1 Departamento de Biología Molecular (Unidad Asociada al CIB-CSIC), Universidad de Cantabria, C. Herrera Oria s/n, 39011 Santander, Spain
2 Division of Molecular Microbiology, Biozentrum of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
Correspondence
Matxalen Llosa
llosam{at}unican.es
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ABSTRACT |
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Present address: Laboratory of Immunogenetics, NIAID/NIH, 12441 Parklawn Drive, Rockville, MD 20852, USA.
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INTRODUCTION |
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In spite of the differences between cT4SS and pT4SS in the nature of the secreted substrate (nucleoprotein vs protein complex), in their biological role (DNA transfer vs virulence), and in the target cell (prokaryotic vs eukaryotic), there is homology among them. Most T4SS are formed by 11 proteins, named VirB1 to VirB11 for the components of the prototypical At T4SS. The overall architecture of the transporter is conserved in the family, as shown by a common gene organization (see Fig. 1), the same membrane topology of each component, and conservation of proteinprotein interactions between the T4SS components. For instance the VirB7VirB9 interaction has been described for the T4SS of At (Baron et al., 1997
; Das et al., 1997
; Spudich et al., 1996
), Bordetella pertussis (Farizo et al., 1996
), Bartonella henselae (Shamaei-Tousi et al., 2004
) and Xanthomonas axonopodis (Alegria et al., 2005
).
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The other components that make up a T4SS can be summarized as follows (for a review and update, see Cascales & Christie, 2003; Llosa & O'Callaghan, 2004
): two highly conserved inner-membrane-bound NTPases, VirB11 and VirB4, are involved in early substrate transfer reactions (Atmakuri et al., 2004
); VirB2 and VirB5 are pilus components (Lai & Kado, 1998
; Schmidt-Eisenlohr et al., 1999
); VirB6 is an integral membrane protein required for T4SS assembly and function (Hapfelmeier et al., 2000
; Jakubowski et al., 2004
); VirB1 is a lytic transglycosylase required for T4SS early assembly (Koraimann, 2003
), although not an essential component in all systems; finally, the function of the outer-membrane associated protein VirB3 is unknown.
Many T4SS, including all cT4SS, have an associated coupling protein (T4CP). T4CPs are proteins anchored to the inner membrane through their N-terminus, so named because they interact both with the secretion substrate and with the secretion machinery (Cabezón et al., 1997; Llosa et al., 2003
). Besides their coupling role, they may act as DNA pumps during conjugation (Gomis-Rüth et al., 2001
; Tato et al., 2005
). Specific proteinprotein interactions between the T4CP and cT4SS components have now been described. TrwB, the T4CP of conjugative plasmid R388, interacts with proteins TrwC and TrwA, which bind to the substrate DNA, and with protein TrwE, a VirB10 homologue (Llosa et al., 2003
). Moreover, T4CPs interact with VirB10 homologues from heterologous cT4SS so that they can deliver their substrate DNA through the heterologous transporter, with efficiencies that correlate with the strength of the corresponding T4CPVirB10 interaction (Llosa et al., 2003
). Interactions of the At T4CP have been described with a protein substrate, protein VirE2, and with the T4SS NTPases VirB4 and VirB11 (Atmakuri et al., 2003
, 2004
).
Functional complementation between homologues from different T4SS has been reported in a few instances: the VirB5 homologues of two cT4SS, TraC protein of conjugative plasmid pKM101 and VirB5 of At T4SS (Schmidt-Eisenlohr et al., 1999); and some VirB1 homologues, but not others, could be exchanged (Hoppner et al., 2004
). The most related T4SS systems described to date are the Trw systems found in the conjugative plasmid R388 and in Bartonella spp. [B. henselae and B. tribocorum (Bt)], which are a cT4SS and a pT4SS respectively. Identities among the Trw components of each system range from 25 to 80 %; the core components share more than 50 % identity. Functional complementation was observed between the TrwD and TrwH components of both systems (Seubert et al., 2003
), underscoring the close relationships between T4SS even when their biological role involves very different processes. Thus, the Trw T4SS of R388 and Bartonella spp. are probably the best candidates to obtain a hybrid c/pT4SS which ideally could be used to deliver DNA into the eukaryotic host cells (Llosa & de la Cruz, 2005
).
In this work we undertook an extended analysis of T4CPT4SS interactions and showed that T4CPs also interact with the VirB10-like component of several pT4SS. This interaction reflects a functional interaction in the case of the Trw T4SS, as shown by functional complementation between the respective T4SS components. We performed a complementation analysis between these two systems in order to obtain information about the building blocks of a T4SS that can be exchanged. Our results support the concept of a core complex of highly conserved components that can be substituted, while the peripheral components are more specific for their host/function.
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METHODS |
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To produce wild-type proteins R388-TrwE (pHP102), Bt-TrwE (pHP100) and Bs-VirB10 (pHP101), the corresponding genes were amplified with oligonucleotides that introduced SalI and EcoRI sites and the digested products were inserted into the same sites of vector pSU24. Expression of the inserted genes was dependent on the vector lac promoter.
Plasmid pFJS134 was constructed by insertion of an EcoRIHindIII fragment from pSU4051 carrying the R388 oriTtrwABC region into the same sites of broad-host-range vector pRL662. Plasmid pFJS193 contains a 3·6 kb fragment from plasmid pSU4814 (Núñez & de la Cruz, 2001) cloned into the EcoRIHindIII sites of pRL662; this fragment contains the mobilization region of CloDF13.
To obtain separately the two putative R388 trw operons present in pSU4058, we used plasmid pSU4105 (Table 2), which carries a Tn5tac1 insertion at coordinate 4101 (GenBank sequence X81123), in the intergenic region between the two proposed transcripts. The insertion lies between trwH and trwI and does not affect any trw gene, and in fact it was the only mutant obtained that retained sensitivity to the pilus-specific phage PRD1 (data not shown). Since the transposon has several restriction sites close to both ends and pSU4058 has HindIII sites at both ends of the R388 trw region, the 5·5 kb EcoRIHindIII fragment and the 4 kb BglIIHindIII fragment from pSU4105 contained the korAtrwI and trwHtrwD regions of R388, respectively. Plasmid pHP111 was constructed by insertion of the EcoRIHindIII fragment from pSU4105 into the same sites of vector pHG329. Plasmid pHP109 carries the R388 trwHtrwD operon in vector pSU18. Since this operon includes a Kil function (Bolland et al., 1990
), the plasmid was constructed in two steps: first, the korA gene from R388 was PCR-amplified and cloned into the EcoRIBamHI sites of vector pSU18, selecting for the orientation that allows KorA expression from the vector lactose promoter; second, the BglIIHindIII fragment from pSU4105 was inserted into the BamHIHindIII sites of the previous construction. These plasmids were maintained in strain D1210, which has a chromosomal lacIq gene, to avoid toxicity by expression of the trw genes from the vector lac promoter.
Two-hybrid assay.
Strain DHM1 was co-transformed with plasmids bearing a T25 and a T18 fusion. Three independent transformants were grown together overnight in liquid medium at 30 °C, then 10 µl samples of these cultures were spread on sectors of X-Gal-containing plates to observe and compare the blue colour. -Galactosidase levels were measured on 100 µl samples as described by Miller (1992)
. All experiments included positive and negative controls. Plasmid pSU4111 (Moncalián et al., 1997
), which carries lacZ under the control of the lactose promoter, produced about 6000 Miller units in this system.
Quantitative mating assays.
Samples (100 µl, or 1 ml in the case of matings involving Bs strains) of overnight cultures of donor and recipient strains were mixed; cells were collected and placed on 0·22 µm filters on prewarmed agar plates for 1 h at 37 °C. When indicated, IPTG (0·5 mM) was added to the agar plate. To induce expression of Bs virB genes by low pH (Boschiroli et al., 2002), the Bs pellets were resuspended in 1 ml MM broth at pH 4·5 (Rouot et al., 2003
), and incubated for 4 h at 37 °C with shaking prior to mating. Plating was done in selective medium for both donor cells and transconjugants. Transfer frequencies are expressed as the number of transconjugants per donor cell. The data reported are the mean of at least two independent assays. Mean values were calculated as simple means of the logarithm of the frequencies obtained, followed by calculation of the anti-logarithm of this mean value.
Transposon mutagenesis.
In vivo insertional mutagenesis with transposon Tn5tac1 was carried out as described by Chow & Berg (1988) on plasmid pSU4058. Selected transposon insertions were then transferred to plasmid pSU1425 by homologous recombination using strain JC7623, as previously described (Llosa et al., 1994
).
Bacteriophage PRD1 sensitivity assays.
Sensitivity to the pilus-specific phage PRD1 was assayed as previously described (Bolland et al., 1990).
Cell extracts for protein analysis.
Bacterial cultures (50 ml) in early stationary phase were harvested at 4500 r.p.m. for 15 min. Bacterial pellets were resuspended in 500 µl PBS buffer containing 1 mM EDTA, 1 mM PMSF and 10 mM benzamidine. The suspensions were transferred to chilled FASTPREP tubes containing glass beads (lysing matrix B) and cells lysed in a FASTPREP FP120 instrument (Bio 101 Thermo Savant) at a speed of 6·0 for two cycles of 30 s. Cell supernatants were harvested by centrifugation at 13 000 r.p.m. for 15 min at 4 °C. Protein concentrations were quantified by the Bradford method using decimal dilutions of BSA as a standard, and kept frozen at 80 °C.
Immunoblot analysis.
Samples (20 µg) of total protein from each of the Bs cell lysates were run in 12 % (w/v) SDS-PAGE. After the run, protein samples were transferred from the SDS-PAGE gel to a PVDF membrane (Bio-Rad) (Towbin et al., 1979), and blocked using 3 % (w/v) bovine serum albumin (Sigma) in TBST. Incubation with primary antibody was performed for 1 h at room temperature using rabbit polyclonal antibodies at the following dilutions: anti-TrwC (Grandoso et al., 1994
), 1 : 10 000; anti-TrwD (Rivas et al., 1997
), 1 : 5000; anti-TrwF (Sastre, 1996
; Seubert et al., 2003
), 1 : 20 000. The secondary antibody was goat anti-rabbit IgG horseradish peroxidase conjugate (1 : 2000 dilution) (Pierce). Blots were then developed with SuperSignal West Dura Extended Duration Substrate (Pierce), and either exposed to Hyperfilm MP (Amersham), or quantified by using the Chemidoc system and Quantity One software (Bio-Rad).
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RESULTS |
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Fig. 2 shows a summary of the results obtained. All interactions were made reciprocal (with each protein fused to the T25 or the T18 Cya domains) and no significant differences were found in any of the pairs (data not shown). The T4CP protein At VirD4 did not interact with any of the other fusion proteins. We do not have a way of testing the integrity of the protein, so no conclusions can be drawn from this negative result. The remaining constructions tested showed interactions with one another. VirB10 homologues interacted with themselves and with each other, as previously observed for the VirB10 members of cT4SS (Llosa et al., 2003
). The main result was the existence of interactions between the conjugative T4CPs (TrwB, TraJ and TaxB) and the VirB10 components of pT4SS (At, Bt and Bs). Interactions with their cognate VirB10 homologues (TrwE, TraF and PilX10) are also shown for comparison, although in the absence of protein quantification the results are considered only qualitative.
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With respect to heterologous complementations, we did not detect significant complementation with Bs-VirB10. However, there was a low but consistent complementation by Bt-TrwE, the closest homologue to R388-TrwE. The transfer efficiency of the R388 trwE mutant when complemented with Bt-TrwE was 100 times lower than when complemented with R388-TrwE. Similar frequencies were obtained in the presence of IPTG (not shown). Thus, the interaction observed between R388-TrwB and Bt-TrwE could reflect a functional interaction since Bt-TrwE can partially substitute for R388-TrwE.
Analysis of the R388 Trw region
The DNA sequence of the R388 T4SS genetic region (GenBank accession no. X81123) includes 11 genes, named trwN to trwD, with homology to virB1 to virB11 respectively, plus four additional ORFs presumably involved in entry exclusion (eex) and regulation functions (korA, orf34 and korB) (Bolland et al., 1990 and our unpublished results). Fig. 1
shows a detailed map of this region. The role of trwN in R388 conjugation remains to be determined, since full transfer efficiency was obtained without this gene (Bolland et al., 1990
). Analysis of the DNA sequence suggested that this region is organized in four operons: korB (which is transcribed in the opposite direction to the rest of the genes), trwNorf34, korAtrwI, and trwHtrwD. This assumption is based first on the arrangement of the genes within each proposed transcript, suggesting translational coupling, while two intergenic regions of more than 100 bp are found between each proposed transcript (225 bp between trwN and korA, and 112 bp between trwI and trwH); second, on the presence of sequences with homology to the consensus promoter 5' of each proposed transcript; and third, the three putative promoter regions are defined by the presence of kor boxes' (shown as dashed vertical lines in Fig. 1
), presumed transcription control sites by the Kor proteins (R. Fernández, C. Revilla, M. P. Garcillán & F. de la Cruz, unpublished), that are shared by the homologous T4SS of plasmid pKM101 and Bt-Trw (More et al., 1996
; Seubert et al., 2003
). From now on we will refer to the two putative operons containing the R388 T4SS genes as trw region 1 (korAtrwI) and trw region 2 (trwHtrwD).
In order to analyse this R388 region in detail, we obtained Tn5tac1 insertion mutants along the R388 DNA segment present in plasmid pSU4058, which includes trw regions 1 and 2. Tn5tac1 insertions have been shown to be non-polar (Llosa et al., 1991), so mutations are expected to affect only the target ORF, unless high levels of expression of the gene are required for its normal functioning. Insertions were obtained in all trw genes except for trwG and trwM. A trwG mutant was obtained from a previous collection of mutants by insertion of interposon
along the trw region (Bolland et al., 1990
). All insertions affecting a trw gene conferred resistance to the pilus-specific phage PRD1. Selected insertions mapping in each gene (shown in Fig. 1
) were transferred to R388 by homologous recombination to test for their conjugation frequency (see relevant plasmid pairs in Table 4
). The trwG mutation was already in a plasmid containing the whole R388 transfer region. The nine mutants with insertions in different trw genes were transfer-deficient, except the mutant in trwH, which conjugated about 1000-fold less efficiently than wild-type (Table 4
). All were complemented to wild-type frequencies by pSU4058, which provides the whole korAtrwD region (Table 4
). We confirmed the non-polar character of these mutants by showing full complementation of mutant pairs in adjacent genes in all cases (e.g. pSU4130+pSU4064, see Table 4
; data not shown).
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We also assayed complementation of the R388 insertion mutants by each putative trw operon separately, as shown in Table 4. All mutants were complemented to some extent; however, complementation levels were significantly lower than when complemented by pSU4058. When both pHP109 and pHP111 plasmids were present, complementation levels were higher, but still about 10 times lower than when complemented by pSU4058.
Functional interactions between R388 cT4SS and Bs pT4SS
In order to detect possible functional relationships between the R388 and Brucella T4SS, a series of matings were performed using Bs strains as donors harbouring R388 or its mobilizable derivatives. R388 conjugated from Bs as efficiently as from Ec (about 101 transconjugants per donor). None of the R388 insertion mutants was mobilized from Bs significantly better than from Ec donors (data not shown), suggesting that individual T4SS components cannot be exchanged between the two T4SS. We did not detect conjugative transfer of a plasmid carrying the R388 oriTtrwABC region (pFJS134) through the intact Bs T4SS. We assayed mobilization of pFJS193, a plasmid containing the mobilization region of plasmid CloDF13, in order to test if Bs-T4SS could be used by a mobilizable plasmid rather than a conjugative system, since the former are more flexible in the use of T4SS. pFJS193 was not mobilized by the Bs T4SS either.
We assayed a Bs virB5 mutant that contains a polar mutation in virB5 (Boschiroli et al., 2002; O'Callaghan et al., 1999
). Thus, in this strain presumably only the VirB1 to VirB4 proteins are produced and no functional T4SS is assembled. Consequently, the strain is avirulent in cellular models of infection (O'Callaghan et al., 1999
). When R388 was mobilized from this strain, the transfer frequency dropped drastically to 106 transconjugants per donor. We determined R388 Trw protein levels in both wild-type and virB5 Bs strains harbouring R388. Fig. 3
shows immunoblots of cell extracts from Bs strains probed with anti-TrwC, anti-TrwD and anti-TrwF antibodies. It can be observed that, while the amount of TrwC (the conjugative relaxase) remains constant in both wild-type and virB5 strains, the levels of the T4SS components TrwD and TrwF are diminished significantly. We calculated a 17-fold decrease in the amount of TrwD in the Bs virB5 mutant compared to wild-type. Quantification of TrwF was not accurate due to non-specific binding of the anti-TrwF antibody to proteins of a size similar to TrwF (Fig. 3
).
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DISCUSSION |
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Our model cT4SS is the R388 Trw system. Its T4SS genetic determinant comprises genes trwLtrwD (Fig. 1). The adjacent gene trwN is a homologue of the VirB1-type components of other T4SS; however, to date there is no evidence that trwN is required for R388 conjugal transfer (Bolland et al., 1990
), so we have excluded this gene from our analysis. Non-polar insertion mutations proved that genes trwD, E, F, G, I, J, K and L are essential for R388 conjugation; an insertion in trwH renders a plasmid that can self-transfer with very low efficiency (Table 4
). Analysis of the DNA sequence strongly suggests that the trwLD genes are expressed from two transcriptional units: korAtrwI and trwHtrwD. The functionality of the promoter upstream of trwH has been shown for the homologous regions in Bt-Trw and conjugative plasmid pKM101 (More et al., 1996
; Seubert et al., 2003
). We have shown that expression of each putative operon from different replicons allows the assembly of a functional T4SS, as tested by high-efficiency mobilization of a plasmid carrying R388 oriTtrwABC (pFJS134).
It was previously reported that conjugative T4CPs could interact with VirB10 homologues from other cT4SS, and moreover the strength of this interaction affected the efficiency of DNA mobilization (Llosa et al., 2003). We extended the analysis of this interaction to the pT4SS of At, Bt and Bs. By two-hybrid analysis we have shown that conjugative T4CPs interact similarly with VirB10 homologues from pT4SS. VirB10 proteins interact with themselves and with each other, from both pT4SS and cT4SS. Thus, these interactions are conserved even in T4SS that do not have any known T4CP, such as Bt and Bs. In the case of Bt, it cannot be excluded that the T4CP from the VirB T4SS is being used by both coexisting T4SS (Schröder & Dehio, 2005
).
The interaction detected between TrwB and Bt-TrwE (the closest TrwE homologue according to the amino acid sequence) may also reflect a functional interaction since Bt-TrwE could partially complement an R388-trwE mutation. This complementation adds evidence to previous work showing that Bt-trwD and Bt-trwH could complement trwD and trwH mutations in R388 (Seubert et al., 2003), underscoring the functional similarity between the R388- and Bt-Trw T4SS. This prompted us to extend the complementation analysis between these two systems. As shown in Table 5
, several R388 trw mutants could conjugate at low efficiency in the presence of pAB2, a cosmid providing the whole Bt-trw region. It is noteworthy that complementation is observed only for genes belonging to R388 trw region 2. The conservation of gene synteny is frequently due to the need for co-expression of gene products that strongly depend on each other for function. Region 2 encodes the more conserved elements of the T4SS apparatus, the core components, which may play a similar function in both systems and could thus be exchanged to a certain extent. On the other hand, the T4SS components encoded in region 1 may be system-specific. For instance, the pilus components (TrwL and TrwJ) could be responsible for specific interactions with the recipient cells.
Possible functional relationships between R388-Trw and Bs-VirB T4SS were also addressed. Bs T4SS did not complement any of the R388 T4SS individual mutants, not surprisingly considering that the similarity between these two T4SS is significantly lower than that between the R388- and Bt-Trw T4SS. However, when a Bs virB5 polar mutant was used as a donor, a strong dominant negative effect was exerted on R388 transfer. Dominant negative effects are typical of proteins that make oligomers. The results suggest that when components of the Bs-T4SS cannot assemble into their own T4SS, they interact with the related R388 Trw proteins, resulting in non-functional heteromultimers. The unassembled Trw proteins are probably destabilized, leading to diminished cellular levels of the R388 T4SS proteins (Fig. 3). The poisoning effect could be mediated for example by VirB2, the homologue to the major pilus component. Pili are made up of a high number of pilin subunits and interference by a low number of heterologous pilin subunits could impede T4SS assembly, thus explaining the strong interference observed.
The existence of interactions between cT4SS and pT4SS components opens up the attractive possibility that DNA substrates recruited by the T4CPs could be coupled to pT4SS, so pathogens could be used as intracellular DNA delivery tools. So far, our attempts to use the pT4SS of both Bs and Bt to mobilize R388 derivatives lacking their cognate T4SS have had no success. Substrate selection by T4SS probably depends on more than a single proteinprotein interaction. Each T4SS component interacts with several other components of the secretion machinery (Ward et al., 2002). Recent results on the At T4SS show that formation of the VirB9VirB10 complex is essential for T-DNA substrate selection and translocation through the distal portion of the secretion channel (Cascales & Christie, 2004a
, b
; Jakubowski et al., 2005
). The partial functional exchanges found between the R388 and Bt T4SS open up the way for mutagenesis experiments in order to obtain mutant T4CPs that better interact with selected pT4SS components or subassemblies. The study of these two highly related T4SS will also help in understanding the similarities and differences between cT4SS and pT4SS.
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ACKNOWLEDGEMENTS |
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Received 4 August 2005;
revised 12 September 2005;
accepted 15 September 2005.
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