Why bacterial conjugation

VirB10 is the largest protein in the core complex, spanning the inner and outer membranes and the periplasmic space Jakubowski et al. VirB10 shares structural similarities with TonB-like proteins Cascales and Christie, a , such as a common bitopic membrane topology and a prolin-rich extended region in the periplasm Evans et al.

These structural features were later confirmed by the crystal structure of the periplasmic domain residues of ComB, the VirB10 homologue in H. In this crystal structure, the VirB10 periplasmic domain was shown to form dimers, confirming data obtained by two-hybrid experiments Ding et al. Therefore, given that there are 14 copies of VirB10 in the core structure Fronzes et al. However, the subunit packing observed in this structure, with the N-termini of each monomer at opposite ends, is not compatible with that observed in the core complex structure.

Such a small size implies that any protein substrate crossing the channel should be in an unfolded state, unless a large aperture of the channel takes place. Interestingly, the crystal structure of the outer membrane part of the core complex, formed by 14 copies of VirB7, VirB9, and the C-terminal domain of VirB10 Chandran et al.

Therefore, the EM and the crystal structures significantly differ in the inner diameter dimensions of the pore. Comparison of the outer membrane cap crystal structure Chandran et al. This conformational change could be due to the fact that the cap crystal structure was obtained after removal by proteolysis of the N-terminal half of VirB In any case, it seems that VirB10 N-terminus plays an essential role in the transmission of the signal from the inner membrane to the rest of the core complex, which could result in the opening of the secretion channel.

These interactions take place in the inner membrane de Paz et al. Interestingly, heteromeric transmembrane interactions in VirB10 have been found to be essential for T-pilus biogenesis but not for substrate secretion Garza and Christie, VirB10 is also able to interact with VirB4. A low-resolution EM structure of the core complex bound to the VirB4-homologue of the conjugative plasmid pKM revealed an intimate relation between VirB4 and the channel complex Wallden et al.

Instead, an association to the complex through the integral membrane protein VirB3 seems a more plausible explanation. It is enticing to speculate that the ATPase activity by VirB4 induces conformational changes in the core complex during pilus biogenesis whereas VirD4 does it during substrate transport. The crystallographic structure of the CTD of a VirB4 homologue in Thermoanaerobacter pseudethanolicus has recently been reported Wallden et al.

The structure turned out to be strikingly similar to the computer generated models of the CTD of A. These three motor proteins seem to have evolved from a common ancestor Iyer et al. The IncX subfamily of VirB4 proteins is characterized by the presence at the N-terminus of an extra region corresponding to a fusion with a VirB3 protein Batchelor et al.

VirB4 has been reported to be an integral cytoplasmic membrane protein Dang and Christie, However, computer predictions of VirB4 topology are negative for transmembrane spans in most of the VirB4 members, excluding those of the IncX branch, which contain a VirB3-like sequence at the N-termini Arechaga et al. Moreover, TraB, the VirB4 homologue in the conjugative plasmid pKM, has been isolated both, as a soluble and as a membrane-associated form Durand et al. It is altogether very likely that VirB3 acts as an anchor of VirB4 to the membrane, assisting it in its VirB2 dislocase function Kerr and Christie, This structure was obtained in its hexameric form, which is likely to be the catalytic active conformation of the enzyme Arechaga et al.

The oligomeric state of VirB4 proteins has been largely under dispute. Initial reports on the oligomeric state of A. On the other hand, the VirB4 homologue in the conjugative plasmid pKM has been reported to be present both as dimer and hexamer, being the hexameric form soluble and catalytically active and the dimeric form inactive and membrane-associated Durand et al.

Moreover, this structure shows not only one but two hexamers attached to the inner membrane domain of the T4SS. The implications of this arrangement in the molecular mechanism are however still unclear.

The linker region between both domains has been proposed to play a key role in enzyme catalysis Hare et al. In fact, although the crystallographic structures of two non-conjugative homologues, HP from Helicobacter pylori Yeo et al. These differences are produced by a large domain swap of the central linker, which is much larger in the Brucella homologue than in the Helicobacter counterpart Hare et al.

Based on biochemical and structural evidence, a common model for the mechanism of action of these secretion ATPases has been proposed Yamagata and Tainer, ; Ripoll-Rozada et al.

According to this model, the VirB11 NTD of the nucleotide-free form of the enzyme would be pivoting over the flexible linker. Binding of magnesium and ATP would lock the enzyme in a closed conformation.

A specific signal for instance, substrate binding or release would unlock this state, resuming the catalytical cycle by releasing the ADP for the next turnover Ripoll-Rozada et al. The biological function of VirB11 is still unknown. These interactions depend on the length of the linker region, as inferred from experiments in which VirB4 was tested for interactions with VirB11 homologues of different linker size Ripoll-Rozada et al.

These interactions seem to be unstable as judged by the fruitless attempts to purify stable VirB4—VirB11 complexes J. VirB11 has also been suggested to play an essential role in the first steps of the DNA translocation pathway Atmakuri et al.

Supporting this dual functionality, mutations in VirB11 uncouple T-pilus production from substrate translocation Sagulenko et al. It has been suggested that VirB11 acts as a molecular switch between pilus biogenesis and substrate transport by replacing VirB4 and VirD4 ATPases at the entrance of the secretion channel Ripoll-Rozada et al.

It is worth noting that VirB11, although widely distributed among conjugative systems, is not found in all T4SS. Therefore, in these systems, the function of VirB11 seems to be dispensable or replaceable by other proteins with similar structure. The coupling function of T4CP is mediated, on one side, by interactions with the relaxosome auxiliary proteins Disque-Kochem and Dreiseikelmann, ; Tato et al.

This transmembrane domain also regulates nucleotide binding affinity Vecino et al. In IncF plasmids, interactions between the coupling protein TraD and the accessory protein TraM have also been reported Lu and Frost, and the structure of the C-terminal tail of TraD bound to the TraM tetramerization domain has been solved Lu et al.

The protein might be involved in resolving G4 secondary structures that arise during conjugative DNA processing. Alternatively, this higher affinity for G-quadruplex might reflect the need of a secondary DNA structure as a loading site for the motor. Further experiments are needed to clarify the in vivo function of G-quadruplex structures in the conjugative process.

However, despite the mounting evidence describing the interactions between T4SS components and the interactions between them and the translocated substrate, there are still a number of questions that remain open. For instance, the involvement of the core complex subunits during substrate transport, the specific role of the three ATPases or the fate of the pilus after the formation of the mating pair are still not fully understood processes.

Last but not least, an important question that remains unanswered is the conformational state, native or unfolded, of the protein substrate during translocation. Here, based on the information available up to date, a detailed description of the events that take place during DNA processing and transport in bacterial conjugation is provided Fig.

Nucleoprotein transport by T4SS. Step 1 — Donor cells contact recipient cells. The cell-to-cell contact, mediated by the pilus, could be triggered by specific factors in the recipient. Step 2 — Upon cell contact, the retraction of the extracellular pilus facilitates the interaction between membranes of donor and recipient cells, resulting in a membrane fusion process.

Simultaneously, or prior to this membrane fusion, the coupling protein drives the relaxosome towards the secretion channel. The relaxase is unfolded and translocated through the channel covalently bound to the DNA.

Conjugative and mobilizable plasmids contain a cognate DNA sequence, oriT , which is recognized by specific auxiliary factors within the relaxosome. Relaxases are usually, but not always, large multi-domain proteins for a recent review see Garcillan-Barcia et al. In all cases, the relaxase domain, which is approximately aa long, is located at the N-terminus of the protein. Occasionally, an extra domain of unknown function is also observed. In most cases, relaxases contain a conspicuous signature consisting of a histidine triad 3H motif Garcillan-Barcia et al.

This metal ion is essential for the relaxase cleavage reaction Boer et al. Crystal structures of the relaxase domains of TrwC of plasmid R Guasch et al. This transesterification reaction results in a covalent linkage between protein and DNA Guasch et al. Then, the helicase domain displaces the old DNA strand, which is replaced by the new synthesized DNA strand, and the covalently bound nucleoprotein is transferred to the recipient cell.

The auxiliary proteins of the relaxosome are involved in the regulation of gene expression. For instance, TraY and TraM in IncF plasmids bind to the F tra operon promoters, which results either in a positive or negative regulation of gene expression Schwab et al. The role of the different auxiliary proteins and the mechanism of cleavage and processing of conjugative DNA is described in further detail in de la Cruz et al. Once the cleavage reaction has ended, the nucleoprotein complex must be recruited to the membrane channel to initiate transfer Llosa et al.

Binding of the coupling protein to the DNA is sequence unspecific but, thanks to the auxiliary proteins that recognize a specific sequence in the oriT Moncalian and de la Cruz, , the coupling protein is able to bind to an specific region within the relaxosome. T4SS-dependent transfer of relaxases to human Schulein et al. TrwC has been shown to display site-specific and recombinase activities in the recipient cell Draper et al. However, the mechanism of transport across the T4SS channel is unknown.

Given the large size of most relaxases over kDa , it is unlikely that they can go through the T4SS channel in a native state conformation and an unfolding mechanism should be envisaged.

Nothing is known about this unfolding mechanism but it would involve the assistance of some kind of unfoldase. Interestingly, and similarly to TrwD Rivas et al. However, experimental evidence of interactions between VirB11 and the relaxase is still missing. The putative role of VirB11 in substrate transport as an acceptor of the nucleoprotein complex from the coupling protein is supported by several lines of evidence, such as the T-DNA immunoprecipitation TrIP assays carried out in A.

However, the mechanism by which the relaxase is transported across the secretion channel is still unclear. In the VirB system of Brucella , this type of signal sequence has been identified in several effectors de Jong et al.

This signal has been localized in some cases at the N-termini, whereas in other cases this signal was located at the C-termini of the translocated protein de Jong et al. In contrast, relaxase translocation signals TSs in conjugative T4SS have been localized to internal positions. The three-dimensional structure of TSA has recently been reported Redzej et al. Based on conservation of the consensus sequence within a RecD2-like domain of the proteins, an extension of these results to other relaxases was proposed Lang et al.

These results seem to indicate that a single substrate can be recruited by two different T4SSs through different signals Alperi et al.

According to a shoot and pump model Llosa et al. The proposed mechanism Cabezon and de la Cruz, is a modified version of the so-called binding change mechanism proposed for the F 1 -ATPase, which involves a sequential binding and hydrolysis of ATP Boyer, After the transfer of a complete copy of a single DNA strand into the recipient cell, the relaxase would recognize the nic site and it would carry out a second transesterification reaction, resulting in re-circularization of the transferred strand in the recipient Draper et al.

One important question that remains unanswered is the extent of the conformational changes that take place in the core complex channel. It is evident that these structural conformational changes should be large enough to permit the nucleoprotein passage across the T4SS.

These dramatic conformational transitions are likely to require energy, which might be provided by one of the two ATPases at the entrance of the secretion channel. Such an energy-induced conformational change in VirB10 is required to form the complex with the outer membrane-associated proteins VirB9 and VirB7 Cascales and Christie, a.

The mechanism resembles that of the TonB-dependent transporters, where TonB also acts as an energy sensor Postle and Kadner, TonB and GspB act as energy sensors of the proton motive force pmf , and in some cases, like in Klebsiella oxytoca , this is the only energy source required Possot et al. The pmf has also been postulated to be the only energy source required for substrate secretion by T3SS Wilharm et al.

Hence, the translocation of substrates across the T4SS might also require both the pmf and ATP hydrolysis as energy sources. Regardless of the source of energy, it has become evident that VirD4 interacts with VirB10 during substrate transport Cascales et al. The interactions between these two proteins would only be possible if the resident VirB4 protein, involved in pilus biogenesis, is displaced from the base of the core channel.

Although much progress has been made in describing the molecular architecture of the T4SS, these efforts have not been accompanied by a better understanding of the dynamics of the secretion mechanism.

In DNA transfer systems, many essential questions remain unsolved, such as the mechanism by which the nucleoprotein complex enters in the recipient cell, overcoming the membrane barrier, or the mechanism by which entry exclusion proteins prevents the entry of a second identical plasmid in the cell Garcillan-Barcia and de la Cruz, Little is known about specific receptors in the recipient cell that allow pilus attachment.

Interactions between the pilus tip and lipopolysaccharides LPS in the recipient cells have been reported Anthony et al. However, recent data have shown that mutations in the LPS pathway had little effect on conjugation and that no nonessential recipient E.

These results suggest that conjugation would take place with little regard to the recipient cell constitution Perez-Mendoza and de la Cruz, Another important question that remains unsolved is the exact role of pili in DNA transfer. Although the most universally accepted idea is that pili only provide cell-to-cell contacts, the possibility that ssDNA travels through the lumen of the pili is still debatable. However, it is difficult to envisage the passage of the protein substrate through the pilus.

It could be also possible, especially in the short-rigid pili encoded by IncP and IncW plasmids, that after engagement of the recipient cell, pili depolymerize facilitating the membrane contacts between donor and recipient cells, leading to membrane fusion. At that stage, T4SS could easily deliver its substrates in the interior of the recipient cell.

This membrane fusion hypothesis would also explain the mechanism of interaction between the entry exclusion factor in the recipient cell and T4SS components in the donor cell Garcillan-Barcia and de la Cruz, Alternatively, a mechanism similar to the T3SS injectisome, in which the pilus works as a needle, shooting up the virulence factors directly into the recipient cell, is also feasible Galan and Wolf-Watz, In summary, there are still challenging questions to be solved to understand the intimate mechanisms of DNA and protein transport in bacterial conjugation.

Understanding these mechanisms is essential to develop strategies in the battle against the dissemination of antibiotic resistance genes. Additionally, a better understanding of the conjugative machinery could be helpful in the development of farfetched biotechnological applications, such as gene therapy Llosa and de la Cruz, ; Schroder et al. Present Address. Google Scholar. Baquero F Coque TM de la Cruz F Ecology and evolution as targets: the need for novel eco-evo drugs and strategies to fight antibiotic resistance Antimicrob Agents Chemother 55 Unveiling the molecular mechanism of a conjugative relaxase: the structure of TrwC complexed with a mer DNA comprising the recognition hairpin and the cleavage site J Mol Biol Identification of a Brucella spp.

Plasmid 60 1 Molecular recognition determinants for type IV secretion of diverse families of conjugative relaxases Mol Microbiol 78 Structure of type IV secretion system Nature 3. Brucella modulates secretory trafficking via multiple type IV secretion effector proteins PLoS Pathog 9 e Perez-Mendoza D de la Cruz F Escherichia coli genes affecting recipient ability in plasmid conjugation: are there any?

BMC Genomics 10 Structure of a translocation signal domain mediating conjugative transfer by type IV secretion systems Mol Microbiol 89 Tzfira T Citovsky V Agrobacterium -mediated genetic transformation of plants: biology and biotechnology Curr Opin Biotechnol 17 Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein Biochim Biophys Acta 9.

Waters VL Conjugative transfer in the dissemination of beta-lactam and aminoglycoside resistance Front Biosci 4 D Crystal structure of the RuvA-RuvB complex: a structural basis for the Holliday junction migrating motor machinery Mol Cell 10 Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search.

Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Towards an integrated model of bacterial conjugation. Oxford Academic. Jorge Ripoll-Rozada. Fernando de la Cruz. Ignacio Arechaga. Select Format Select format. Permissions Icon Permissions. Open in new tab Download slide. Google Scholar Crossref. Search ADS. Cell-cell interactions in conjugating Escherichia coli : role of F pili and fate of mating aggregates. Google Scholar PubMed. Pilus biogenesis at the outer membrane of Gram-negative bacterial pathogens.

A translocation motif in relaxase TrwC specifically affects recruitment by its conjugative type IV secretion system. The VirB5 protein localizes to the T-pilus tips in Agrobacterium tumefaciens.

Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex. The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids. Comparison of proteins involved in pilus synthesis and mating pair stabilization from the related plasmids F and R insights into the mechanism of conjugation. Ecology and evolution as targets: the need for novel eco-evo drugs and strategies to fight antibiotic resistance. Nucleotide sequences and comparison of two large conjugative plasmids from different Campylobacter species.

Unveiling the molecular mechanism of a conjugative relaxase: the structure of TrwC complexed with a mer DNA comprising the recognition hairpin and the cleavage site. An update from the Infectious Diseases Society of America. TraY proteins of F and related episomes are members of the Arc and Mnt repressor family. Topological analysis of a putative virB8 homologue essential for the cag type IV secretion system in Helicobacter pylori.

Structure-function analysis of Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase domains. Genetic evidence of a coupling role for the TraG protein family in bacterial conjugation. Functional characterization of the multidomain F plasmid TraI relaxase-helicase. Biogenesis, architecture, and function of bacterial type IV secretion systems.

Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation. Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion. Horizontal gene transfer and the origin of species: lessons from bacteria. A novel cytology-based, two-hybrid screen for bacteria applied to protein-protein interaction studies of a type IV secretion system.

Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells. Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.

Maturation of IncP pilin precursors resembles the catalytic Dyad-like mechanism of leader peptidases. Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins. Sequence-imposed structural constraints in the TonB protein of E. The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface.

To efficiently counteract the problems associated with antibiotic resistance it is therefore necessary to understand the mobile genetic elements—conjugative plasmids CPs and integrative conjugative elements ICEs —that are the vehicles for transfer of antibiotic resistance genes from the large communal gene pool to human pathogenic bacteria. In the following sections we will give an overview on the current knowledge of bacterial conjugation. As will be evident, it is a widely distributed, if not ubiquitous phenomenon in the bacterial world.

Special emphasis will be given to regulatory mechanisms ensuring timely and spatially controlled expression of tra genes. Furthermore, we consider recent advancements in understanding population dynamics and coevolution of CPs and host cells. In the context of this manuscript intelligence is understood as cell-cell communication and complex regulatory systems producing cellular responses that maximize successful DNA transmission and at the same time do not impose a burden or fitness cost to the whole population of CP carrying cells.

Bacterial conjugation is a cell-cell contact dependent DNA transfer event. Approximately 10—20 proteins fewer in Gram positive bacteria, see below constitute the building blocks of the T4SS dedicated to ssDNA and protein transfer. Other genetic elements such as mobilizable plasmids or genomic islands can be mobilized by either of these self-transmissible elements Smillie et al.

Unlike in true bacterial conjugation where DNA is transferred directly from a donor to a recipient cell, Neisseria gonorrhoeae secretes ssDNA contact-independently via a T4SS encoded by a genomic island Ramsey et al.

Historically, research on bacterial conjugation focused on the F-plasmid and related CPs from Gram negative bacteria Willetts and Skurray, ; Frost et al. For structural studies on the T4S machinery plasmid pKM has had a pivotal role since it was possible to determine the 3D structure of a ring like core T4S complex composed of mers of three proteins which span the periplasmic space from the inner to the outer membrane Rivera-Calzada et al.

From genomic sequencing projects and bioinformatics analyses it became evident that the most abundant self transmissible elements are ICEs that are maintained chromosomally similarly to temperate bacteriophages and can be transferred via a plasmid intermediate Wozniak et al. Figure 1. Before transfer can occur, tra genes must be expressed and a T4SS assembled. After cell-cell contact formation, transfer competent donor cells initiate a rolling circle type replication from circular dsDNA and translocate ssDNA via the T4S machinery into recipient cells.

A CPs can autonomously replicate due to the presence of rep genes. Only a subset of proteins typically found in Gram negative bacteria is also present in the Gram positives which led to the concept of minimized T4SS that are present in Gram positive bacteria Zhang et al.

Major differences arising from the specific architecture of the cell envelope of diderms vs. Presumably, at this stage, the Dtr complex is docked to the T4S complex which has been pre-assembled in the cell envelope Zechner et al.

The T4S complex consists of i ATPases fueling assembly of the T4S apparatus and DNA transfer, ii translocon proteins of the inner membrane, iii core proteins spanning the cell envelope, and iv pilus proteins or adhesins.

The Dtr complex physically interacts with the T4S apparatus mainly via protein-protein interactions especially via one of the ATPases of the T4S complex being a substrate receptor Bhatty et al. In order to start translocating ssDNA, a productive and stable mating pair between a donor and a suitable recipient cell has to be formed.

This includes initial contact via the pilus or adhesins, pilus retraction in F conjugation Clarke et al. It is not known whether the pilus additionally functions as a device delivering ssDNA by penetration of the recipient cell envelope. Upon an elusive signal, ssDNA with the relaxase covalently bound to the 5 prime end of the ssDNA is transported through the conjugation channel the T4S apparatus and reaches the cytoplasm of the recipient where the DNA is recircularized presumably via the co-transported relaxase to regenerate a circular ssDNA which can be replicated to dsDNA in the recipient Zechner et al.

Overall, conjugative DNA replication is similar to the replication of ssDNA phages in which ssDNA in that case termed the plus strand is generated by rolling circle replication from a dsDNA intermediate, packaged into viral proteins and then released into the environment, ready to infect novel recipient cells.

Strategies to secure successful gene transfer in natural environments require sensing mechanisms that ensure that tra genes Dtr and T4S genes are turned ON at the right time and at the right place. As a general rule, the default status of transfer genes is OFF. Transfer gene expression followed by assembly of the T4S machinery in the cell envelope and formation of the Dtr complex transforms donors into transfer competent cells.

Molecular regulatory networks and switches coupled to positive feedback loops ensure that once a certain threshold which is defined at the single cell level is reached, tra genes are expressed and the system is turned ON. Negative feedback loops ensure that—after the expression burst—the system returns to the default OFF state for a general scheme see Figure 2A.

Some of the well-studied conjugation systems that display these features, with a focus on how tra genes are turned ON, are described in the following sections. Figure 2. Two models for the development of transfer competence in single cells A and populations B are shown. In single cells A tra genes are turned ON by a variety of stimuli. A positive feedback loop ensures that, once initiated, cells proceed to transfer competence, involving formation of the Dtr complex and assembly of the T4S apparatus.

In transfer competent cells, tra genes are switched OFF, mediated by a negative feedback loop. Eventually, transfer competence is lost by transition to unfavorable conditions. Recipient sensing by donors is achieved through a processed and secreted inhibitory peptide, termed PhrI, that is then transported back into the cells. A similar case with a Rap protein counteracting an Xre-type transcriptional repressor exists in case of the native B. In both examples, the presence of recipients is sensed by an inhibitory peptide that is secreted by cells containing the conjugative element.

A requirement for this system to work is that the recipient cells surrounding donors possess an uptake system for the secreted inhibitory peptide, thereby reducing its concentration in donors. A variation of this theme is present in the two well-studied pheromone-inducible CPs of Enterococcus faecalis , pAD1 and pCF10, respectively.

As in many other cases, the repressor TraA of pAD1 does not control tra genes directly, but an activator protein which, when it escapes repressor control, is positively auto-activated and efficiently transforms the donor cell into the transfer competent state Clewell, If donor cell densities are too high, the inhibitory quorum sensing molecule iCF10 keeps donor cells in the OFF state.

Similarly, iCF10 is also responsible for shutting off the tra genes after an initial burst caused by the inducing factor Chatterjee et al. The low transfer frequency observed for SXT transfer and repressor inactivation is presumably maintained by a subpopulation of cells that inherently express SOS genes McCool et al.

Agrobacteria harboring Ti plasmids are not only capable of transforming plant cells by T-DNA transfer but also contain a specific set of DNA transfer genes for conjugation. Tra genes are not transcribed unless a specific transcriptional activator, TraR is expressed. First of all, traR transcription is dependent on opines, amino sugars specifically produced by transformed plant tumor cells.

Opines can be taken up and used as nutrients only by agrobacteria harboring the Ti plasmid. Opines, specifically nopalines, inactivate a Ti plasmid encoded repressor AccR that controls several genes on the Ti plasmid.

Among the genes controlled by AccR is the gene for the transcriptional activator TraR. As a consequence, Ti plasmid encoded tra genes are only turned ON inside crown galls where opines are produced by plant cells at high cell densities reviewed in White and Winans, In the Ti plasmid system, induction of tra genes is therefore dependent on signal molecule mediated repressor inactivation and activator production, which, once initiated, is enhanced by a positive feedback loop provided by AHL synthesis , presumably resulting in a burst of tra gene expression in individual cells harboring the Ti plasmid.

The system can be turned OFF by anti-activators TraM and TrlR under the control of TraR—negative feedback loop and may be modulated by lactonases that can specifically hydrolyze the AHL molecule in response to plant signals Haudecoeur and Faure, Besides the Ti plasmid and two chromosomes, Agrobacterium tumefaciens C58 also harbors another large conjugative plasmid, pAT.

Notwithstanding the lack of obvious signaling molecules involved in F-conjugation module mediated DNA transfer, sensing environmental conditions in combination with the physiological status of the potential donor cell affects the behavior of the cell through a network of regulatory elements for a detailed description see Frost and Koraimann, CPs with F-like conjugation modules are mainly found in the Enterobacteriaceae including pathogenic Escherichia , Salmonella , and Klebsiella species.

Typically, the plasmid encoded transcriptional activator of tra genes, TraJ, is under the negative control of two fertility inhibition elements, FinO and FinP.

In populations of donor cells—under optimal conditions promoting growth and cell division—only in few cells 1—10 out of potential donor cells TraJ escapes this negative FinOP mediated control and promotes transcription of tra genes together with the host encoded transcriptional activator ArcA-P Strohmaier et al. Similarly to other conjugation systems described in this review, a negative feedback-loop exists that mediates shut-off of tra gene expression via the DNA binding protein TraY which has an activating role at low concentrations but can inhibit tra gene expression at higher concentrations.

Other factors that contribute to the shut-off of tra gene transcription or modulate and fine tune this system are extracellular and cellular stress response elements, including the CpxAR two component system, proteases, and the chaperone protein GroEL Zahrl et al. From studies of many different conjugation systems it has become evident that, even under controlled laboratory conditions, the transition to transfer competence does not occur in all cells of a population Figure 2B.

One example in which this phenomenon has been illustrated nicely is the demonstration of discontinuous patches of gene transfer between donor and recipient cells at the edge of bacterial colonies on semi-solid agar surfaces Reisner et al.

Higher magnifications of these zones revealed that these patches correspond to infrequently occurring gene transfer events from some cells of the donor cell population to recipient cells but not from all. This phenomenon was observed in the case of a derivative of the naturally repressed F-like plasmid R1 with an intact FinOP repressor system see above. These findings are consistent with the observation in liquid media where tra gene expression is low in the presence of plasmid R1 compared to a de-repressed mutant.

Since the first observation published by Ghigo that the presence of CPs in bacterial populations induces the formation of biofilms it has become increasingly evident that these microbial communities are hot-spots for social interactions and horizontal gene transfer HGT.

In short, CPs promote biofilm formation and, vice versa, biofilms promote conjugation Molin and Tolker-Nielsen, ; Madsen et al. The underlying gene regulatory mechanisms, however, are largely unknown because tra gene expression studies in biofilms are difficult to perform due to the dynamic nature of biofilms and the associated inherent heterogeneity of cells.

Which donor cells in a biofilm community actually progress via activation of tra gene expression to transfer competent cells is unknown. What can be observed at the single cell level by sophisticated genetic constructs and fluorescence microscopy, however, is the transfer of plasmid DNA into recipient cells and the spread of the CP in the recipient population.

In one case, the transfer of the pWW0 TOL plasmid from Pseudomonas putida donor to recipient microcolonies on semi-solid agar surfaces was investigated. Intriguingly, time-lapse microscopic images revealed that spreading of the CP in the recipient originated from one transfer event between cells contacting each other at the edges of donor and recipient microcolonies.

This single transfer event was followed by limited, cell division dependent, spreading of the CP in the recipient colony. Again, similarly to plasmid R1 see above , not all donor cells that were in contact with recipient cells initially transferred the CP, indicating regulatory mechanisms that maintain the OFF state in most of the cells of the donor microcolony Seoane et al. Regulatory mechanisms including negative autoregulation by a transcriptional repressor of tra genes that could account for a shut-off after an initial burst have indeed been demonstrated for the pWW0 plasmid Lambertsen et al.

In analogy to the microcolony situation, limited invasion of recipient cells in E. In theory, CPs should, once established in a bacterial host, represent a burden and generate a fitness disadvantage, resulting eventually in the elimination of the plasmid from a population. This, however, as evidenced by the persistence of these elements, seems not to be the case.

Are there advantages conferred to the host by the CP in the absence of selection for genes that are carried by the CP? There are several studies in which the apparent paradox of the persistence of CPs in the bacterial world has been investigated Modi and Adams, ; Dahlberg and Chao, ; Dionisio et al. One interesting result of such studies was that, CPs such as R1 and RP4 were not lost from bacterial populations even after more than generations of growth without selective pressure.

This was attributed to the fact that such plasmids have a controlled replication system and low copy numbers as well as active partitioning and plasmid stability systems that prevent plasmid loss.

An initial minimal fitness cost that was imposed on the E. Coevolution induced changes were observed in both the evolved host cells and plasmids. Interestingly, evolved plasmid R1 had slightly lower transfer rates in the evolved host than in the ancestral host Dahlberg and Chao, These findings were corroborated by a similar study where it was found that an evolved plasmid R1 even conferred a relative fitness advantage to the original E. In addition, the original R1 plasmid had no fitness cost in the evolved E.

These results and other studies have led to the proposal that CP mediated bacterial conjugation is a coevolutionary process Harrison and Brockhurst, Although not measured directly, the data of Dahlberg and Chao suggest that a major cost of CP carriage is the expression of tra genes. In line with this proposition is the fact that the activation of tra genes in case of plasmid R1 causes the up-regulation of extracytoplasmic and cytoplasmic stress regulons Zahrl et al.

In addition, F plasmid tra gene expression and T4S system assembly causes increased sensitivity to bile salts Bidlack and Silverman, In any case, due to the regulatory regime that keeps tra genes OFF in the majority of donors, metabolic burden and exposure to pilus specific bacteriophages is not evenly distributed within a population but instead restricted to a small fraction of the population.

In this way, possible detrimental and cell threatening effects associated with tra gene expression are limited to a few cells within a population whereas all cells retain beneficial genes and the potential for HGT. An intrinsically beneficial feature contributing to the persistence of CPs within bacterial populations may be their well documented ability to promote formation of biofilms see above. Interestingly, besides the specific regulatory mechanisms discussed in this review that operate to control tra gene expression, there is a general silencing mechanism in enterobacteria that mediates silencing of laterally acquired genes by H-NS and related proteins Navarre et al.

Although the molecular details of how regulatory networks control tra gene expression are different in the conjugation systems presented in this review, there is a common theme: As a default, tra genes are OFF and whenever positive stimuli are present, not the whole population transits to the ON stage but only a fraction of the cells carrying a conjugative element.

In this way the metabolic burden fitness cost imposed by expression of tra genes and assembly of a cell envelope localized DNA secretion machine a T4SS is carried not by the whole population but distributed to only a few cells within a population. Further studies at the single cell level are needed to reveal whether the transformation of only a fraction of a donor cell population into transfer competent cells is due to a stochastic process or depends on different physiological states such as metabolic conditions, cellular fitness and cell age.

Moreover, positioning of individual cells in structured communities microcolonies or biofilms may influence transition to transfer competence. Undoubtedly intelligent strategies exist to minimize or even eliminate fitness costs associated with the carriage of conjugative elements.

Populations harboring CPs and presumably ICEs can grow and divide largely unaffected by the presence of these elements. At the same time, some cells within a population do become transfer competent and thereby secure the spread and persistence of conjugation modules in many different bacterial species, among them pathogens causing disease in humans, animals, and plants.

Thus, genes carried on the conjugative element, which are beneficial for the host cell in particular habitats e. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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