The ATPase Activity of the DNA Transporter TrwB Is Modulated by Protein TrwA

Tato, Irantzu; Matilla, Inmaculada; Aréchaga, Ignacio; Zunzunegui, Sandra; Cruz, Fernando de la; Cabezón, Elena

doi:10.1074/jbc.m703464200

Cited by 71 publications

(32 citation statements)

References 35 publications

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“…These proteins, by their capacity to direct this interaction, are crucial for the specificity of mobilization. It has been shown with the IncW plasmid R388 that membrane protein TrwB is responsible for coupling the relaxosome with the DNA transport apparatus (6,47). In general, the coupling proteins are specific (31).…”

Section: Discussionmentioning

confidence: 99%

Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn 1549 , a Member of a New Family of Conjugative Elements

Tsvetkova¹,

Marvaud²,

Lambert³

2010

J Bacteriol

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Conjugative transfer from Clostridium symbiosum to enterococci of Tn1549, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (oriT) in the element. Transcription analysis of Tn1549 indicated that orf29, orf28, orfz, and orf27 were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to orf29-orf30 of Tn1549 was mobilized in Escherichia coli recA::RP4::⌬nic provided that orf28 and orf29 were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in trans, but a cis-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp oriT was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp oriT fragment. The minimal oriT contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOB p superfamily. The nick site was identified within oriT by an oligonucleotide cleavage assay. Closely related oriTs linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn1549 is a member of a MOB p clade. Interestingly, the Tn1549-derived constructs were mobilized by RP4 in E. coli, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn1549 to be mobilized by a heterologous transfer system.Tn1549 is a 34-kb transposon which confers vancomycin resistance in Enterococcus spp. (22). This entirely sequenced element, which harbors the vanB operon, is transferred passively among enterococci by conjugative plasmids or by the exchange of large chromosomal fragments (9,17,22). Tn1549 has also been reported in Gram-positive anaerobes (18, 46), and transfer from Clostridium symbiosum to Enterococcus has been demonstrated (28). This element is likely to be a conjugative transposon consisting of three functional modules involved in transposition, transfer, and antibiotic resistance. The transposition module encodes an excisionase and an integrase which cooperatively direct excision of the element. Excision is followed by circularization, resulting in a heteroduplex at the joined ends according to the tyrosine recombinase model of the Tn916 prototype. Analysis of Enterococcus transconjugants indicated that acquisition of VanB-type resistance was due to transposition of Tn1549 (28). However, it remains to be established if Tn1549 is a conjugative or a mobilizable transposon, since a single retransfer event has been obtained but in the presence of Tn916 (28). In both instances, whether active or passive transfer is being considered, presence of a transfer origin (oriT) is requi...

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Section: Discussionmentioning

confidence: 99%

Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn 1549 , a Member of a New Family of Conjugative Elements

Tsvetkova¹,

Marvaud²,

Lambert³

2010

J Bacteriol

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“…NTDs are thought to mediate the coupling of T4CPs with cognate T4SS channels (14,(44)(45)(46)(47). We therefore engineered T4CPs consisting of the NTD from TraJ pKM101 joined to ⌬NTD variants of VirD4 At , VirD4 Ap , and VirD4 Ap (Fig.…”

Section: Methodsmentioning

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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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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“…The structural similarity between TrwB and well known molecular motors, such as F 1 -ATPase (8) or T7 gene 4 helicase (9), suggests that TrwB also operates as a motor, using energy released from ATP hydrolysis to pump DNA through its central channel (10). Recent biochemical data indicated that TrwB ATPase activity can be activated by DNA and/or by protein TrwA, a tetrameric protein that binds specifically at the origin of transfer of the plasmid (oriT) and facilitates TrwB assembly on DNA (11). The TrwA-TrwB system resembles other DNA-dependent molecular motors, such as the RuvA-RuvB recombination system, that target specific DNA structures for assembly as hexamers to pump DNA (12).…”

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confidence: 99%

The Conjugative DNA Translocase TrwB Is a Structure-specific DNA-binding Protein

Matilla

Alfonso

Rivas

et al. 2010

Journal of Biological Chemistry

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TrwB is a DNA-dependent ATPase involved in DNA transport during bacterial conjugation. The protein presents structural similarity to hexameric molecular motors such as F 1 -ATPase, FtsK, or ring helicases, suggesting that TrwB also operates as a motor, using energy released from ATP hydrolysis to pump single-stranded DNA through its central channel. In this work, we have carried out an extensive analysis with various DNA substrates to determine the preferred substrate for TrwB. Oligonucleotides with G-rich sequences forming G4 DNA structures were the optimal substrates for TrwB ATPase activity. The protein bound with 100-fold higher affinity to G4 DNA than to single-stranded DNA of the same sequence. Moreover, TrwB formed oligomeric protein complexes only with oligonucleotides presenting such a G-quadruplex DNA structure, consistent with stoichiometry of six TrwB monomers to G4 DNA, as demonstrated by gel filtration chromatography and analytical ultracentrifugation experiments. A protein-DNA complex was also formed with unstructured oligonucleotides, but the molecular mass corresponded to one monomer protein bound to one oligonucleotide molecule. Sequences capable of forming G-quadruplex structures are widespread through genomes and are thought to play a biological function in transcriptional regulation. They form stable structures that can obstruct DNA replication, requiring the action of specific helicases to resolve them. Nevertheless, TrwB displayed no G4 DNA unwinding activity. These observations are discussed in terms of a possible role for TrwB in recognizing G-quadruplex structures as loading sites on the DNA.Bacterial plasmids are key players in bacterial evolution. Not only do they participate in the slow processes of speciation that shape bacterial genomes, but they also mediate the much more rapid and medically important processes of dissemination of antibiotic resistance and bacterial virulence (1). Among the three classical mechanisms of horizontal gene transfer (conjugation, transformation, and transduction), conjugation is undoubtedly the most salient, at least in many human pathogens, from Gram-negative Enterobacteriaceae and Pseudomonas to Gram-positive Enterococcus, to cite just three examples. Bacterial conjugative systems contain an essential integral membrane protein called the Type IV coupling protein (T4CP). This protein is involved in DNA transport, coupling the relaxosome (a DNA-protein complex) to the secretion channel (a multiprotein complex that spans the inner and outer membranes of the donor cell) (2, 3). TrwB, the T4CP of conjugative plasmid R388, is a DNA-dependent ATPase (4). It is a member of the FtsK/SpoIII family of DNA translocases (5, 6). The crystallographic structure of TrwB⌬N70, the cytoplasmic domain of TrwB, reveals a hexamer with a six-fold symmetry and a central channel of about 20 Å in diameter (7). The structural similarity between TrwB and well known molecular motors, such as F 1 -ATPase (8) or T7 gene 4 helicase (9), suggests that TrwB also operates as a motor, u...

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The ATPase Activity of the DNA Transporter TrwB Is Modulated by Protein TrwA

Cited by 71 publications

References 35 publications

Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn 1549 , a Member of a New Family of Conjugative Elements

Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn 1549 , a Member of a New Family of Conjugative Elements

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

The Conjugative DNA Translocase TrwB Is a Structure-specific DNA-binding Protein

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