DNA Substrate-Induced Activation of the Agrobacterium VirB/VirD4 Type IV Secretion System

The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface.     

Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.The Agrobacterium tumefaciens VirB/VirD4 type IV secretion system (T4SS) delivers effector proteins and DNA to plant cells during infection (1, 14). The 11 VirB proteins and VirD4 substrate receptor mediate assembly of the envelope-spanning translocation channel, whereas the VirB proteins independently of VirD4 are required for polymerization of the extracellular T pilus (6, 32, 46). These T4SS subunits include the three ATPases VirD4, VirB4, and VirB11; a trans-envelope core complex comprised of VirB7, VirB9, and VirB10; subunits involved in assembly or spatial positioning of the core complex (VirB1, VirB6, and VirB8); and other structural components (VirB2 pilin, VirB3, and pilus-associated VirB5) (1, 14, 43, 48, 55, 70). The VirB/VirD4 subunits are conserved among many Gram-negative bacterial T4SSs, and recent structures of homologs of VirD4, VirB5, VirB8, VirB10, and VirB11 and a VirB7/VirB9/VirB10 machine subassembly are supplying exciting new information about T4SS machine architectures (11, 28, 29).The pilin subunit VirB2 is a component of both the secretion channel and T pilus (39, 47, 48). Its role in substrate transfer was established with a modified chromatin immunoprecipitation (ChIP) assay termed transfer DNA (T-DNA) immunoprecipitation (TrIP), wherein the pilin (but not the T pilus) was shown to form formaldehyde-cross-linkable contacts with the translocating T-DNA substrate (10). TrIP studies with virB mutant strains also supplied evidence that VirB2 occupies a distal portion of the translocation channel near or at the outer membrane (OM) (10). Complementary genetic studies identified mutations in several VirB subunits, including VirB6, VirB9, VirB10, and VirB11, that selectively block T pilus production without affecting substrate transfer (39, 40, 41, 62). These Tra⁺ Pil⁻ “uncoupling” mutations do not bypass the requirement for VirB2 production for substrate transfer, as the further deletion of virB2 from the Tra⁺ Pil⁻ mutant strains renders these strains transfer defective (39, 41, 62). Therefore, VirB2 pilin, but not an intact T pilus, is required for passage of substrates to target cells.The pathways culminating in the integration of VirB2 into the two terminal organelles, the secretion channel and T pilus, are fundamentally poorly understood. The early VirB protein-independent reactions involve insertion of the 12.3-kDa propilin into the inner membrane (IM); cleavage of a long, 47-residue signal sequence, presumably by LepB signal peptidase; and covalent joining of the N-terminal Gln48 and C-terminal Ser121 to form the mature, cyclic pilin (24). This unusual head-to-tail cyclization reaction was also shown for the VirB2 homolog, TrbC (24/51% sequence identity/similarity) of plasmid RP4 (24, 34, 44). Other VirB2 homologs, such as F plasmid TraA (19/47% identity/similarity) (67), remain linear although their N termini are modified by N acetylation (54).Prevailing models suggest that mature forms of conjugative pilins accumulate in the IM as pools for use in assembly of the channel/pilus upon receipt of an unknown morphogenetic signal(s). The IM-integrated VirB2, TraA_F, and TrbC_RP4 pilins likely adopt similar topologies, as deduced from similar predicted secondary structures and results of reporter fusion studies with periplasmically active alkaline phosphatase (PhoA) (5, 22, 56). Two hydrophobic domains are thought to orient across the IM so that a small, intervening hydrophilic loop is cytoplasmic and the hydrophilic N and C termini are periplasmic. Detailed studies confirming this overall topology are lacking, and limited information exists regarding the nature of pilin interactions with other T4SS subunits (36, 51). Furthermore, little is known about the mechanism or energetic requirements for dislocation of membrane-integrated forms of conjugative pilins during machine morphogenesis.In A. tumefaciens, mutations in the Walker A nucleoside triphosphate (NTP) binding site motifs of the VirB4 and VirB11 ATPases render cells defective for substrate transfer and pilus production, indicating that NTP energy consumption by both ATPases is essential for assembly of the two terminal organelles (6, 7, 58, 62, 68). VirB4-like subunits are signatures of all T4SSs described to date, whereas VirB11-like proteins are common but not ubiquitous among the T4SSs (1). Some T4SSs, such as the conjugation machines encoded by Escherichia coli F-like plasmids, lack VirB11 homologs, and yet their conjugative pili extend and retract dynamically by a mechanism(s) dependent on VirB4 homologs (18, 65). On the basis of these observations, it is reasonable to propose that the VirB4-like subunits catalyze early reactions associated with assembly of conjugative pili.Here, we used the scanning cysteine accessibility method (SCAM) (9) to define the IM topology of cyclized VirB2. We then assayed for contributions of VirB subunits to the pilin structural organization. We present biochemical evidence for VirB4-mediated dislocation of VirB2 pilin from the membrane and also for a contribution by VirB11 in modulating pilin tertiary or quaternary structure. We discuss our findings in the context of recent advances in our understanding of T4SS machine assembly and architecture.     

Interactions between Brucella suis VirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems

The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.Brucellosis is a major worldwide zoonosis primarily affecting developing countries and causing them severe economic losses (7). Bacteria of the genus Brucella, the causative agent, are gram-negative facultative intracellular pathogens of various wild and domestic mammals, as well as humans, where it causes a very debilitating disease known as Malta fever (48). In addition, these bacteria are also a focus of concern as possible biological warfare agents (23).The key aspect of Brucella virulence is its ability to survive and proliferate within professional and nonprofessional phagocytes (7). Once phagocytosed, this bacteria subverts the vesicular traffic in the host cell to establish a niche in a compartment derived from the endoplasmic reticulum, where it multiplies (3, 34, 39, 40, 41). Several factors have been reported to be essential for the virulence of this bacterium (16, 17, 18, 19, 21, 22, 29, 31). Strikingly, we (36) and others (44) have demonstrated the presence in Brucella of a type IV secretion system (T4SS) that is encoded by the virB operon and whose integrity is required for virulence (8, 14, 19). Several other species of gram-negative bacteria have been found to rely on the presence of a T4SS for full virulence (10, 13). Both extracellular (Helicobacter) and intracellular (Legionella, Bartonella) pathogens use their T4SSs to inject effector proteins directly into the target cell, where they affect the biology of the cell. Bordetella pertussis uses its T4SS to secrete the pertussis toxin into the extracellular medium, where it is taken up by cells. The T4SS of Agrobacterium tumefaciens translocates both effector proteins and a nucleoprotein complex into target plant cells through a mechanism reminiscent of bacterial conjugation through T4SS.The A. tumefaciens VirB T4SS, which is considered the T4SS paradigm, is composed of 11 different proteins named VirB1 to VirB11 plus VirD4. These proteins can be functionally subdivided in three different groups. The proteins VirB4, VirB11, and VirD4 are inner membrane ATPases with a large cytoplasmic domain and are believed to provide the energy required for T4SS assembly and for the translocation of effectors. VirB2 and VirB5 form an extracellular bacterial appendage believed to anchor the bacteria to the host cell (4, 25). Finally, VirB3 and VirB6 to VirB10 are believed to form a channel-like structure spanning both the inner and outer membranes of the bacteria.Among these structural proteins, VirB8 has been shown to play a key role in the assembly of the T4SS. Recent studies with A. tumefaciens demonstrated that VirB8 acts as a nucleation center required to recruit VirB9 and VirB10 into clusters in the outer membrane (15, 30) and to localize VirB proteins at the cell pole (26). VirB8 is a protein spanning the bacterial inner membrane, with the first 67 amino acids forming a short cytoplasmic tail, followed by a single hydrophobic transmembrane (TM) domain. The carboxy-terminal moiety of the protein, of 172 amino acids, is believed to be entirely periplasmic. Recently, the three-dimensional structures of the periplasmic domains of VirB8 from Brucella suis (46) and A. tumefaciens (5) have been determined. Using these structural data, site-directed mutagenesis has been performed on the periplasmic part of B. suis VirB8, showing that changes in amino acids that inhibit the dimerization of VirB8 or its interactions with VirB4 or VirB10 also affect T4SS assembly and B. suis virulence (37). Among all of the homologs of B. suis VirB8, the closest are the proteins TraJ, encoded in the tra operons of broad-host-range plasmids pSB102 and pIPO2 (43, 45). The TraJ protein from pSB102 shares more than 50% identity with B. suis VirB8 at the amino acid level, and this percentage increases to more than 60% when only the periplasmic domain is considered. In a previous study, we have taken advantage of this close similarity between VirB8 and TraJ to examine the possibility of a functional heterologous complementation of VirB8 by TraJ in BS1008, a B. suis mutant carrying an in-frame deletion of the virB8 gene (38). From our results, it appeared that the protein TraJ was unable to complement BS1008. As the major similarities between VirB8 and TraJ were found in their respective periplasmic domains, we constructed chimeric genes encoding proteins in which the major part of the periplasmic domain of one protein (amino acids 77 to 241 of TraJ and amino acids 76 to 239 of VirB8) was replaced with the corresponding part of the other protein (these proteins are described in Fig. ​Fig.1)1) and studied the ability of these chimeric proteins to restore the virulence of BS1008. The TraJB8 chimera, where the periplasmic part of VirB8 replaces the corresponding part of TraJ, was also unable to complement BS1008. In contrast, B8TraJ, the reverse chimera in which the periplasmic part of TraJ replaces the corresponding part of VirB8, partially restored the virulence of BS1008. These results show that, when fused to the cytoplasmic and TM parts of VirB8, the periplasmic part of TraJ can functionally replace the corresponding part of VirB8 in T4SS assembly. In contrast, the cytoplasmic and TM parts of TraJ cannot replace the corresponding part of VirB8, whether fused to the periplasmic part of TraJ or VirB8. To further elucidate these points, we undertook a more detailed study of the interactions of these proteins by bacterial two-hybrid (BACTH) analysis. Here we show that the proteins VirB8, B8TraJ, TraJB8, and TraJ display strikingly different abilities to interact with themselves, as well as with VirB8 itself. Further, the TM domain of these proteins plays a crucial role in determining the strength of these interactions. Finally we show that, when overexpressed in a wild-type B. suis strain, these various proteins are able to modulate its virulence, even leading to complete loss of virulence. These data give interesting clues concerning the mechanisms of type IV secretion.Open in a separate windowFIG. 1.Schematic representation of the proteins used in this study. Light and dark gray parts represent protein domains from VirB8 and TraJ, respectively. All proteins were synthesized as fusion proteins with the T18 or T25 subunit of the B. pertussis adenylate cyclase domain fused to the amino-terminal end of the VirB8- and/or TraJ-containing part. In the case of the periplasmic domains of VirB8 (VirB8p) and TraJ (TraJp), synthesis of these proteins as fusions with the subunits of B. pertussis adenylate cyclase likely targets them to the bacterial cytoplasm, as suggested by fractionation studies. The rightmost column indicates whether the corresponding proteins have (+) or have not (−) been detected in Western blotting experiments.     

Type IV secretion system core component VirB8 from Brucella binds to the globular domain of VirB5 and to a periplasmic domain of VirB6

Type IV secretion systems are macromolecular assemblies in the cell envelopes of bacteria that function in macromolecular translocation. Structural biology approaches have provided insights into the interaction of core complex components, but information about proteins that undergo transient interactions with membrane components has not been forthcoming. We have pursued an unbiased approach using peptide arrays and phage display to identify interaction partners and interaction domains of type IV secretion system assembly factor VirB8. These approaches identified the globular domain from the VirB5 protein to interact with VirB8. This interaction was confirmed in cross-linking, pull-down, and fluorescence resonance energy transfer (FRET)-based interaction assays. In addition, using phage display analysis, we identified different regions of VirB6 as potential interaction partners of VirB8. Using a FRET-based interaction assay, we provide the first direct experimental evidence of the interaction of a VirB6 periplasmic domain with VirB8. These results will allow us to conduct directed structural biological work and structure-function analyses aimed at defining the molecular details and biological significance of these interactions with VirB8 in the future.     

Structure and Function of the Autolysin SagA in the Type IV Secretion System of Brucella abortus

A recent genetic study with Brucella abortus revealed the secretion activator gene A (SagA) as an autolysin component creating pores in the peptidoglycan (PGN) layer for the type IV secretion system (T4SS) and peptidoglycan hydrolase inhibitor A (PhiA) as an inhibitor of SagA. In this study, we determined the crystal structures of both SagA and PhiA. Notably, the SagA structure contained a PGN fragment in a space between the N- and C-terminal domains, showing the substrate-dependent hinge motion of the domains. The purified SagA fully hydrolyzed the meso-diaminopimelic acid (DAP)-type PGN, showing a higher activity than hen egg-white lysozyme. The PhiA protein exhibiting tetrameric assembly failed to inhibit SagA activity in our experiments. Our findings provide implications for the molecular basis of the SagA-PhiA system of B. abortus. The development of inhibitors of SagA would further contribute to controlling brucellosis by attenuating the function of T4SS, the major virulence factor of Brucella.     

Conservation of the Type IV Secretion System throughout Wolbachia evolution

The Type IV Secretion System (T4SS) is an efficient pathway with which bacteria can mediate the transfer of DNA and/or proteins to eukaryotic cells. In Wolbachia pipientis, a maternally inherited obligate endosymbiont of arthropods and nematodes, two operons of vir genes, virB3-B6 and virB8-D4, encoding a T4SS were previously identified and characterized at two separate genomic loci. Using the largest data set of Wolbachia strains studied so far, we show that vir gene sequence and organization are strictly conserved among 37 Wolbachia strains inducing various phenotypes such as cytoplasmic incompatibility, feminization, or oogenesis in their arthropod hosts. In sharp contrast, extensive variation of genomic sequences flanking the virB8-D4 operon suggested its distinct location among Wolbachia genomes. Long term conservation of the T4SS may imply maintenance of a functional effector translocation system in Wolbachia, thereby suggesting the importance for the T4SS in Wolbachia biology and survival inside host cells.     

TgaA,a VirB1-Like Component Belonging to a Putative Type IV Secretion System of Bifidobacterium bifidum MIMBb75

Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1-like gene, named tgaA. Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.     

In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.     

The Rcs Two-Component System Regulates Expression of Lysozyme Inhibitors and Is Induced by Exposure to Lysozyme

The Escherichia coli Rcs regulon is triggered by antibiotic-mediated peptidoglycan stress and encodes two lysozyme inhibitors, Ivy and MliC. We report activation of this pathway by lysozyme and increased lysozyme sensitivity when Rcs induction is genetically blocked. This lysozyme sensitivity could be alleviated by complementation with Ivy and MliC.In gram-negative bacteria, the cell envelope represents an important functional compartment that extends from the cytoplasmic membrane to the outer membrane and supports a number of essential processes, such as solute transport, protein translocation, and respiratory energy generation (15). In addition, the cell envelope accommodates the bacterial peptidoglycan layer, a distinct and structurally vital element of the cell. Most recently, Laubacher and Ades (10) have demonstrated that the Rcs phosphorelay system of Escherichia coli, originally described as regulator of capsule synthesis, is activated by β-lactam antibiotics that inhibit penicillin-binding proteins and consequently interfere with peptidoglycan synthesis. Moreover, mutational activation of the Rcs pathway provided significant protection against these antibiotics, indicating that members of this regulon can prevent or repair the peptidoglycan damage caused by β-lactam antibiotics (10).Interestingly, ivy and ydhA, two genes encoding specific lysozyme inhibitors, were found to reside under this Rcs regulon (8, 10). Ivy (inhibitor of vertebrate lysozyme, formerly known as YkfE) was discovered in 2001 as the first bacterial lysozyme inhibitor (1, 14), while the inhibitory activity of YdhA was only recently revealed by our research group (3). Although Ivy and YdhA are both able to inhibit c-type lysozymes, such as human lysozyme and hen egg white lysozyme (HEWL), they are structurally unrelated (1, 16). Interestingly, YdhA belongs to a group of proteins with a common conserved COG3895 domain that are widely spread among the Proteobacteria (3, 16). Unlike Ivy, which resides in the periplasm, YdhA is a lipoprotein and was therefore renamed MliC (membrane-bound lysozyme inhibitor of c-type lysozyme) (3).Given the elementary observation that the two currently known lysozyme inhibitors of E. coli are both part of the Rcs regulon that can in turn be induced by antibiotic-mediated peptidoglycan stress, we wondered whether Rcs induction could also result from exposure to lysozyme itself. To test this, we introduced a tolA knockout from MG1655 tolA (3) into strain DH300 that is equipped with a genomic rprA-lacZ fusion able to report Rcs activation (12), in order to increase outer membrane permeability for HEWL (Table ​(Table11 lists all strains). A stationary-phase culture of the resulting strain, designated LC100, was diluted 1/100 in 4 ml fresh LB medium with different final concentrations of HEWL (0, 5, 10, 25, and 50 μg/ml), and after 2.5 h of further growth at 37°C, β-galactosidase activity was measured (13). Interestingly, rprA-lacZ was significantly induced at HEWL concentrations of >10 μg/ml, up to 4.4-fold at 50 μg/ml (Fig. ​(Fig.1A).1A). This induction could be completely abolished upon the additional introduction of a knockout of rcsB (strain LC102), the response regulator required to activate gene expression in the Rcs pathway. Moreover, knocking out rcsF (strain LC101), the outer membrane lipoprotein sensor that triggers the Rcs pathway upon antibiotic-mediated peptidoglycan stress (10), also resulted in a loss of lysozyme induction. As a comparison, rprA-lacZ induction in DH300 treated with amdinocillin (Sigma-Aldrich, Bornem, Belgium), as previously described (10), resulted in a 16-fold increase in β-galactosidase activity (Fig. ​(Fig.1B).1B). Please note that the difference in basal β-galactosidase levels between LC100 and DH300 (Fig. 1A and B) is probably due to the tolA mutation in LC100, which is known to result in a higher basal expression of the Rcs pathway (5). These data clearly demonstrate that the Rcs phosphorelay can indeed be activated by exposure to lysozyme and that this induction is mediated by the outer membrane sensor rcsF. This also implies that the Rcs pathway responds to different types of peptidoglycan stress, as β-lactam antibiotics block the formation of peptide side-chain cross-links by binding irreversibly to the transpeptidases, while lysozyme hydrolyzes the heteropolysaccharide backbone.Open in a separate windowFIG. 1.Induction of the Rcs pathway in LC100 (tolA::Kn Rcs⁺) with different HEWL concentrations (0 to 50 μg/ml) (A) and in DH300 (Rcs⁺) with (+) or without (−) amdinocillin treatment (B). Rcs induction is measured as β-galactosidase activity originating from a genomic rprA-lacZ reporter fusion and expressed in Miller units (13). Error bars indicate standard deviations of results from three replicate experiments. The corresponding RcsB⁻ strain (LC102) and the RcsF⁻ strain (LC101) showed rprA-lacZ inductions of <10 Miller units when subjected to lysozyme treatments and are therefore not shown.

TABLE 1.

Bacterial strains and plasmids used in the study

Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems

The translocation of DNA across biological membranes is an essential process for many living organisms. In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells. The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals. In Gram-negative bacteria, the conjugation machines are composed of a cell envelope-spanning secretion channel and an extracellular pilus. These dynamic structures (i) direct formation of stable contacts-the mating junction-between donor and recipient cell membranes, (ii) transmit single-stranded DNA as a nucleoprotein particle, as well as protein substrates, across donor and recipient cell membranes, and (iii) mediate disassembly of the mating junction following substrate transfer. This review summarizes recent progress in our understanding of the mechanistic details of DNA trafficking with a focus on the paradigmatic Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation systems.