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1 Department of Medical Microbiology and Immunology, 1-63 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
2 National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada
3 Department of Medical Microbiology, Stanford University, CA 94305, USA
Correspondence
Diane E. Taylor
diane.taylor{at}ualberta.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The conjugative pilus initiates plasmid transfer from the donor cell by recruitment of recipient cells. A membrane-spanning protein complex called the mating pair formation (Mpf) complex, which constitutes the greater part of the conjugative type IV secretion system (T4SS), enables the production of conjugative pili, and facilitates the transfer of plasmid DNA from donor to recipient bacteria (for reviews, see Cascales & Christie, 2003, 2004; Lawley et al., 2003a). Plasmid DNA is processed to a transfer intermediate in the cytoplasm by the relaxosome, a DNA–protein complex containing the relaxase and accessory proteins (Furste et al., 1989). The T4SS also includes the coupling protein at the cytoplasmic membrane, and by serving as the link between the relaxosome and Mpf complex, the transferring DNA strand contacts the cell envelope and then proceeds onwards to the recipient cell.
The incompatibility group HI1 plasmid (IncHI1) R27 is a large (180 kb) self-transmissible resistance plasmid (Sherburne et al., 2000). There has been some confusion over the years as to the origin of R27, but Datta & Olarte (1974) have clearly distinguished it as a plasmid characteristic of Salmonella enterica serovar Typhi. However, R27 and a related plasmid Tp117 have been isolated from Salmonella typhimurium strain 1M1407 (Lawn et al., 1967; Smith et al., 1973). The proteins required for R27 transfer are located within two separate transfer regions, Tra1 and Tra2 (Lawley et al., 2002, 2003b). Encoded within Tra1 are the relaxosome, the coupling protein and part of the Mpf complex, and the remainder of the Mpf is encoded in Tra2. Interactions between the relaxosome and coupling protein have been identified in the IncW, IncP and IncF plasmid families (Disque-Kochem & Dreiseikelmann, 1997; Llosa et al., 2003; Schroder et al., 2002). The coupling protein then interacts with the Mpf/T4SS at the cytoplasmic membrane through the TraB/VirB10 protein (Gilmour et al., 2003; Llosa et al., 2003). The R27 coupling protein is termed TraG, and this family of proteins has been structurally characterized. The hexameric ring structure of the IncW plasmid-coupling protein TrwB resembles the tertiary structure of a number of P-loop ATPases, such as the F1 ATPase, suggesting that this complex is actively involved in DNA translocation (Gomis-Ruth et al., 2001).
Three other essential genes encoded in the Tra1 region (traI, traH and traJ) are not required for the production of the H-conjugative pilus, and consequently are not components of the Mpf complex (Lawley et al., 2002). The TraI protein shares homology with the relaxase family of proteins, whereas TraH contains a DNA-binding motif and an N-terminal coiled-coil domain (Lawley et al., 2002). TraJ homologues from a variety of genomic sources have been identified (Gilmour et al., 2004), and an alignment of these homologues has indicated that the predicted four transmembrane (TM) domains are well-conserved among this family of proteins. A conserved module of traI–traG–traJ has been identified in each of the genomic sources that encode a TraJ homologue (Gilmour et al., 2004). Few of the TraJ-encoding genomes encode R27-like Mpf genes, suggesting that the module of traI–G–J genes can be inherited independently from other transfer determinants.
The objective of the present study was to identify additional R27-encoded interaction partners for the coupling protein TraG. Here, we identified an interaction between TraJ and TraG, and propose that because of co-localization at the cytoplasmic membrane, binary heterologous interaction, and co-inheritance with conserved domain organization of known DNA translocases, TraJ is a protein which associates with the coupling protein TraG and is involved in DNA translocation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. All strains were grown in Luria–Bertani (LB) broth, Lennox formulation (Difco) at 37 °C with shaking at 200 r.p.m., unless otherwise stated. Antibiotics used in this broth were as follows: ampicillin (100 µg ml–1), kanamycin (50 µg ml–1), tetracycline (10 µg ml–1), rifampicin (50 µg ml–1), nalidixic acid (20 µg ml–1) and trimethoprim (25 µg ml–1). For the bacterial two-hybrid (BTH) assay, conjugative transfer proteins were fused to catalytic domain fragments of adenylate cyclase from Bordetella pertussis by cloning into the BTH vectors pKT25 and pUT18C (Karimova et al., 1998). Plasmids encoding fused proteins were co-transformed into competent BTH101 cells, processed and analysed as previously described (Gilmour et al., 2003). Liquid conjugative matings and complementation experiments with a derepressed derivative of R27, which contains an insertion in the htdA gene that causes a derepressed phenotype, were performed as described previously (Lawley et al., 2002; Taylor & Levine, 1980). Complementation experiments with the F plasmid were performed similarly, with the exception that donor and recipient cells were grown at 37 °C, and mating experiments were performed at 37 °C for 16 h. For solid matings, conjugative transfer of RP4 and R388 plasmids was performed as described by Bradley et al. (1980).
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. PCR products were amplified with terminal restriction enzymes added within the primer sequences (Table 2). Amplified DNA was initially cloned into pGEM-T (Promega), digested with appropriate enzymes, and subcloned into the expression vector pMS119EH/pMS119HE or the adenylate cyclase fusion vectors pKT25 and pUT18C. Restriction enzyme endonucleases were used according to the manufacturer's specifications, and digested products were resolved on agarose gels.
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, containing pMS119EH encoding each tagged coupling protein, was grown at 28 °C to OD600 0.5–0.7. The bacteria were induced for 1 h at 28 °C with IPTG at a final concentration of 0.4 mM. Whole-cell lysates were resolved on 10 % SDS-PAGE, and following transfer to nitrocellulose membranes, a primary mouse anti-His6 mAb (Invitrogen) and a secondary goat anti-mouse–horseradish peroxidase (HRP) antibody conjugate were used to probe for protein synthesis.
Immunoprecipitation.
Immunoprecipitation of R27 transfer proteins was performed as described by Couturier et al. (2006). Briefly, E. coli DH5 cells containing the plasmid pUT18C-TraJ were co-transformed with pBAD33-TraGFLAG or pBAD33-TrhBFLAG for the interaction studies. Cells were induced at mid-logarithmic growth phase (OD600 0.5–0.7) with 0.4 mM IPTG and 0.2 % arabinose for 1 h prior to cell lysis. Cells were harvested by centrifugation and resuspended in a threefold volume of lysis buffer [150 mM PBS, 7.15 % (w/v) sucrose, 1 % Nonidet P-40, 1 mM PMSF, 1/66 vol. 15 µg ml–1 lysozyme). The cells were frozen and thawed three times, and lysed cells were removed by centrifugation at 14 000 r.p.m. for 10 min. The supernatant was removed and anti-FLAG M2 agarose (Sigma) beads were added. The beads had been incubated at 4 °C overnight with rotation in a 5 % BSA solution to avoid non-specific precipitation. The supernatant and beads were incubated at 4 °C with rotation for 2 h. The supernatant was removed after centrifugation at 14 000 r.p.m. for 30 s, and the beads were washed three times with lysis buffer. The immunoprecipitate fraction was collected by resuspending the beads in 50 µl 1xLaemmli sample buffer (5 % 
-mercaptoethanol). Upon resolution of the cleared lysate and immunoprecipitation by 10 % SDS-PAGE, proteins were transferred to nitrocellulose and probed with the anti-adenylate cyclase mAb 3D1 (List Biological Laboratories). This mAb is specific for the distal portion of the adenylate cyclase catalytic domain (aa 377–399) (Lee et al., 1999).
Membrane preparations.
Overnight culture (20 ml) of E. coli DH5 (pUT18C-TraJ) with ampicillin was used to inoculate 500 ml LB–Lennox broth. Cells were grown to mid-exponential phase at 28 °C and 225 r.p.m., and induced with 0.4 mM IPTG for 20 min. Bacterial cells were harvested by centrifugation (7000 g, 7 min), resuspended in 10 ml buffer A [10 mM Tris/HCl, pH 7.6, 5 mM MgCl2, one Complete Protease Inhibitor Cocktail tablet (Roche), 1 mM PMSF, 100 U micrococcal nuclease, 1 mg RNase]. One milligram of lysozyme was added to cells for 30 min on ice, and cells were lysed using sonication for 3 min (Fisher model 300 sonicator). Unlysed cells were removed by centrifugation (twice at 6800 g for 10 min), and this preparation was called the whole-cell lysate. The cytoplasmic/periplasmic fraction was obtained by ultracentrifugation (100 000 g for 2 h at 4 °C) and the supernatant fraction was transferred to a new tube. The resulting pellet containing the membrane fraction was resuspended in 1 ml buffer B [55 % sucrose (w/v), 10 mM Tris/HCl, pH 7.6, 5 mM EDTA].
Immunodetection of proteins.
Protein preparations were boiled for 10 min and separated using 10 % SDS-PAGE, transferred to nitrocellulose membranes, and blocked overnight in 1 % (w/v) skimmed milk and 0.1 % Tween 20 in PBS. Membranes were probed with the anti-adenylate cyclase mAb 3D1, anti-DnaK mouse antibody (Stressgen) or anti-CpxA rabbit antibody (Raivio et al., 1999) for 1 h at room temperature, washed, and the secondary anti-mouse IgG or anti-rabbit IgG (Sigma) was applied. Detection was performed using the ECL kit (Amersham Biosciences) according to the manufacturer's instructions.
Phylogenetic analysis.
Protein sequences were aligned using CLUSTAL_W (Gonnet matrix, gap penalty=10, extension penalty=0.2) (Thompson et al., 1994), and phylogenetic trees were constructed using the unweighted pair group method with arithmetic mean (UPMGA) algorithm of MEGA3 (Kumar et al., 2004). To test the statistical significance of the tree, 500 bootstrap samples were generated from the alignment data.
Web-based computer programs.
PSI-BLAST (http://www.ncbi.nlm.nih.gov/blast/), ScanProsite (http://au.expasy.org/tools/scanprosite/), TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/), CLUSTAL_W (http://www.ebi.ac.uk/clustalw/) and EMBOSS pairwise local alignment (Blosum40 matrix, gap penalty=10, extension penalty=0.5; http://www.ebi.ac.uk/emboss/align/) were used.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Elegant studies have identified similar interactions between TrwB (coupling protein) and TrwE (VirB10 homologue) from the IncW plasmid R388, as well as VirB10 homologues in IncN and IncX plasmids (Llosa et al., 2003). In this study, BTH technology was used to investigate TraG and potential interactions with non-Mpf proteins TraJ, TraH and TraI, located contiguously in the Tra1 region of R27 (Lawley et al., 2002).
In vivo detection of the R27 TraG and TraJ interaction
The R27 relaxase gene traI, non-Mpf gene traH and non-Mpf gene traJ (encoding a putative membrane-associated transfer protein) were each cloned into the BTH vector pUT18C. The R27 coupling protein gene traG was cloned in the appropriate reading frame of the pKT25 vector, and the resulting gene product TraGAC25 was a fusion between the 78 kDa coupling protein and the C-terminal 25 kDa region of the adenylate cyclase catalytic domain. Screening for pairwise interactions using the BTH system did not identify an interaction that involved R27 relaxase TraI or TraH. However, co-production of R27 TraJAC18 with TraGAC25 allowed for functional complementation of the B. pertussis adenylate cyclase domains, which were initially screened by plating transformed BTH101 cells onto media containing X-Gal. More than 99 % of the colonies containing TraJAC18 with TraGAC25 constructs were blue within 48 h of plating. The BTH positive control (vectors containing leucine zippers) showed similar blue colonies, whereas the BTH101 cells containing empty BTH vectors were white. A standard Miller assay was used to quantify -galactosidase activity (Fig. 1). The in vivo interaction between TraJ and TraG was found to produce comparable levels of -galactosidase activity to those of the BTH leucine zipper-positive control (Fig. 1).
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), and to ensure that this interaction was not a non-specific interaction of membrane-spanning domains caused by overexpression of R27 transfer proteins, the R27 virB10 homologue trhB was cloned into the BTH vector pUT18C. The VirB10 family of proteins are cytoplasmic membrane-associated conjugative proteins that have been shown to interact with coupling proteins (Gilmour et al., 2003; Llosa et al., 2003). When TraJAC18 was co-produced with TrhBAC25 in BTH101, >99 % of the colonies were white after 48 h incubation on media containing X-Gal. The inability of TrhB to interact with TraJ implies that the productive TraG–TraJ interaction was not simply due to predicted co-localization of transfer proteins at the cytoplasmic membrane.
Immunoprecipitation of a TraG and TraJ complex
To confirm the interaction between the R27 coupling protein and TraJ, we purified R27-tagged protein complexes using an immunoprecipitation technique. The C termini of TraG and TrhB were fused with a FLAG octapeptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys tag. Cell lysates were prepared after co-production of TraJAC18 and TraGFLAG, and in the presence of Sepharose beads conjugated with anti-FLAG antibody, the TraJAC18 construct was precipitated by TraGFLAG (Fig. 2). The mAb 3D1, which recognizes an epitope in the 18 kDa adenylate cyclase fragment of B. pertussis (Lee et al., 1999), was used to detect precipitated TraJAC18. When TraGFLAG was omitted from the immunoprecipitation experiment, no TraJAC18 was recovered by the anti-FLAG bead matrix (Fig. 2), ensuring that TraJ was not bound non-specifically by a component of the immunoprecipitation reaction. Additionally, to ensure that the TraG and TraJ interaction was not due to overproduction of two membrane-associated proteins, TrhB was included in an immunoprecipitation experiment with TraJ. Although TraJAC18 was soluble and detectable in the cleared lysate prior to immunoprecipitation, TrhBFLAG did not precipitate TraJAC18 (Fig. 2). Furthermore, the inability of TraJ to interact with TrhB confirmed that the interaction of TraJAC18 and TraGFLAG was not due to non-specific interaction between the 18 kDa adenylate cyclase domain and TraGFLAG. The precipitation of a TraG–TraJ complex confirmed the BTH data, which showed that the R27 coupling protein interacted with the non-Mpf protein TraJ.
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220 aa), and contain four conserved hydrophobic TM regions (Gilmour et al., 2004). To determine if the predicted TM regions correlated with localization of TraJ to the cellular membrane, a fractionation study was performed on E. coli containing pUT18C-TraJ. Separation of the cytoplasmic membrane and outer membranes (M fractions) from the soluble cytoplasmic and periplasmic fractions (S fractions), and subsequent Western blot analysis revealed that TraJAC18 associated exclusively with the membrane fraction (Fig. 3A).
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replication (Liberek et al., 1988), and an anti-DnaK antibody identified this protein exclusively in the S fraction (Fig. 3B
). CpxA is a component of the Cpx pathway involved in the envelope stress response (see Raivio, 2005, for a review), and anti-CpxA antibody detected this protein exclusively in the M fraction (Fig. 3C).
Immunofluorescent analysis of the R27 coupling protein TraG has previously demonstrated a similar membrane localization (Gunton et al., 2005). Fluorescently labelled R27 coupling protein was visualized as discrete foci in the periphery of the cell. The discovery that TraJ is membrane bound and able to interact with the membrane-associated R27 coupling protein indicates that TraJ may act as an interacting partner for the coupling protein during conjugation.
Non-cognate coupling protein interactions with the IncH Mpf
Complementation of conjugative plasmids containing coupling protein mutations by non-cognate coupling proteins has indicated that the Mpf–coupling protein interaction is non-specific (Cabezon et al., 1994; Hamilton et al., 2000; Llosa et al., 2003). Furthermore, recent studies on the coupling proteins and VirB10 homologues from the IncW R388 plasmid, IncN plasmid pKM101 and IncX plasmid R6K have demonstrated that each coupling protein is able to interact with cognate and heterologous VirB10 proteins (Llosa et al., 2003). To expand upon these studies, the coupling proteins from the conjugative plasmids R27 (IncH), RP4 (IncP), F (IncF) and R388 (IncW) were cloned into the pUT18C BTH vector. When each coupling protein was co-produced in BTH101 cells with the R27 Mpf construct TrhBAC25, there were successful interactions between each coupling protein and the VirB10-homologue TrhB (Fig. 4A). The values obtained in the BTH studies of the interactions between the coupling proteins and TrhB were similar to those produced by the BTH leucine-zipper positive control. This seemed remarkable, given the low level of overall similarity shared between members of the coupling protein family. The coupling proteins from the RP4, R388 and F plasmids had similar levels of sequence identity with TraGR27: TraGRP4, 19.6 % identity over an alignment length of 749 aa; TraDF, 19.5 % identity over an alignment length of 845 aa; and TrwBR388, 20.1 % identity over an alignment length of 750 aa.
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). These data suggest that the coupling protein–TraJ interaction is specific to conjugative systems that encode traJ.
Because the coupling proteins TraGRP4, TraDF and TrwBR388 did not interact with TraJ in a BTH screen, and because TraJ is essential for conjugation (Lawley et al., 2002), it is unlikely that these non-cognate coupling proteins would be functionally interchangeable with the R27 coupling protein TraGR27 in context with the remainder of the R27 conjugative apparatus. To investigate this, E. coli DY330R cells containing an R27 traG insertional mutant were transformed with the expression vector pMS119 expressing C-terminal His-tagged TraGR27, TraGRP4, TraDF and TrwBR388. Immunoblot analysis confirmed expression of each coupling protein (data not shown), and RP4, R388 and F plasmids encoding coupling protein mutations were successfully complemented by the constructs expressing their cognate coupling proteins (data not shown). The TraG coupling protein from R27 was the only protein that restored conjugative transfer of the R27 traG mutant.
Functional co-inheritance of TraJ and TraG homologues
Bioinformatic analyses of the IncHI2 plasmid R478 have revealed that traG and traJ homologues are encoded by a number of genomic sources, including plasmids, conjugative elements and chromosomes (Gilmour et al., 2004). Similar genomic analyses were repeated, and adjacent determinants encoding proteins similar to TraG and TraJ were observed in 38 gamma- and beta-proteobacteria strains, including many members of the order Burkholderiales (Table 3). The newly observed TraJ homologues were all of a similar size (201–252 aa), and typically encoded four or five predicted TM domains. Phylogenetic analysis of TraJ homologues encoded within this co-inherited module revealed that the majority of the plasmid-encoded determinants clustered on a different node from the chromosomal determinants (Fig. 5). Not all of the presented sequences were fully annotated, so the genomic location (chromosome versus plasmid) was uncertain in some instances, although the majority of TraJ homologues encoded from a chromosomal source had an additional 30 aa at the N terminus of the protein, and the traJ and traG coding sequences overlapped (Table 3). The protein sequences similar to TraG were also examined, and a tree with very similar topology to that of TraJ was observed, except that IncP, IncF and IncW coupling proteins, and Ti plasmid-coupling plasmid orthologue VirD4 were included, and these were divergent from the IncH TraG-like sequences (Fig. 6). This comparison of coupling protein homologues indicates that the IncP, IncW and IncF, and Ti plasmid sequences are distinct from the IncH-type coupling proteins, and the requirement for TraJ as an interacting partner may partly explain the divergence of the IncH-type coupling proteins.
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). The diversity of this TraJ–TraG interaction was further characterized by using the BTH technology to determine if non-cognate TraJ homologues interacted with each coupling protein homologue. As expected, the highly related IncH plasmids R27 and R478 encoded TraJ homologues that were interchangeable in interactions with their coupling proteins (Fig. 6). The R391 plasmid-encoded TraJ and TraG homologues were not able to interact with the respective proteins from the other three genomic sources. Intriguingly, the coupling protein homologue from the P. fluorescens genome showed a strong interaction with TraJ from R27. In contrast, there was no evidence of an interaction with the opposite configuration, in which TraG from R27 was tested with the P. fluorescens TraJ homologue.
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; Wu et al., 1995). These DNA translocase ATPases are essential for coordinating chromosome segregation with cell division (FtsK; Weiss, 2004), or for mediating DNA partitioning during the process of sporulation (SpoIIIE; Wu & Errington, 1994). The similarity to conjugative coupling proteins is located primarily within the region surrounding the Walker A and B boxes, which are essential for nucleoside triphosphate binding and hydrolysis (Walker et al., 1982). At the N terminus of the DNA translocases, R27-encoded TraJ is 27 % identical over 110 aa, and has 20.1 % identity over an alignment length of 249 aa to Streptococcus pneumoniae SpoIIIE (GenBank accession no. NP_345365). This region of the DNA translocases contains between four and five TM domains that target SpoIIIE and FtsK to the site of cell division (Bath et al., 2000; Sharp & Pogliano, 2003; Wang & Lutkenhaus, 1998). This coincides with the well-conserved TM domains of TraJ, and an alignment of R27-encoded TraJ and TraG to FtsK of Shewanella oneidensis illustrates the domains with which the conjugative transfer proteins share homology (TraJ, 21.4 % identity over an alignment length of 243 aa; TraG, 21.1 % identity over an alignment length of 913 aa) (Fig. 8).
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ACKNOWLEDGEMENTS |
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Edited by: C. W. Chen
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 16 August 2006;
revised 16 October 2006;
accepted 16 October 2006.
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