Towards an integrated model of bacterial conjugation

Cabezón, Elena; Ripoll‐Rozada, Jorge; Peña, Alejandro; Cruz, Fernando de la; Aréchaga, Ignacio

doi:10.1111/1574-6976.12085

Cited by 187 publications

(215 citation statements)

References 199 publications

(233 reference statements)

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“…The T4CPs are a unique receptor superfamily associated with nearly all T4SSs (2,16). T4CPs fulfill the complex tasks of serving as docking sites for cognate DNA or protein substrates, energizing substrate transfer, and productively engaging with translocation channels (17,19,22,30,47,80,81).…”

Section: Discussionmentioning

confidence: 99%

“…One T4SS-associated ATPase, designated the type IV coupling protein (T4CP), was the focus of interest by virtue of a large body of evidence that T4CPs function as the substrate specificity checkpoints for cognate T4SSs (13)(14)(15)(16). T4CP monomers consist minimally of three distinct domains: (i) an N-terminal transmembrane domain (NTD) implicated in establishment of critical contacts with one or more subunits of the T4SS translocation channel; (ii) a conserved nucleotide-binding domain (NBD) that is thought to provide energy through ATP hydrolysis for substrate translocation; and (iii) a sequence-variable, all-alpha domain (AAD) that participates in DNA and possibly effector protein substrate recognition (Fig.…”

mentioning

confidence: 99%

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Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

Whitaker¹,

Berry²,

Rosenthal³

et al. 2016

J Bacteriol

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Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis. A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4 At ) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4 At and the TraJ/VirD4 At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ⌬CTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4 At 's CTD to the VirE2 effector, although other VirD4 At domains bound this substrate in vitro. We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCEBacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.T he type IV secretion systems (T4SSs) display striking versatility among the known bacterial translocation systems in their capacity to translocate DNA and protein substrates to bacterial or eukaryotic target cells (1, 2). Members of one major T4SS subfamily, the conjugation machines, mediate transfer of mobile DNA elements and are responsible for widespr...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

Whitaker¹,

Berry²,

Rosenthal³

et al. 2016

J Bacteriol

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“…While VirB4 together with VirB11 are required for pilus biogenesis and substrate translocation, VirD4 plays the role of a coupling protein, responsible for the recruitment of the substrate to the T4S system channel (Cabezon et al , 1997, 2015; Cascales & Christie, 2004). VirB11 has been reported as a hexamer in different systems (Machon et al , 2002; Savvides et al , 2003; Hare et al , 2006).…”

Section: Introductionmentioning

confidence: 99%

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

et al. 2017

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Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA. T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1‐11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1‐11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein–protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.

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“…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)(4)(5). Many Gram-negative pathogens additionally have adapted T4SSs for translocation of effector proteins to eukaryotic target cells during the course of infection (6,7).…”

mentioning

confidence: 99%

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

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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...

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Towards an integrated model of bacterial conjugation

Cited by 187 publications

References 199 publications

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery

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

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