein contacts.…”

Section: Discussionmentioning

confidence: 85%

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

et al. 2015

View full text Add to dashboard Cite

Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AAD PcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. IMPORTANCEFor conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are largely undefined. Here, we supply genetic and biochemical evidence that a helical bundle, designated the all-alpha domain (AAD), of T4SS receptors functions as a substrate specificity determinant. We show that AADs from two substrate receptors, Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC, bind DNA without sequence or strand preference but specifically bind the cognate relaxases responsible for nicking and piloting the transferred strand through the T4SS. We propose that interactions of receptor AADs with DNA-processing factors constitute a basis for selective coupling of mobile DNA elements with type IV secretion channels. Bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to target cells, generally by a mechanism requiring direct cell-to-cell contact (1). These systems are phylogenetically widely distributed among many Gram-negative and -positive bacterial species (2, 3). In both cell types, the T4SSs function as conjugation machines by delivering DNA substrates to bacterial recipient cells (3-5). Many G...

show abstract

Section: Discussionmentioning

confidence: 85%

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

et al. 2015

View full text Add to dashboard Cite

show abstract

“…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).…”

mentioning

confidence: 99%

“…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). The relaxase of pSK41 is termed the NES nicking enzyme in S. aureus, and it is a single-tyrosine relaxase (1-6, 8, 9).…”

mentioning

confidence: 99%

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

et al. 2016

View full text Add to dashboard Cite

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.

show abstract

“…During recent years, the way in which a complex network of plasmid and host encoded factors regulate tra gene expression in F-like plasmids has been intensively studied (Frost & Koraimann, 2010;Wong et al, 2012). Of note, all of the F-like plasmids characterized to some degree (such as R1, R100, pSLT, p307) carry a repressor system (FinOP) that disables expression of the tra operon in the majority of cells (.99 %) within a population.…”

Section: Introductionmentioning

confidence: 99%

Silencing and activating type IV secretion genes of the F-like conjugative resistance plasmid R1

et al. 2013

View full text Add to dashboard Cite

Expression of DNA transfer (tra) genes of F-type conjugative plasmids is required for the assembly of a functional type IV secretion machinery and subsequent plasmid DNA transfer from donor to recipient cells. Transcription of tra genes depends on the activation of a single promoter, designated P Y , by the plasmid encoded TraJ protein. We here determine plasmid specificity of TraJ proteins from various subgroups of F-like plasmids and find that plasmid R1 conjugation and P Y promoter activation can be achieved only by its cognate activator and by TraJ of the Salmonella plasmid pSLT and not by F or R100 TraJ proteins. In addition, we characterize the P Y promoter of plasmid R1. We show that TraJ binds to P Y DNA in vivo and that H-NS acts as a silencer of the P Y promoter. In the natural plasmid context, H-NS silences transfer gene expression and horizontal plasmid DNA transfer. In contrast to what was found for the F plasmid, lack of H-NS did not abolish the requirement for ArcA and TraJ to reach full tra gene expression and DNA transfer activity. We propose that, besides a passive de-silencing activity, both ArcA and TraJ play a direct role in synergistically stimulating tra operon transcription and subsequent DNA transfer.

show abstract

Relaxosome function and conjugation regulation in F‐like plasmids – a structural biology perspective

Cited by 80 publications

References 138 publications

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

Silencing and activating type IV secretion genes of the F-like conjugative resistance plasmid R1

Contact Info

Product

Resources

About