Search

Two B/O plasmids were sequenced. The kanamycin resistance plasmid R805a was found in a Salmonella Typhi strain from a 1972 typhoid fever outbreak in Mexico City. pCERC6, which confers resistance to ampicillin, streptomycin and sulphamethoxazole, was found in a commensal E. coli isolated from a healthy adult in Sydney in 2008. Both plasmids contain the same gene for RNAI, the incompatibility determinant, as the reference B/O plasmid pMU707 and the recently reported plasmid p838B-R, which came from a commensal E. coli isolated in Sydney in 2004. However, three different repA alleles are associated with the IncB/O rnaI. Whereas the repA gene of p838B-R is identical to that of pMU707, R805a and pCERC6 each carry a different repA. Comparison of the plasmid backbones revealed that R805a has a different oriT-nikAB region to that in pCERC6 and p838B-R, encoding different NikA relaxosome accessory factors and NikB relaxases. The different nikA genes were associated with oriT sites that differed in the sequences of their long, imperfect inverted repeats. A further 11 publicly available complete plasmid sequences contain the same B/O rnaI, with either the repA of pMU707/p838B-R or of R805a, and the oriT-nikAB region of p838B-R/pCERC6 or of R805a. All four possible combinations were seen. Extensive variation was also observed in the traY-excA entry exclusion region, and in the locations of mobile elements, including ones carrying antibiotic resistance genes. Plasmids that contain the same rnaI gene would be considered the same by a number of typing methods, but this study has unveiled previously unnoticed mosaicism in the backbones of one such group. This highlights the importance of considering entire plasmid backbones for the typing and tracking of specific lineages.

Bacterial type IV secretion systems (T4SSs) mediate the conjugative transfer of mobile genetic elements (MGEs) and their cargoes of antibiotic resistance and virulence genes. Here, we report that the pED208-encoded T4SS (TrapED208) translocates not only this F plasmid but several plasmid-encoded proteins, including ParA, ParB1, single-stranded DNA-binding protein SSB, ParB2, PsiB, and PsiA, to recipient cells. Conjugative protein translocation through the TrapED208 T4SS required engagement of the pED208 relaxosome with the TraD substrate receptor or coupling protein. T4SSs translocate MGEs as single-stranded DNA intermediates (T-strands), which triggers the SOS response in recipient cells. Transfer of pED208 deleted of psiB or ssb, which, respectively, encode the SOS inhibitor protein PsiB and single-stranded DNA-binding protein SSB, elicited a significantly stronger SOS response than pED208 or mutant plasmids deleted of psiA, parA, parB1, or parB2. Conversely, translocation of PsiB or SSB, but not PsiA, through the TrapED208 T4SS suppressed the mating-induced SOS response. Our findings expand the repertoire of known substrates of conjugation systems to include proteins with functions associated with plasmid maintenance. Furthermore, for this and other F-encoded Tra systems, docking of the DNA substrate with the TraD receptor appears to serve as a critical activating signal for protein translocation. Finally, the observed effects of PsiB and SSB on suppression of the mating-induced SOS response establishes a novel biological function for conjugative protein translocation and suggests the potential for interbacterial protein translocation to manifest in diverse outcomes influencing bacterial communication, physiology, and evolution. IMPORTANCE Many bacteria carry plasmids and other mobile genetic elements (MGEs) whose conjugative transfer through encoded type IV secretion systems (T4SSs), or “mating” channels, can lead to a rapid intra- and interspecies proliferation of genes encoding resistance to antibiotics or heavy metals or virulence traits. Here, we show that a model IncF plasmid-encoded T4SS translocates not only DNA but also several proteins intercellularly. The repertoire of translocated proteins includes the plasmidic SOS inhibitor protein PsiB, single-stranded DNA-binding protein SSB, and several partitioning proteins. We demonstrate that intercellular transmission of PsiB and SSB suppresses the SOS response, which is triggered in recipient cells upon acquisition of the single-stranded DNA transfer intermediate during mating. Our findings identify a new biological function for conjugative protein translocation in mitigating potentially deleterious consequences to plasmid and genome integrity resulting from SOS-induced recombination and mutation events.

23 p.-5 fig.-2 tab., Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site-and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named rel(LS20), is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the Rel(LS20) shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research., Work in the Meijer lab was funded by the Spanish government through grant Bio2013- 41489-P of the Ministry of Economy and Competitiveness, and through grant Bio2016- 77883-C2-1-P of the Ministry of Economy, Industry and Competitiveness; the former grant also funded AMA and CGC. The Spanish government also supported DRB, JRLO, and CA.DRB was funded by grant Bio2016-77883-C2-2-P of the Ministry of Economy, Industry and Competitiveness, and JRLO and CA were supported by grant BFU2014-52070-C2-2-P of the Ministry of Economy and Competitiveness to CA.LJW's work was supported by Wellcome Trust grant WT098374AIA to Jeff Errington.

Transfer of genetic material is the main mechanism underlying the spread of antibiotic resistance and virulence factors within the bacterial community. Conjugation is one such process by which the genetic material is shared from one bacterium to another. The DNA substrate is processed and prepared for transfer by a multi-protein complex called the relaxosome .The relaxosome of plasmid R1 possesses the most crucial enzyme TraI which, both nicks and unwinds the dsDNA substrate. TraI comprises 1765 residues and multiple functional domains, including those catalyzing the DNA trans-esterase (relaxase) on the dsDNA designated for a conjugative transfer and DNA helicase activities. Structural and functional studies have been reported for most of the TraI except the C-terminal domain spanning from residue 1630 to 1765. This region is the least understood part of TraI and is thought to be highly disordered and flexible. This region, being intrinsically disordered, is hypothesized to be serving as an interacting platform for other proteins involved in this DNA transfer initiation mechanism. In this work, we report the 1H, 13C, 15N resonance assignment of this region as well as the secondary structure information based on the backbone chemical shifts.

Dissemination of multiresistance has been accelerating among pathogenic bacteria in recent decades. The broad host-range conjugative plasmids of the IncA/C family are effective vehicles of resistance determinants in Gram-negative bacteria. Although more than 150 family members have been sequenced to date, their conjugation system and other functions encoded by the conserved plasmid backbone have been poorly characterized. The key cis-acting locus, the origin of transfer (oriT), has not yet been unambiguously identified. We present evidence that IncA/C plasmids have a single oriT locus immediately upstream of the mobI gene encoding an indispensable transfer factor. The fully active oriT spans ca. 150-bp AT-rich region overlapping the promoters of mobI and contains multiple inverted and direct repeats. Within this region, the core domain of oriT with reduced but detectable transfer activity was confined to a 70-bp segment containing two inverted repeats and one copy of a 14-bp direct repeat. In addition to oriT, a second locus consisting of a 14-bp imperfect inverted repeat was also identified, which mimicked the function of oriT but which was found to be a recombination site. Recombination between two identical copies of these sites is RecA-independent, requires a plasmid-encoded recombinase and resembles the functioning of dimer-resolution systems.

Conjugation is the process by which bacteria exchange genetic materials in a unidirectional manner from a donor cell to a recipient cell. The discovery of conjugation signalled the dawn of genetics and molecular biology. In Gram‐negative bacteria, the process of conjugation is mediated by a large membrane‐embedded machinery termed “conjugative type IV secretion (T4S) system”, a large injection nanomachine, which together with a DNA‐processing machinery termed “the relaxosome” and a large extracellular tube termed “pilus” orchestrates directional DNA transfer. Here, the focus is on past and latest research in the field of conjugation and T4S systems in Gram‐negative bacteria, with an emphasis on the various questions and debates that permeate the field from a mechanistic perspective.

During conjugative plasmid transfer, one plasmid strand is cleaved and transported to the recipient bacterium. For F and related plasmids, TraI contains the relaxase or nickase activity that cleaves the plasmid DNA strand. F TraI36, the F TraI relaxase domain, binds a single-stranded origin of transfer (oriT) DNA sequence with high affinity and sequence specificity. The TraI36 domain from plasmid R100 shares 91% amino acid sequence identity with F TraI36, but its oriT DNA binding site differs by two of eleven bases. Both proteins readily distinguish between F and R100 binding sites. In earlier work, two amino acid substitutions in the DNA binding cleft were shown to be sufficient to change the R100 TraI36 DNA-binding specificity to that of F TraI36. In contrast, three substitutions could make F TraI36 more "R100-like", but failed to completely alter the specificity. Here we identify one additional amino acid substitution that completes the specificity swap from F to R100. To our surprise, adding further substitutions from R100 to the F background were detrimental to binding instead of being neutral, indicating that their effects were influenced by their structural context. These results underscore the complex and subtle nature of DNA recognition by relaxases and have implications for the evolution of relaxase binding sites and oriT sequences.

Conjugation, the process by which plasmid DNA is transferred from one bacterium to another, is mediated by type IV secretion systems (T4SSs). T4SSs are versatile systems that can transport not only DNA, but also toxins and effector proteins. Conjugative T4SSs comprise 12 proteins named VirB1-11 and VirD4 that assemble into a large membrane-spanning exporting machine. Before being transported, the DNA substrate is first processed on the cytoplasmic side by a complex called the relaxosome. The substrate is then targeted to the T4SS for export into a recipient cell. In this review, we describe the recent progress made in the structural biology of both the relaxosome and the T4SS.

Bacterial conjugation is the process by which a conjugative element (CE) is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer (oriT), where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis. We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1LS20 and aux2LS20, and which we show are essential for conjugation. Both Aux1LS20 and Aux2LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1LS20 and Aux2LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriTLS20, although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1LS20 and/or Aux2LS20 are located upstream of almost 400 relaxase genes of the RelLS20 family (MOBL) of relaxases. Thus, Aux1LS20 and Aux2LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

Summary Relaxases play essential roles in conjugation, the main process by which bacteria exchange genetic material, notably antibiotic resistance genes. They are bifunctional enzymes containing a trans-esterase activity, which is responsible for nicking the DNA strand to be transferred and for covalent attachment to the resulting 5′-phosphate end, and a helicase activity, which is responsible for unwinding the DNA while it is being transported to a recipient cell. Here we show that these two activities are carried out by two conformers that can both load simultaneously on the origin of transfer DNA. We solve the structure of one of these conformers by cryo electron microscopy to near-atomic resolution, elucidating the molecular basis of helicase function by relaxases and revealing insights into the mechanistic events taking place in the cell prior to substrate transport during conjugation., Graphical Abstract, Highlights • Relaxases process and transfer plasmid DNAs during conjugation • The TraI relaxase binds the origin of transfer DNA as a dimer • TraI can adopt a closed and open conformation, and both co-exist in the dimer • The presented ssDNA-bound TraI structure reveals the closed “helicase” conformation, To achieve genetic exchange during bacterial conjugation, two relaxase monomers collaborate, adopting distinct structural conformations to provide the two necessary enzymatic activities for processing the DNA.

Background: The complement of factors involved in mobilization of the Helicobacter pylori disease-associated tfs4 genomic island are presently unknown. Results: tfs4 encodes a VirD2-like relaxase with distinctive DNA binding and nicking activity. Conclusion: Tfs4 VirD2 probably initiates mobilization of tfs4 by specific interaction at a chromosomal transfer origin sequence. Significance: Tfs4 VirD2-mediated mobilization of tfs4 may increase pathogenic potential of H. pylori strains., Four different type IV secretion systems are variously represented in the genomes of different Helicobacter pylori strains. Two of these, encoded by tfs3 and tfs4 gene clusters are contained within self-transmissible genomic islands. Although chromosomal excision of tfs4 circular intermediates is reported to be dependent upon the function of a tfs4-encoded XerD tyrosine-like recombinase, other factors required for transfer to a recipient cell have not been demonstrated. Here, we characterize the functional activity of a putative tfs4-encoded VirD2-like relaxase protein. Tfs4 VirD2 was purified as a fusion to maltose-binding protein and demonstrated to bind and nick both supercoiled duplex DNA and oligonucleotides in vitro in a manner dependent upon the presence of Mg2+ but independently of any auxiliary proteins. Unusually, concentration-dependent nicking of duplex DNA appeared to require only transient protein-DNA interaction. Although phylogenetically distinct from established relaxase families, site-specific cleavage of oligonucleotides by Tfs4 VirD2 required the nick region sequence 5′-ATCCTG-3′ common to transfer origins (oriT) recognized by MOBP conjugative relaxases. Cleavage resulted in covalent attachment of MBP-VirD2 to the 5′-cleaved end, consistent with conventional relaxase activity. Identification of an oriT-like sequence upstream of tfs4 virD2 and demonstration of VirD2 protein-protein interaction with a putative VirC1 relaxosome component indicate that transfer initiation of the tfs4 genomic island is analogous to mechanisms underlying mobilization of other integrated mobile elements, such as integrating conjugative elements, requiring site-specific targeting of relaxase activity to a cognate oriT sequence.

Summary Cointegrate plasmids are useful models for the study of plasmid evolution if their evolutionary processes can be replicated under laboratory conditions. pBMB0228, a 17 706 bp native plasmid originally isolated from Bacillus thuringiensis strain YBT-1518, carries two nematicidal crystal protein genes, cry6Aa and cry55Aa. In this study, we show that pBMB0228 is in fact a cointegrate of two plasmids and contains two functional replication regions and two functional mobilization regions. Upon introduction into B. thuringiensis strain BMB171, pBMB0228 spontaneously resolves into two constituent plasmids via recombination at its oriT1 and oriT2 sites. The resolution does not require conjugation but can be promoted by conjugation. We further confirm that the resolution is mediated by oriT site-specific recombination requiring Mob02281 or Mob02282. Additionally, the two constituent plasmids of pBMB0228 are mobilizable, and can fuse back via oriT site-specific integration after entering into the same cell by conjugation. Our study confirms that native plasmid can reversibly interconvert between a cointegrate structure and its constituent plasmids. This study provides insight into the evolution of cointegrate plasmids, linking plasmid evolution with conjugation and the oriT site-specific recombination function of relaxase.

NAH7 and pWW0 from gammaproteobacterial Pseudomonas putida strains are IncP-9 conjugative plasmids that carry the genes for degradation of naphthalene and toluene, respectively. Although such genes on these plasmids are well-characterized, experimental investigation of their conjugation systems remains at a primitive level. To clarify these conjugation systems, in this study, we investigated the NAH7-encoded conjugation system by (i) analyzing the origin of its conjugative transfer ( oriT )-containing region and its relaxase, which specifically nicks within the oriT region for initiation of transfer, and (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 oriT ( oriT N ) region was located within a 430-bp fragment, and the strand-specific nicking ( nic ) site and its upstream sequences that were important for efficient conjugation in the oriT N region were identified. Unlike many other relaxases, the NAH7 relaxase exhibited unique features in its ability to catalyze, in a conjugation-independent manner, the site-specific intramolecular recombination between two copies of the oriT N region, between two copies of the pWW0 oriT ( oriT W ) region (which is clearly different from the oriT N region), and between the oriT N and oriT W regions. The pWW0 relaxase, which is also clearly different from the NAH7 relaxase, was strongly suggested to have the ability to conjugatively and efficiently mobilize the oriT N -containing plasmid. Such a plasmid was, in the presence of the NAH7Δ nic derivative, conjugatively transferable to alphaproteobacterial and betaproteobacterial strains in which the NAH7 replication machinery is nonfunctional, indicating that the NAH7 conjugation system has a broader host range than its replication system. IMPORTANCE Various studies have strongly suggested an important contribution of conjugative transfer of catabolic plasmids to the rapid and wide dissemination of the plasmid-loaded degradation genes to microbial populations. Degradation genes on such plasmids are often loaded on transposons, which can be inserted into the genomes of the recipient bacterial strains where the transferred plasmids cannot replicate. The aim was to advance detailed molecular knowledge of the determinants of host range for plasmids. This aim is expected to be easily and comprehensively achieved using an experimental strategy in which the oriT region is connected with a plasmid that has a broad host range of replication. Using such a strategy in this study, we showed that (i) the NAH7 oriT -relaxase system has unique properties that are significantly different from other well-studied systems and (ii) the host range of the NAH7 conjugation system is broader than previously thought.

All plasmids that spread by conjugative transfer encode a relaxase. That includes plasmids that encode the type IV secretion machinery necessary to mediate cell to cell transfer, as well as mobilizable plasmids that exploit the existence of other plasmids' type IV secretion machinery to enable their own lateral spread. Relaxases perform key functions in plasmid transfer by first binding to their cognate plasmid as part of a multiprotein complex called the relaxosome, which is then specifically recognized by a receptor protein at the opening of the secretion channel. Relaxases catalyze a site- and DNA-strand-specific cleavage reaction on the plasmid then pilot the single strand of plasmid DNA through the membrane-spanning type IV secretion channel as a nucleoprotein complex. In the recipient cell, relaxases help terminate the transfer process efficiently and stabilize the incoming plasmid DNA. Here, we review the well-studied MOBF family of relaxases to describe the biochemistry of these versatile enzymes and integrate current knowledge into a mechanistic model of plasmid transfer in Gram-negative bacteria., Work in the authors’ laboratories was supported by Austrian Science Fund (FWF) grants P24016 and W901 DK Molecular Enzymology (ELZ) and BioTechMed-Graz (ELZ) and by the Spanish Ministry of Economy and Competitiveness grants BFU2014-55534-C2-1-P (FdlC) and BFU2014-55534-C2-2-P (GM).

Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination.

During conjugation, a single strand of DNA is cleaved at the origin of transfer ( oriT ) by the plasmid-encoded relaxase. This strand is then unwound from its complement and transferred in the 5′-to-3′ direction, with the 3′ end likely extended by rolling-circle replication. The resulting, newly synthesized oriT must then be cleaved as well, prior to recircularization of the strand in the recipient. Evidence is presented here that the R1162 relaxase contains only a single nucleophile capable of cleaving at oriT , with another molecule therefore required to cleave at a second site. An assay functionally isolating this second cleavage shows that this reaction can take place in the donor cell. As a result, there is a flux of strands with free 3′ ends into the recipient. These ends are susceptible to degradation by exonuclease I. The degree of susceptibility is affected by the presence of an uncleaved oriT within the strand. A model is presented where these internal oriT s bind and trap the relaxase molecule covalently bound to the 5′ end of the incoming strand. Such a mechanism would result in the preferential degradation of transferred DNA that had not been properly cleaved in the donor.

Summary The tra operon of the prototypical F plasmid and its relatives enables transfer of a copy of the plasmid to other bacterial cells via the process of conjugation. Tra proteins assemble to form the transferosome, the transmembrane pore through which the DNA is transferred, and the relaxosome, a complex of DNA-binding proteins at the origin of DNA transfer. F-like plasmid conjugation is characterized by a high degree of plasmid specificity in the interactions of tra components, and is tightly regulated at the transcriptional, translational and post-translational levels. Over the past decade, X-ray crystallography of conjugative components has yielded insights into both specificity and regulatory mechanisms. Conjugation is repressed by FinO, an RNA chaperone which increases the lifetime of the small RNA, FinP. Recent work has resulted in a detailed model of FinO/FinP interactions and the discovery of a family of FinO-like RNA chaperones. Relaxosome components include TraI, a relaxase/helicase, and TraM, which mediates signalling between the transferosome and relaxosome for transfer initiation. The structures of TraI and TraM bound to oriT DNA reveal the basis of specific recognition of DNA for their cognate plasmid. Specificity also exists in TraI and TraM interactions with the transferosome protein TraD.

Integrative and conjugative elements (ICEs, also known as conjugative transposons) are mobile elements that are found integrated in a host genome and can excise and transfer to recipient cells via conjugation. ICEs and conjugative plasmids are found in many bacteria and are important agents of horizontal gene transfer and microbial evolution. Conjugative elements are capable of self-transfer and also capable of mobilizing other DNA elements that are not able to self-transfer. Plasmids that can be mobilized by conjugative elements are generally thought to contain an origin of transfer ( oriT ), from which mobilization initiates, and to encode a mobilization protein (Mob, a relaxase) that nicks a site in oriT and covalently attaches to the DNA to be transferred. Plasmids that do not have both an oriT and a cognate mob are thought to be nonmobilizable. We found that Bacillus subtilis carrying the integrative and conjugative element ICE Bs1 can transfer three different plasmids to recipient bacteria at high frequencies. Strikingly, these plasmids do not have dedicated mobilization- oriT functions. Plasmid mobilization required conjugation proteins of ICE Bs1 , including the putative coupling protein. In contrast, plasmid mobilization did not require the ICE Bs1 conjugative relaxase or cotransfer of ICE Bs1 , indicating that the putative coupling protein likely interacts with the plasmid replicative relaxase and directly targets the plasmid DNA to the ICE Bs1 conjugation apparatus. These results blur the current categorization of mobilizable and nonmobilizable plasmids and indicate that conjugative elements play a role in horizontal gene transfer even more significant than previously recognized.

Highlights ► Mechanism of protein transfer by plasmid R1 conjugative T4 system requires relaxosome. ► Protein translocation depends on concomitant DNA transfer. ► Required functions of T4 coupling protein correlate for protein and DNA transfer., Bacterial conjugation disseminates genes among bacteria via a process requiring direct cell contact. The cell envelope spanning secretion apparatus involved belongs to the type IV family of bacterial secretion systems, which transport protein as well as nucleoprotein substrates. This study aims to understand mechanisms leading to the initiation of type IV secretion using conjugative plasmid paradigm R1. We analyze the general requirements for plasmid encoded conjugation proteins and DNA sequence within the origin of transfer (oriT) for protein secretion activity using a Cre recombinase reporter system. We find that similar to conjugative plasmid DNA strand transfer, activation of the R1 system for protein secretion depends on binding interactions between the multimeric, ATP-binding coupling protein and the R1 relaxosome including an intact oriT. Evidence for DNA independent protein secretion was not found.

Bacteria communicate with each other through contact-independent and-dependent signaling mechanisms. Sensory perception of both types of signals is needed for conjugative transfer of mobile DNA elements via type IV secretion systems (T4SSs) to bacterial or eukaryotic target cells. While the regulatory circuitries coupling extracellular quorum and environmental signals to transcription of T4SS genes are increasingly understood, it remains fundamentally unknown how a potential recipient cell stimulates donor conjugative DNA transfer upon contact. In this issue, Zechner and colleagues report use of the male-specific bacteriophage R17, a phage that binds conjugative pili elaborated by IncF plasmid R1, to define requirements for phage-contact-mediated T4SS activation and phage penetration. They report that R17 penetrates only through T4SS channels engaged for delivery of their plasmid cargo to recipient cells. Engagement requires docking of catalytically-active relaxase TraI bound at oriT with the TraD substrate receptor (also termed the T4CP). The data, together with recent ultrastructural and biochemical findings, support an intriguing new model that the T4CP cumulatively senses an intracellular signal (substrate docking) and an extracellular signal (pilus bound by phage or a recipient cell) to coordinate a late stage morphogenetic or gating reaction that enables bidirectional transmission of nucleoprotein substrates through the T4SS.

Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV couplingprotein (T4CP). TrwB is the T4CP of the conjugative plasmid R388. In earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate. In contrast a soluble truncated mutant TrwBΔN70 binds uniformly all nucleotides tested. In this work, TrwB has been successfully reconstituted into liposomes. The non-membranous portion of the protein is almost exclusively oriented towards the outside of the vesicles. Functional analysis of TrwB proteoliposomes demonstrates that when the protein is inserted into the lipid bilayer the affinity for adenine and guanine nucleotides is enhanced as compared to that of the protein purified in detergent or to the soluble deletion mutant, TrwBΔN70. The proteinspecificity for adenine nucleotides is also increased. No ATPase activity has been found in TrwB reconstituted in proteoliposomes. This result suggests that the N-terminal transmembrane segment of this T4CP interferes with its ATPase activity and can be taken to imply that the TrwB transmembrane domain plays a regulatory role in its biological activity., This work was supported with funds from the Spanish Ministerio de Educación y Ciencia (grant no. BFU2007-62062), from the Diputación Foral de Bizkaia (DIPE07/16) and from LSHM-CT-2005_019023 (European VI Framework Program). R.L.S. was a postdoctoral scientist supported by a CSIC I3P postdoctoral fellowship. A.J.V. and S.A. were pre-doctoral students supported by the Basque Government; B.U.U. was pre-doctoral student supported by the University of the Basque Country.

Bacteroides are Gram-negative anaerobes indigenous to the intestinal tract of humans, and they are important opportunistic pathogens. Mobile genetic elements, such as conjugative transposons (CTns), have contributed to an increase in antibiotic resistance in these organisms. CTns are self-transmissible elements that belong to the superfamily of i ntegrative and c onjugative e lements (ICEs). CTn 341 is 52 kb; it encodes tetracycline resistance and its transfer is induced by tetracycline. The mobilization region of CTn 341 was shown to be comprised of a three-gene operon, mobABC , and the transfer origin, oriT . The three genes code for a nicking accessory protein, a relaxase, and a VirD4-like coupling protein, respectively. The Mob proteins were predicted to mediate the formation of the relaxosome complex, nick DNA at the oriT , and shuttle the DNA/protein complex to the mating-pore apparatus. The results of mutational studies indicated that the three genes are required for maximal transfer of CTn 341 . Mob gene transcription was induced by tetracycline, and this regulation was mediated through the two-component regulatory system, RteAB. The oriT region of CTn 341 was located within 100 bp of mobA , and a putative Bacteroides consensus nicking site was observed within this region. Mutation of the putative nick site resulted in a loss of transfer. This study demonstrated a role of the mobilization region for transfer of Bacteroides CTns and that tetracycline induction occurs for the mob gene operon, as for the tra gene operon(s), as shown previously.

The mechanisms controlling progression of conjugative DNA processing from a preinitiation stage of specific plasmid strand cleavage at the transfer origin to a stage competent for unwinding the DNA strand destined for transfer remain obscure. Linear heteroduplex substrates containing double-stranded DNA binding sites for plasmid R1 relaxosome proteins and various regions of open duplex for TraI helicase loading were constructed to model putative intermediate structures in the initiation pathway. The activity of TraI was compared in steady-state multiple turnover experiments that measured the net production of unwound DNA as well as transesterase-catalyzed cleavage at nic . Helicase efficiency was enhanced by the relaxosome components TraM and integration host factor. The magnitude of stimulation depended on the proximity of the specific protein binding sites to the position of open DNA. The cytoplasmic domain of the R1 coupling protein, TraDΔN130, stimulated helicase efficiency on all substrates in a manner consistent with cooperative interaction and sequence-independent DNA binding. Variation in the position of duplex opening also revealed an unsuspected autoinhibition of the unwinding reaction catalyzed by full-length TraI. The activity reduction was sequence dependent and was not observed with a truncated helicase, TraIΔN308, lacking the site-specific DNA binding transesterase domain. Given that transesterase and helicase domains are physically tethered in the wild-type protein, this observation suggests that an intramolecular switch controls helicase activation. The data support a model where protein-protein and DNA ligand interactions at the coupling protein interface coordinate the transition initiating production and uptake of the nucleoprotein secretion substrate.

TraI relaxase-helicase is the central catalytic component of the multiprotein relaxosome complex responsible for conjugative DNA transfer (CDT) between bacterial cells. CDT is a primary mechanism for the lateral propagation of microbial genetic material, including the spread of antibiotic resistance genes. The 2.4-{angstrom} resolution crystal structure of the C-terminal domain of the multifunctional Escherichia coli F (fertility) plasmid TraI protein is presented, and specific structural regions essential for CDT are identified. The crystal structure reveals a novel fold composed of a 28-residue N-terminal {alpha}-domain connected by a proline-rich loop to a compact {alpha}/{beta}-domain. Both the globular nature of the {alpha}/{beta}-domain and the presence as well as rigidity of the proline-rich loop are required for DNA transfer and single-stranded DNA binding. Taken together, these data establish the specific structural features of this noncatalytic domain that are essential to DNA conjugation.

F plasmid-mediated bacterial conjugation requires interactions between a relaxosome component, TraM, and the coupling protein TraD, a hexameric ring ATPase that forms the cytoplasmic face of the conjugative pore. Here we present the crystal structure of the C-terminal tail of TraD bound to the TraM tetramerization domain, the first structural evidence of relaxosome-coupling protein interactions. The structure reveals the TraD C-terminal peptide bound to each of four symmetry-related grooves on the surface of the TraM tetramer. Extensive protein-protein interactions were observed between the two proteins. Mutational analysis indicates that these interactions are specific and required for efficient F conjugation in vivo. Our results suggest that specific interactions between the C-terminal tail of TraD and the TraM tetramerization domain might lead to more generalized interactions that stabilize the relaxosome-coupling protein complex in preparation for conjugative DNA transfer.

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence ( oriT ). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCΔN103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCΔN103 bound pCF10 but not pcfG or Δ oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCΔN103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

The TraM protein encoded by plasmid R1 has three functions in conjugative transfer: (i) it positively controls transfer gene expression, (ii) it stimulates efficient site-specific ssDNA cleavage at the oriT in vivo and (iii) it couples the relaxosome to the envelope-bound transport complex. Plasmid R1 contains two binding regions for TraM, sbmA and sbmB, either of which comprises several minimal TraM targets. SELEX of a randomized minimal (18 bp) TraM binding sequence was used to search for sequences that bind TraM most efficiently. This approach resulted in a sequence with a modular structure with two 7 bp consensus motifs 5' T1G2A3N4T5C6R7 and 5' T1G2A3N4T5Y6R7 spaced by a 4 bp linker with the consensus sequence 5' Y1A2/C2T3/G3A4. EMSAs revealed that evolution in vivo was for maximal binding affinity. R1 derivatives with mutated sbmA and sbmB sites based on one in vitro selected sequence were found to be either 5-fold impaired (mut sbmA) in conjugation or could not be transferred by conjugation anymore (mut sbmB). Transcriptional lacZ fusions demonstrated that the introduced sbmB mutations affected promoter activity severely and abrogated autoregulation by TraM, thereby explaining this drastic effect on conjugal transfer. The implications of these findings are discussed in the context of the role of TraM.

Integrative and conjugative elements (ICEs), also known as conjugative transposons, are mobile genetic elements that can transfer from one bacterial cell to another by conjugation. ICE Bs1 is integrated into the trnS-leu2 gene of Bacillus subtilis and is regulated by the SOS response and the RapI-PhrI cell-cell peptide signaling system. When B. subtilis senses DNA damage or high concentrations of potential mating partners that lack the element, ICE Bs1 excises from the chromosome and can transfer to recipients. Bacterial conjugation usually requires a DNA relaxase that nicks an origin of transfer ( oriT ) on the conjugative element and initiates the 5′-to-3′ transfer of one strand of the element into recipient cells. The ICE Bs1 ydcR ( nicK ) gene product is homologous to the pT181 family of plasmid DNA relaxases. We found that transfer of ICE Bs1 requires nicK and identified a cis -acting oriT that is also required for transfer. Expression of nicK leads to nicking of ICE Bs1 between a GC-rich inverted repeat in oriT , and NicK was the only ICE Bs1 gene product needed for nicking. NicK likely mediates conjugation of ICE Bs1 by nicking at oriT and facilitating the translocation of a single strand of ICE Bs1 DNA through a transmembrane conjugation pore.

Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBΔN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBΔN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBΔN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly.

Whole-genome transcriptional profiling experiments were performed to identify the complete set of TraR-regulated genes in isogenic A. tumefaciens strains containing an octopine-type or nopaline-type Ti plasmid. Most of the known TraR-regulated genes as well as a number of new inducible genes were identified. Surprisingly, some known members of this regulon showed both weaker induction and weak levels of expression than we had predicted based upon earlier studies. In particular, traA was expressed at surprisingly weak levels. Genetic analysis showed that the traAFBH operon is repressed by formation of a putative relaxosome at oriT consisting the TraA, TraC and TraD. These proteins also repressed the divergent traCDGyci operon. TraA was essential for oriT processing, and both TraC and TraD were necessary for the efficient processing, although some processing occurred in their absence. Likewise, Ti plasmid conjugation required TraA, TraF and TraG, and occurred at reduced levels in the absence of TraC or TraD. TraA preferentially acted in cis in repressing the traA and traC promoters and in the processing of oriT, which explains the very high activity of plasmid-borne traA-lacZ fusions reported in previous studies.