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Published in final edited form as: Mol Microbiol. 2004 Dec;54(5):10.1111/j.1365-2958.2004.04345.x. doi: 10.1111/j.1365-2958.2004.04345.x

Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion

Krishnamohan Atmakuri 1, Eric Cascales 1, Peter J Christie 1,*
PMCID: PMC3869561  NIHMSID: NIHMS537354  PMID: 15554962

Summary

Bacteria use type IV secretion systems (T4SS) to translocate DNA (T-DNA) and protein substrates across the cell envelope. By transfer DNA immunoprecipitation (TrIP), we recently showed that T-DNA translocates through the Agrobacterium tumefaciens VirB/D4 T4SS by forming close contacts sequentially with the VirD4 receptor, VirB11 ATPase, the inner membrane subunits VirB6 and VirB8 and, finally, VirB2 pilin and VirB9. Here, by TrIP, we show that nucleoside triphosphate binding site (Walker A motif) mutations do not disrupt VirD4 substrate binding or transfer to VirB11, suggesting that these early reactions proceed independently of ATP binding or hydrolysis. In contrast, VirD4, VirB11 and VirB4 Walker A mutations each arrest substrate transfer to VirB6 and VirB8, suggesting that these subunits energize this transfer reaction by an ATP-dependent mechanism. By co-immunoprecipitation, we supply evidence for VirD4 interactions with VirB4 and VirB11 independently of other T4SS subunits or intact Walker A motifs, and with the bitopic inner membrane subunit VirB10. We reconstituted substrate transfer from VirD4 to VirB11 and to VirB6 and VirB8 by co-synthesis of previously identified ‘core’ components of the VirB/D4 T4SS. Our findings define genetic requirements for DNA substrate binding and the early transfer reactions of a bacterial type IV translocation pathway.

Introduction

The bacterial type IV secretion systems (T4SS) translocate DNA and protein macromolecules to a diverse range of bacterial and eukaryotic cell types. These systems are classified on the basis of an ancestral relatedness to bacterial conjugation systems (Christie, 2004; Llosa and O’Callaghan, 2004). Agrobacterium tumefaciens uses an archetypal T4SS to translocate oncogenic T-DNA and effector proteins to susceptible plant cells, ultimately inciting Crown Gall disease on a variety of plant hosts (Zhu et al., 2000). This T4SS, composed of the VirD4 type IV coupling protein (T4CP) and the VirB mating pair formation (Mpf) proteins, shares a common ancestry and functional properties with the conjugation systems of the pKM101 (IncN), R388 (IncW), RP4 (IncP), F (IncF) and R27 (IncH) plasmids of Gram-negative bacteria (Christie, 2004; Llosa and O’Callaghan, 2004). The VirB/D4 T4SS and related systems encode an extracellular pilus for establishing donor – recipient cell contacts, and a transenvelope ‘mating channel’. Although conjugative pili have been extensively characterized (Eisenbrandt et al., 1999; Lai et al., 2000; Lawley et al., 2003), at present there is little mechanistic understanding about how T4SS convey substrates across the bacterial cell envelope.

To better define the architecture and dynamic action of the VirB/D4 T4SS, we recently developed an in vivo formaldehyde cross-linking – immunoprecipitation assay, termed transfer DNA immunoprecipitation (TrIP) (Cascales and Christie, 2004). This assay permits isolation of DNA substrate–channel subunit complexes formed during substrate translocation. Results of the initial TrIP studies showed that the DNA transfer intermediate establishes close contacts with six T4SS subunits: VirD4, VirB11, VirB6, VirB8, VirB2 and VirB9. Further studies of T4SS mutants, coupled with available data describing the subcellular locations of the VirD4 and VirB proteins, led to formulation of a DNA translocation pathway described in terms of a temporally and spatially ordered series of substrate–channel subunit interactions. In the first step of the pathway, the DNA substrate is recruited to the T4SS by the VirD4 T4CP, a member of a family of ATP-binding subunits ubiquitously associated with bacterial conjugation systems (Llosa et al., 2002). VirD4 next transfers the substrate to VirB11, an ATPase localized at the cytoplasmic face of the inner membrane. In turn, the substrate is delivered to two inner membrane subunits, the polytopic VirB6 and bitopic VirB8 proteins. A third ATP-binding subunit, VirB4, does not interact with the DNA substrate, but is required for substrate transfer to the VirB6 and VirB8 subunits. Finally, VirB6 and VirB8 function co-ordinately to deliver substrate to the periplasmic and outer membrane-associated proteins, VirB2 and VirB9, for passage to the cell exterior (Cascales and Christie, 2004).

By TrIP, we showed that the three energetic components, VirD4, VirB11 and VirB4, mediate successive early steps of the postulated T-DNA translocation pathway – VirD4 T4CP binds substrate and transfers it to VirB11, whereas VirB4 co-ordinates substrate transfer to VirB6 and VirB8 (Fig. 1). These findings raised a fundamental question, namely, do the energetic components mediate the early substrate transfer reactions through utilization of ATP? Here, we present evidence that VirD4, VirB11 and VirB4 interact with each other, and that they promote substrate transfer by both ATP-independent and -dependent mechanisms. Additionally, through TrIP analyses of strains synthesizing various subsets of VirB proteins, we show that in addition to the energetic components, subunits of a transenvelope ‘core’ structure must also be synthesized for transfer of substrate from VirD4 to VirB11 and then to VirB6 and VirB8. We discuss our findings in the context of two alternative models depicting how the transfer intermediate is conveyed across the inner membrane.

Fig. 1.

Fig. 1

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Effects of Walker A mutations on substrate transfer through the VirB/D4 T4SS.

A. Effects of null mutations and Walker A mutations of the three energetic subunits on substrate transfer through the postulated translocation pathway.

B. T-strand interactions with T4SS subunits from strains producing Walker A mutant proteins as shown by TrIP. Strains: wild type (WT), A348; ΔB4(B4KQ), PC1004(pKA102) producing VirB4K439Q; ΔB11(B11KQ), PC1011(pSR40) producing VirB11K175Q; ΔD4(D4KQ), Mx355(pKA101) producing VirD4K152Q. Strains were treated in vivo with formaldehyde before lysis. Antibodies listed on the left were used to immunoprecipitate (IP) the cognate Vir protein and associated DNA substrate; S, supernatant after precipitation; P, precipitate. Samples were assayed for T-strand (T) and the pTi control fragment (C) by PCR amplification and agarose gel electrophoresis.

C. Results of quantitative TrIP (QTrIP) assay. Bars in the histogram represent the amount of T-strand recovered with antibodies to a given T4SS subunit (listed on the x-axis) from a mutant strain (identified above each histogram panel) relative to that recovered with the same antibody from the isogenic WT strain (normalized to 1.0). Strains: ΔB4, PC1004; ΔB4(B4), PC1004(pKA93) producing VirB4; ΔB4(B4KQ), PC1004(pKA102) producing VirB4K439Q; ΔB11, PC1011; ΔB11(B11), PC1011(pSR45) producing VirB11; ΔB11(B11KQ), PC1011(pSR40) producing VirB11K175Q; ΔD4, Mx355; ΔD4(D4), Mx355(pKA42) producing VirD4; ΔD4(D4KQ), Mx355(pKA101) producing VirD4K152Q. Note that the VirD4 antibodies precipitated T-strand from Mx355(pKA42) or Mx355(pKA101) at levels approximately three- or twofold higher than from the WT strain. These strains overproduce the VirD4 proteins as a result of gene expression from a pBBR1-based replicon, whose copy number is estimated to be several-fold higher than the native pTi plasmid (Kovach et al., 1994).

Results

Effects of Walker A mutations on T-DNA transfer and complex formation

Strains lacking VirD4, VirB11 or VirB4 display substrate transfer arrests at three successive steps of the postulated T-DNA translocation pathway, as shown with the TrIP assay (Fig. 1A) (Cascales and Christie, 2004). Moreover, previous studies have shown that mutations of invariant residues within the nucleoside triphosphate binding site (Walker A motif) of each of these energetic components block T-DNA transfer to recipient cells (Berger and Christie, 1993; Stephens et al., 1995; Kumar and Das, 2002). Similar mutations in homologues of these VirB proteins also abolish gene transfer and, additionally, the purified mutant proteins show defects in ATP binding and/or hydrolysis in vitro (Krause et al., 2000; Rabel et al., 2003; Schroder and Lanka, 2003). By use of the TrIP assay and a more sensitive and quantitative version of the TrIP assay, here designated QTrIP, we confirmed first that the ΔvirD4, ΔvirB11 and ΔvirB4 mutations completely block substrate binding to the T4CP and substrate transfer to VirB11, VirB6 and VirB8 respectively (Fig. 1C, left). Second, expression of wild-type virD4, virB11 and virB4 in trans restored substrate transfer to all channel subunits at or above wild-type levels (Fig. 1C, middle). We next determined the effects of Walker A mutations on substrate transfer through the T4SS (Fig. 1B and C, right).

Remarkably, the VirD4K152Q mutant protein retained the capacity to interact with the T-DNA and to efficiently transfer the substrate to native VirB11. The VirB11K175Q mutant also efficiently bound substrate delivered from native VirD4. These findings, together with well-documented effects of Walker A mutations on ATP utilization cited above, suggest that VirD4 substrate binding and substrate transfer to VirB11 proceed independently of ATP binding and/or hydrolysis by these subunits. In contrast, all three Walker A mutant proteins – VirD4K152Q, VirB11K175Q and VirB4K439Q – completely blocked substrate transfer to VirB6 and VirB8 (Fig. 1B and C, right). Three other mutant proteins deleted of conserved Walker A motif residues, VirD4ΔGK (K. Atmakuri, unpubl.), VirB11ΔGKT (Rashkova et al., 2000) and VirB4ΔGKT (Berger and Christie, 1993), also arrested substrate transfer at the same step of the translocation pathway (data not shown). Results of these TrIP studies thus indicate that all three energetic subunits – VirD4, VirB11 and VirB4 – contribute via ATP-dependent mechanisms to substrate transfer to VirB6 and VirB8 (Fig. 1A).

VirD4 interacts with VirB4 and VirB11 ATPases and bitopic VirB10

The energetic subunits probably interact to mediate the early substrate transfer reactions yet, surprisingly, studies to date have identified only a VirB4–VirB11 interaction by yeast two-hybrid analysis (Ward et al., 2002). We thus assayed for complex formation by immunoprecipitation using N,N-Dimethyldodecylamine N-oxide (DDAO) detergent-solubilized cell extracts as starting material. The initial studies confirmed that DDAO treatment efficiently solubilized the 11 VirB subunits and VirD4 (Fig. 2A, and data not shown). Antibodies to VirD4, VirB11 or VirB4 each co-precipitated a presumptive complex of VirD4, VirB11 and VirB4 from wild-type cell extracts. Additionally, antibodies to VirD4 precipitated VirB10 and, reciprocally, antibodies to VirB10 co-precipitated VirB10 and VirD4. The preimmune anti-sera did not precipitate VirD4 or VirB proteins, establishing the specificity of these interactions (Fig. 2A). We were unable to detect other VirB proteins (VirB2, B3, B5, B6, B7, B8, B9) in material precipitated with the VirD4 antibodies (data not shown).

Fig. 2.

Fig. 2

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Co-immunoprecipitation of complexes composed of VirD4 T4CP, VirB10, VirB4 and VirB11.

A. Co-immunoprecipitation of complexes from wild-type cell extracts. Material immunoprecipitated (IP) with preimmune serum (Pre) or antibodies to the proteins listed (αD4, etc.) were analysed by Western blotting for the presence of Vir proteins listed on the right. (*) VirB10 undergoes proteolysis to an ≈40 kDa species; other unlabelled bands correspond to the cross-reactive heavy chain IgG. Total DDAO-solubilized membrane proteins (MP, right) from the WT strain show positions of the Vir proteins. Molecular mass markers (M) and sizes in kilodaltons (kDa) are shown on the left.

B. Co-immunoprecipitation of VirD4 and VirB proteins with VirD4 antibodies from extracts of strains deleted of virD4 (ΔD4), virB4 (ΔB4), virB10 (ΔB10) or virB11 (ΔB11).

We next assayed for VirD4 complex formation in strains missing one of the putative partner proteins (Fig. 2B). Interestingly, the VirD4 antibodies co-precipitated VirD4 and two of the VirB4, VirB10 and VirB11 proteins from extracts of strains bearing non-polar deletion mutations of virB4, virB10 or virB11. As expected, a control experiment with a virD4 null mutant established that the VirD4 antibodies did not non-specifically precipitate the VirB proteins. Together, these findings suggest that the VirD4 interactions with VirB4, VirB10 and VirB11 are not mediated through a specific contact with a single partner. Homologues of VirD4 and VirB10 in the R388 (Llosa et al., 2003) and R27 (Gilmour et al., 2003) plasmid conjugation systems have been shown to interact, and our further studies of the VirD4–VirB10 interaction are being reported elsewhere.

ATP-binding subunits interact independently of other T4SS subunits

Next, we assayed for VirD4 partner interactions in the absence of other T4SS subunits. As shown in Fig. 3A, VirD4 antibodies precipitated VirD4 from extracts of the ΔvirB operon mutant PC1000, and co-precipitated VirD4 plus VirB4 and/or VirB11 from extracts of isogenic strains expressing virB4 and/or virB11 from IncP or pBBR1 replicons. In reciprocal tests, the VirB4 or VirB11 antibodies did not precipitate VirD4 from PC1000 extracts, but co-precipitated VirD4 plus one or both VirB proteins from extracts of the isogenic strains expressing virB4 and/or virB11. VirD4, VirB4 and VirB11 therefore interact in pairwise fashion in the absence of other T4SS subunits.

Fig. 3.

Fig. 3

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ATP-binding subunits interact independently of other T4SS subunits and energization.

A. Co-immunoprecipitation of VirD4, VirB4 and VirB11 in the absence of other T4SS subunits. Material immunoprecipitated (IP) with antibodies to the proteins listed (αD4, etc.) were analysed by Western blotting for the presence of Vir proteins listed on the right. Unlabelled bands correspond to the cross-reactive heavy chain IgG. Total DDAO-solubilized membrane proteins (MP, right) from the WT strain show positions of the Vir proteins. Strains: ΔvirB (PC1000), producing B4 (pKA93); B11 (pSR45); B4 and B11 (pYJB61). Molecular mass markers (M) and sizes in kilodaltons (kDa) are shown on the left.

B. Immunoprecipitation with VirD4 antibodies of proteins from strains producing combinations of native or Walker A mutants. Left blots: ΔvirB, PC1000 producing VirD4, and either B4KQ (VirB4K439Q from pKA102) or B11KQ (VirB11K175Q from pSR40). Right blots: ΔD4 ΔvirB (KA2001), producing D4KQ (VirD4K152Q from pKA101); D4KQ and B4 (pKA101, pTAD214); D4KQ and B11 (pKA101, pSR45); D4KQ and B4 and B11 (pKA101, pYJB61).

C. Co-immunoprecipitation of VirB4 and VirB11 with antibodies to each protein in the absence of VirD4 ΔB4, PC1004; ΔB11, PC1011; ΔD4, Mx355; ΔD4 ΔvirB, KA2001, producing B4 and B11 (pYJB61).

In view of the above TrIP findings (Fig. 1), we also assayed for complex formation among the native and Walker A mutant forms of all three energetic subunits. Interestingly, the VirD4 antibodies co-precipitated native VirD4 and either VirB4K439Q or VirB11K175Q from extracts of PC1000 expressing alleles for these proteins (Fig. 3B). Additionally, these antibodies co-precipitated VirD4K152Q and one or both of the VirB4 or VirB11 native proteins from a ΔvirD4 ΔvirB mutant (KA2001, see Experimental procedures) expressing the corresponding alleles (Fig. 3B). Thus, native VirD4 stably interacts with the VirB11 and VirB4 Walker A mutants and, conversely, native forms of these VirB subunits interact with the VirD4 Walker A mutant.

Finally, we gained evidence for a VirB11–VirB4 interaction independently of VirD4 or other T4SS subunits. Figure 3C shows that antibodies to VirB11 or VirB4 co-precipitated both proteins from extracts of Mx355, a virD4 mutant expressing all virB genes, as well as extracts of the ΔvirB ΔvirD4 mutant expressing virB4 and virB11 from an IncP replicon. These findings complement the two-hybrid data showing that VirB4 and VirB11 interact in yeast (Ward et al., 2002).

The VirD4–VirB11 interaction: necessary but not sufficient for substrate transfer from VirD4 to VirB11

We have shown that VirD4 binds substrate independently of contributions from the VirB subunits (Cascales and Christie, 2004). Next, we tested whether the VirD4–VirB11 complex identified above (Fig. 3A) supports substrate transfer in the absence of other machine components. Interestingly, however, results of the TrIP assays showed that the corresponding strain KA1001 did not carry out this transfer reaction (Fig. 4A and B). We thus sought to determine whether an additional VirB protein or protein complex might stimulate this transfer step by engineering strains to produce various combinations of VirB subunits.

Fig. 4.

Fig. 4

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in vivo reconstitution of substrate transfer from VirD4 to VirB11.

A. T-strand interactions with T4SS subunits from strains producing subsets of VirB proteins as shown by TrIP. Strains: WT, A348; 11, KA1001; 7–9, 11, KA1009; 7, 10, 11, KA1008. Strains were treated and samples were analysed as in Fig. 1. Precipitation of T-strand substrate (T) and the pTi control fragment (C) was detected by PCR amplification and agarose gel electrophoresis.

B. Summary of strains analysed by TrIP for reconstitution of T-strand transfer from VirD4 to VirB11. KA10XX strains (see Experimental procedures) produce the VirB proteins listed at the top, as confirmed by Western blot analysis (Fig. 4C). (+) T-strand amplification product detected by agarose gel electrophoresis; (−) no detectable amplification product.

C. VirB and VirD4 protein accumulation in A348 (WT), PC1000 (ΔvirB) and the KA10XX strains engineered to produce the VirB proteins listed vertically. Equivalent amounts of total membrane proteins from strains induced for vir gene expression to an OD600 = 0.5 in ABIM were analysed for Vir protein content by SDS-PAGE and Western blotting with antibodies to the VirB and VirD4 proteins listed on the right. (*) a VirB10 proteolytic product. Molecular mass markers (M) and sizes in kilodaltons (kDa) are shown on the left.

Remarkably, strain KA1003 producing VirD4 and VirB7–VirB11 efficiently transferred substrate to VirB11 (Fig. 4B). Moreover, we identified two strains producing alternative combinations of these VirB proteins that also carried out this transfer reaction. Both strains produced VirD4, VirB11 and the VirB7 lipoprotein, whereas KA1009 additionally synthesized VirB8 and VirB9 and KA1008 synthesized VirB10 (Fig. 4A and B). These and other strains analysed in this study accumulated T4SS subunits at detectable levels (Fig. 4C), although there was some evidence for VirB protein turnover in strains lacking VirB6 or VirB7 in agreement with previous findings (Hapfelmeier et al., 2000; Jakubowski et al., 2003; Liu and Binns, 2003). Taken together, these findings strongly suggest that in vivo reconstitution of substrate transfer to VirB11 requires synthesis of specific subsets of T4SS components.

Reconstitution of substrate transfer to VirB6 and VirB8

Finally, we sought to reconstitute substrate transfer from VirB11 to VirB6. Strain KA1010 producing VirD4 and VirB6 through VirB11 failed to transfer substrate beyond VirB11 (Fig. 5A and B), despite the abundant accumulation of these T4SS subunits (Fig. 5C). However, the isogenic strain KA1011 producing these proteins plus VirB4 efficiently transferred substrate to VirB6 as well as to VirB8, consistent with data presented in Fig. 1 demonstrating the importance of VirB4 for this transfer step(s) (Fig. 5A and B). This combination of T4SS subunits did not transfer substrate to VirB9, as expected from the initial TrIP studies establishing the importance of VirB2, VirB3 and VirB5 for the latter substrate transfer reactions (Cascales and Christie, 2004).

Fig. 5.

Fig. 5

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in vivo reconstitution of substrate transfer to VirB6 and VirB8.

A. T-strand interactions with T4SS subunits from strains producing subsets of VirB proteins as shown by TrIP. Strains: WT, A348; 6–11, KA1010; 4, 6–11, KA1011; 4, 6–9, 11, KA1012; 4, 6, 7, 10, 11, KA1013; 4, 6–8, 10, 11, KA1014; 4, 6–8, 11; KA1015.

B. Summary of strains analysed by TrIP for reconstitution of T-strand transfer from VirD4 to VirB6 and VirB8.

C. VirB and VirD4 protein accumulation in strains producing the VirB proteins listed vertically. Samples were prepared and analysed as described in the legend to Fig. 4C. Immunoblots were developed with antibodies to the VirB and VirD4 proteins listed on the right. (*) a VirB10 proteolytic product. Molecular mass markers (M) and sizes in kilodaltons (kDa) are shown on the left.

We also assayed for substrate transfer by strains producing subsets of these VirB proteins. Strain KA1012 producing VirD4, VirB11, VirB4 and VirB6 through VirB9 delivered substrate to VirB6 and to VirB8, but not to VirB9 (Fig. 5A and B). In contrast, strain KA1013 producing the three energetic subunits, VirB6, VirB7 and VirB10, did not transfer substrate beyond VirB11. VirB6 and VirB8 appear to function co-ordinately (Cascales and Christie, 2004), and thus we postulated that the transfer defect of strain KA1013 resulted from the absence of VirB8. Confirming this prediction, strain KA1014 producing the energetic subunits, VirB6, VirB7, VirB8 and VirB10, efficiently transferred substrate to VirB6 and VirB8, but not to VirB9 (Fig. 5A and B). Also, as expected on the basis of results presented in Fig. 4, strain KA1015 producing VirB4, VirB6, VirB7, VirB8 and VirB11 failed to deliver substrate beyond VirD4. All strains examined in these studies accumulated T4SS subunits at abundant levels (Fig. 5C).

These in vivo reconstitution studies, and data presented in Fig. 2, together indicate that the early T-DNA translocation reactions leading to formation of the VirB6 and VirB8 substrate contacts minimally require catalytically active forms of the energetic subunits – VirD4, VirB11 and VirB4 – plus VirB6, VirB7, VirB8 and either VirB9 or VirB10.

Discussion

Our findings advance a mechanistic understanding of early stages of bacterial type IV secretion. The first step of T-DNA transfer involves substrate recruitment to the secretory apparatus. The proposal that the T4CP mediates this reaction originated from genetic studies of chimeric T4CP/Mpf machines (Cabezon et al., 1994; Cabezon et al., 1997; Sastre et al., 1998; Hamilton et al., 2000), and gained further support from in vitro biochemical studies demonstrating T4CP interactions with DNA, relaxases and other substrate processing factors (Disque-Kochem and Dreiseikelmann, 1997; Moncalian et al., 1999; Schroder et al., 2002; Schroder and Lanka, 2003). The TrIP assay permits tests of substrate binding in vivo, and results of the initial studies confirmed that the VirD4 T4CP functions as the T-DNA receptor for the VirB/D4 T4SS (Cascales and Christie, 2004). Here, by TrIP, we further showed that a VirD4 Walker A mutant binds the T-DNA substrate in vivo. Surprisingly, this mutant protein also executes substrate transfer to VirB11, the first identified transfer step of this translocation pathway. Elsewhere, we have reported that VirD4 also binds the VirE2 protein substrate independently of other T4SS subunits in vivo (Atmakuri et al., 2003). As an extension of this line of study, we recently determined that the VirD4K152Q mutant also retains VirE2 binding activity (K. Atmakuri, unpubl. data). Thus, several lines of evidence support a general proposal that T4CPs function as receptors for DNA and protein substrates independently of ATP utilization.

The early studies of chimeric T4CP/Mpf machines further suggested that the T4CPs physically link the DNA processing machinery to the transport apparatus (Llosa et al., 2002). The recent evidence for TraGR27 and TrwBR388 T4CP interactions with the VirB10-like TrhBR27 and TrwER388, respectively, strongly support this proposed function (Gilmour et al., 2003; Llosa et al., 2003). We confirmed the VirD4 T4CP–VirB10 interaction and further supplied evidence for VirD4 complex formation with the VirB11 and VirB4 ATPases. The VirD4 interaction with VirB11 is of special interest in view of earlier genetic studies that resulted in isolation of an unusual class of virB11 dominant alleles (Zhou and Christie, 1997). These alleles encode mutant proteins that ‘uncouple’ VirB11’s contributions to substrate transfer and to pilus biogenesis (Sagulenko et al., 2001). Previous work has shown that VirD4 is required for substrate transfer and not for pilus production (Lai et al., 2000). Thus, we postulated that the ‘uncoupling’ mutations do not affect the VirB11–VirD4 interaction but instead disrupt interactions with other VirB subunits required for pilus biogenesis (Sagulenko et al., 2001). The present findings firmly establish that the VirD4–VirB11 complex mediates the first transfer step of the translocation pathway. Moreover, the finding that the Walker A mutant forms of VirD4 and VirB11 do not detectably disrupt contacts between the energetic subunits, DNA substrate binding, or DNA transfer to VirB11 strongly suggests that ATP binding and/or hydrolysis does not regulate these early transfer reactions.

Recently solved structures of VirD4 and VirB11 homologues suggest how these machine components might interact at the inner membrane. The TrwBR388 T4CP is a ring-like homohexamer, similar in structure to the ball-stem structure of F1-ATPase (Gomis-Ruth et al., 2001; Gomis-Ruth et al., 2002). In contrast, HP0525 of the Helicobacter pylori Cag T4SS, the VirB11 structural prototype, presents as a ring-shaped, double-stacked hexamer structurally similar to members of the AAA ATPase super-family, e.g. p97 (Yeo et al., 2000). The three forms, apoprotein and ATP- and ADP-bound HP0525, differ dramatically in their structures suggesting that the hexamer undergoes dynamic conformational changes with cycles of ATP binding and ADP release (Savvides et al., 2003). The simplest model for how these homohexamers interact depicts the T4CP as positioned adjacent to VirB11 and other Mpf proteins at the inner membrane (Krall et al., 2002). Our TrIP findings are compatible with such a structural relationship, and further suggest that the observed nucleotide-induced conformational changes regulate a step in the translocation pathway downstream of the VirD4 to VirB11 substrate transfer reaction.

Quite remarkably, we were able to reconstitute substrate transfer from VirD4 to VirB11 only by co-production of these energetic subunits, the VirB7 lipoprotein, and either VirB8 and VirB9 or VirB10. Thus, despite the ability of VirD4 and VirB11 to form a stable complex in the absence of other T4SS subunits, this interaction is not productive. The importance of VirB7 for this transfer step is consistent with the initial TrIP studies showing that a ΔvirB7 mutation abolishes substrate transfer from VirD4 to VirB11 (Cascales and Christie, 2004). Previous studies also have identified several biochemical properties of VirB7, VirB8, VirB9 and VirB10 that suggest how these subunits might contribute to this transfer step (see Christie, 2004; Llosa and O’Callaghan, 2004). First, VirB7 forms disulphide bridges with itself or VirB9. These contacts stabilize both proteins as well as other T4SS subunits. Second, both VirB7 dimer species localize predominantly at the outer membrane, whereas the heterodimer interacts via VirB9 protein contacts with the bitopic inner membrane proteins VirB8 and VirB10. Finally, various pairwise interactions between the VirD4, VirB11, VirB8, VirB9 and VirB10 proteins have now been detected by yeast two-hybrid or biochemical assays (this study; Das and Xie, 2000; Krall et al., 2002; Ward et al., 2002).

These properties, and evidence that the VirB7 through VirB10 subunits are highly conserved among many T4SS (Cascales and Christie, 2003), support a proposal that these components assemble as a cell envelope-spanning ‘core’ structure early during machine biogenesis. Indeed, the notion that such a ‘core’ structure is biologically active was suggested from the finding that natural competence by H. pylori is mediated by homologues of the VirB ‘core’ proteins (Hofreuter et al., 2003), as well as the intriguing discovery that A. tumefaciens recipient strains producing the ‘core’ components acquire DNA in matings with donor cells at appreciably higher frequencies than isogenic strains lacking these components (Liu and Binns, 2003). Results of our reconstitution studies indicate that such a ‘core’ structure is both necessary and sufficient for establishment of a productive VirD4–VirB11 interaction in vivo. Although further studies are needed to understand how alternative subsets of ‘core’ subunits mediate this transfer step, at this juncture we favour a working model whereby the outer membrane VirB7–VirB9 and VirB7–VirB7 dimers interact with C-terminal domains of VirB8 and VirB10 respectively. In turn, these interactions modulate the nature of contacts between the bitopic proteins and the energetic subunits at the inner membrane.

Finally, we propose that results of our studies can be explained in the context of two mechanistic models depicting how DNA substrates are translocated across the inner membrane (Fig. 6). One model, termed the ‘shoot-and-pump’ model, was set forth by Llosa et al. (2002). As adapted for the VirB/D4 T4SS in accordance with the TrIP data, this model postulates that VirD4 recruits the T-DNA to the secretion apparatus (step I). Next, the T4CP transfers only the relaxase component of the relaxase-T-strand transfer intermediate to VirB11 (step II). VirB11 then unfolds the relaxase through a chaperone activity and co-ordinates with other VirB proteins, e.g. VirB4, VirB6 and VirB8, to deliver the relaxase across the inner membrane (shoot). Simultaneously, VirD4 uses ATP energy to drive the T-strand across the membrane (pump). Upon transfer, the relaxase-T-strand particle enters a secretion channel composed of the VirB2 and VirB9 subunits for transit through the periplasm to the cell exterior (Llosa et al., 2002).

Fig. 6.

Fig. 6

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Steps depicting early stages of DNA translocation through the A. tumefaciens VirB/D4 T4SS. VirD4 T4CP recruits the T-DNA (step I) and delivers substrate to VirB11 (step II). Next, two models are envisioned (step III). A ‘ping-pong’ model depicts VirB11 as a chaperone that unfolds the relaxase and retrotransfers the unfolded substrate back to VirD4. VirD4 then translocates the unfolded relaxase-T-strand complex across the inner membrane where it then accesses the VirB channel through a vestibule formed by a periplasmic loop of VirB6 (see Jakubowski et al., 2004). An alternative ‘shoot-and-pump’ model postulates that VirB11 interacts with other VirB proteins to transfer the relaxase component of the transfer intermediate across the inner membrane at the same time as VirD4 mediates transfer of the T-strand component (adapted from Llosa et al., 2002). For both models, the early transfer reactions (steps I and II) proceed independently of ATP utilization, whereas successive reactions depicted by the two models (step III) are regulated by ATP through the co-ordinated activities of VirD4, VirB11 and VirB4.

The second model, here designated the ‘ping-pong’ model, depicts the T4CP as the sole translocase for DNA-trafficking type IV systems (Fig. 6). As with the shoot-and-pump model, VirD4 T4CP recruits the T-DNA (step I) and transfers the relaxase component to the VirB11 chaperone (step II; ping). Then, instead of mediating relaxase transfer through the VirB Mpf channel, VirB11 retrotransfers the unfolded relaxase-T-strand particle back to VirD4 (pong). Finally, VirD4 uses ATP energy to translocate the DNA–protein substrate across the inner membrane. At this point, we favour the ‘ping-pong’ model in part because we find it difficult to conceptualize how two translocases function simultaneously to deliver a single substrate, the relaxase-T-strand particle, across the inner membrane. Additionally, elsewhere we describe results of TrIP studies showing that a central periplasmic loop of the polytopic VirB6 protein is essential for formation of VirB6’s substrate contacts, whereas insertions and deletions of cytoplasmic loops or transmembrane segments do not affect the substrate interactions (Jakubowski et al., 2004). Thus, in concert with the ‘ping-pong’ model, it is possible that the central loop of VirB6 forms a vestibule in the periplasm through which the substrate passes for transit to the cell exterior.

Upon substrate transfer to VirB11, the two models differ mechanistically and predict different functions for the energetic components. For example, according to the shoot-and-pump model, VirD4 is a DNA-specific translocase, whereas VirB4 might use the energy of ATP hydrolysis to co-ordinate passage of the relaxase through an inner membrane channel composed of VirB6 and VirB8, in line with an early suggestion based on VirB6 topology studies (Das and Xie, 1998). In contrast, according to the ‘ping-pong’ model, VirD4 translocates a nucleoprotein complex, whereas VirB4 might use the energy of ATP hydrolysis to co-ordinate substrate retrotransfer from VirB11 to VirD4, or substrate delivery from VirD4 to VirB6 and VirB8 at the periplasmic face of the inner membrane. VirB6 and VirB8 thus do not function as inner membrane channel subunits but rather mediate substrate transfer from VirD4 to the portion of the secretion channel extending through the periplasm. Future studies of VirD4, VirB11 and VirB4 functions should supply a detailed mechanistic understanding of how ATP energizes substrate transfer through the VirB/D4 T4SS.

Experimental procedures

Bacterial strains and induction conditions

Escherichia coli strain DH5α served as the host for plasmid constructions. All strains of A. tumefaciens were derived from wild-type strain A348 (Zhu et al., 2000). Table 1 lists the strains used in the reconstitution studies, with plasmid sources provided or constructions described below. Strain PC1000 is deleted of the virB operon (Fernandez et al., 1996), PC1004 is deleted of virB4 (Berger and Christie, 1993), PC1011 is deleted of virB11 (Berger and Christie, 1994) and Mx355 is a virD4 null mutant (Stachel and Nester, 1986). Strain KA2001, deleted of the virB operon and virD4, was constructed as follows. A 1.32 kb NdeI–SnaBI fragment of pMY1153 (Atmakuri et al., 2003) containing 0.74 kb of virD2 and 0.58 kb of virD3 was cloned into NdeI- and EcoRV-digested pBSIIKS+NdeI to yield pKA108. Next, a 0.8 kb EcoRV–NdeI (made blunt) fragment containing 0.18 kb of virD4 and 0.62 kb of virD5 was cloned into the EcoRV–SmaI sites of pKA108 to obtain pKA112. Finally, a 3.5 kb BamHI fragment containing the nptII and sacB genes from pBB50 (Berger and Christie, 1994) was cloned into the corresponding site of pKA112 to yield pKA126. This plasmid was then delivered into A. tumefaciens PC1000 strain to obtain KA2001 by marker exchange–eviction mutagenesis as previously described (Berger and Christie, 1994). A. tumefaciens vir genes were induced in AB induction medium (ABIM) as previously described (Zhou and Christie, 1999). When appropriate, 0.25 mM isopropyl-β-d-thiogalactoside (IPTG) was added to cultures to induce vir genes placed under the control of the Plac promoter.

Table 1.

Strains used for the reconstitution studies.

Straina VirB/VirD4
composition		 Plasmid
compositionb 		Co-integrate plasmidsc
NHR (reference) BHR (reference)
A348 B1–B11, D4
(wild type)
PC1000 D4
(ΔvirB operon)
Mx355 B1–B11
(virD4::Tn3HoHo1)
PC1004 B1–B3, B5–B11, D4
(ΔvirB4)
PC1011 B1–B10, D4
KA2001d –
(ΔvirB ΔvirD4)
Strains used to test for substrate transfer to VirB11
KA1001e B11, D4 pSR45
KA1002 B4, B11, D4 pYJB61 pSR1 (Rashkova et al., 1997) pTAD214 (Dang et al., 1999)
KA1003 B7–B11, D4 pKA117
pZL36 (Liu and Binns, 2003)		 pSR1 pBBR1 (Kovach et al., 1994)
KA1004 B8–B11, D4 pKA118
pED9 (Ward et al., 1990)		 pPC985 (Berger and Christie, 1994) pBBR1
KA1005 B7, B11, D4 pKA119
pSR45		 pPC974 (Berger and Christie, 1994) pBBR1
KA1006 B7, B8, B11, D4 pKA120
pSR45		 pPC975 (Berger and Christie, 1994) pBBR1
KA1007 B7, B9, B11, D4 pKA121
pSR45		 pPC974 pKA103
KA1008 B7, B10, B11, D4 pKA119
pED10 (Ward et al., 1990)		 pPC974 pBBR1
KA1009 B7–B9, B11, D4 pKA122
pSR45		 pJW283 (Berger and Christie, 1994) pBBR1
Strains used to test for substrate transfer to VirB6
KA1010 B6–B11, D4 pKA117
pSJ610 (Jakubowski et al., 2003)		 pSR1 pBBR1
KA1011 B4, B6–B11, D4 pKA123
pSJ610		 pSR1 pKA93
KA1012 B4, B6–B9, B11, D4 pKA124
pSR45		 pKA116 pKA93
KA1013 B4, B6, B7, B10, B11, D4 pKA125
pED10		 pSJ974 pKA93
KA1014 B4, B6–B8, B10, B11, D4 pKA130
pED10		 pKA129 pKA93
KA1015 B4, B6–B8, B11, D4 pKA130
pSR45		 pKA129 pKA93
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a

All strains used in this study are A348 derivatives and carry the pTiA6NC plasmid.

b

Strains carried broad host range plasmids, constructed either by cloning vir genes of interest into IncP or pBBR1MCS2 (Inc group unspecified, compatible with IncP) plasmids, or by construction of co-integrate plasmids. Source of plasmid is in parentheses; other plasmids were constructed in this study as described in Experimental procedures.

c

Co-integrate plasmids were constructed by ligation of a narrow host range (NHR) plasmid (pUC or pBluescript derivative) to a broad host range (BHR) IncP replicon unless indicated as pBBR1.

d

Strain KA2001 is PC1000ΔD4 constructed in this study.

e

The KA10XX strains are PC1000 derivatives.

virD4, virB11 and virB4 expression plasmids

The virB or virD4 genes were expressed in A. tumefaciens by cloning relevant restriction fragments from narrow-host-range (NHR) ColE1 plasmids into the broad-host-range (BHR) IncP plasmids pXZ151 (Zhou and Christie, 1999) or pSW172 (Chen and Winans, 1991), or pBBR1MCS2 which is compatible with the IncP plasmids (Kovach et al., 1994). Alternatively, a ColE1 plasmid carrying the vir gene(s) of interest was ligated to an IncP or pBBR1MCS2 BHR plasmid for maintenance in A. tumefaciens. Plasmid pKA42 is pBBR1MCS2 expressing PvirB-virD4, obtained as a 2.7 kb XbaI–XhoI fragment from plasmid pKA9 (Atmakuri et al., 2003). Plasmid pKA83 is a pKA9 derivative expressing PvirB-virD4K152Q, constructed by QuikChangeR Mutagenesis protocol (Stratagene). The primers for mutagenesis were 5′-CGCGCCAACACGAGCTGGCCAAGGCGTCGGAATCGTAATTCCAACGC-3′ and 5′-TTACGATTCCGACGCCTTGGCCAGCTCGTGTTGGCGCGACGACAA-3′. Plasmid pKA101 (BHR) is pBBR1MCS2 expressing PvirB-virD4K152Q, obtained as a 2.7 kb XbaI–XhoI fragment from pKA83.

Plasmid pKA93 is pBBR1MCS2 expressing PvirB-virB4, obtained as a 2.9 kb XbaI–XhoI fragment from NHR plasmid pTAD944 (Dang et al., 1999). Plasmid pTAD214 is pSW172 expressing PvirB-virB4 (Dang et al., 1999). Plasmid pBB11 expresses Plac-virB4K439Q (Berger and Christie, 1993). pKA96 is pTAD944 derivative expressing PvirB-virB4K439Q obtained by cloning a 2.24 kb SphI–NotI fragment of pBB11. Plasmid pKA102 is pBBR1MCS2 expressing PvirB-virB4K439Q, obtained as a 2.9 kb XbaI–XhoI fragment from pKA96. Plasmids pSR1 and pSR40 (IncP) express PvirB-virB11 and PvirB-virB11K175Q respectively (Rashkova et al., 1997). Plasmid pSR45 (IncP) is pXZ151 (Zhou and Christie, 1999) expressing PvirB-virB11 obtained as a 1.6 kb XbaI–XhoI fragment from pSR1.

Plasmids expressing subsets of virB genes

The following plasmids were used for the reconstitution studies. The ColE1 plasmids described below are pUC or pBlue-script derivatives; the IncP plasmids are derivatives of pSW172, pXZ151, or pTJS75 (Zhou and Christie, 1997). Plasmids pPC974 and pPC985 are ColE1 plasmids expressing PvirB-virB7 and PvirB-virB8 respectively (Berger and Christie, 1994). Plasmid pKA103 is a pBBR1MCS2 plasmid expressing Plac-virB9 obtained as a 1.2 kb XbaI–XhoI fragment from pXZ91. Plasmid pXZ91 is a ColE1 plasmid expressing Plac-virB9 obtained as a 1.06 kb EcoRI–BamHI fragment from pXZ90 (Zhou and Christie, 1997). pXZ9104 is a ColE1 plasmid expressing PvirB-virB10 obtained as 1.14 kb NdeI–XhoI fragment from pPC9108 (Berger and Christie, 1994). Plasmid pSR1 is a ColE1 plasmid expressing PvirB-virB11 (Rashkova et al., 1997). Plasmid pSJ974 is a ColE1 plasmid expressing PvirB-virB6-virB7 obtained as a 1.2 kb NarI–NcoI fragment from pBB20 (Berger and Christie, 1994). Plasmid pKA129 is a ColE1 plasmid expressing Plac-virB6-virB8 obtained by deleting a 0.85 kb SphI–XhoI fragment from pKA116. Plasmid pKA116 is a ColE1 plasmid expressing Plac-virB6-virB9 obtained as a 2.8 kb SacI–XbaI fragment from pSJ610. Plasmid pSJ610 is pXZ151 (IncP) expressing PvirB-virB6-virB10 (Jakubowski et al., 2003). Plasmid pPC975 is a ColE1 plasmid expressing Plac-virB7-virB8 (Berger and Christie, 1994). Plasmid pJW283 is a ColE1 plasmid expressing Plac-virB7-virB9 (Berger and Christie, 1994). Plasmid pZL36 is an IncP plasmid expressing PvirB-virB7-virB10 (Liu and Binns, 2003). Plasmids pED9 and pED10 are IncP plasmids expressing PvirB-virB9-virB11 and PvirB-virB10-virB11 respectively (Ward et al., 1990).

Transfer DNA immunoprecipitation (TrIP) assay

The TrIP assay and the quantification of the T-strand immunoprecipitated as a protein–DNA complex were performed essentially as previously described (Cascales and Christie, 2004). Briefly, 6 ml of A. tumefaciens cells were induced for 14–16 h by shaking at 19°C in ABIM medium with 200 μM acetosyringone. Upon harvesting, the cells were washed once with an equal volume of 20 mM sodium phosphate buffer pH 6.8. Cells were resuspended in the same buffer with formaldehyde (FA) at a final concentration of 0.1%, incubated for 20 min at 18°C with shaking, then FA was added in 0.2% increments to reach a final concentration of 1% over a 15 min period. Cells were then incubated for 40 min at room temperature (RT) without shaking. Cells were pelleted and solubilized by resuspension in 200 μl of TES buffer (50 mM Tris.Cl pH 6.8, 2 mM EDTA, 1% β-mercaptoethanol, 1% SDS).

For immunoprecipitation, protein A-Sepharose CL-4B (Pharmacia) (30 μl bed volume) was incubated with 1.1 ml of the detergent-solubilized material for 60 min at RT and centrifuged at 5000 g to remove Protein A-Sepharose and nonspecifically bound proteins. The supernatant was incubated overnight at 4°C with antibody coupled to Protein A-Sepharose CL-4B. The beads were pelleted by centrifugation and the remaining supernatant was analysed in parallel with material eluted from the beads. The beads were washed twice with NP1 buffer supplemented with 1% Triton X-100 and once with NP1 buffer supplemented with 0.1% Triton X-100. Immunoprecipitates were eluted by incubation for 20 min at 96°C in 20 μl of 10 mM Tris.Cl pH 6.8. Polymerase chain reaction (PCR) was performed as described previously (Cascales and Christie, 2004).

To compare levels of T-strand by quantitative TrIP (QTrIP), immunoprecipitates were subjected to 20 cycles of PCR amplification using the primers specific for gene 7 of the T-DNA. On the 21st cycle, a single round of PCR amplification was performed with addition of 1.0 mCi of [32P]-dGTP (Amersham Biosciences), as described previously (Cascales and Christie, 2004). PCR products were column-purified with the Qiaquick PCR purification kit (Qiagen) to remove unincorporated nucleotides. Aliquots of the eluted material were mixed with 3.5 ml of scintillation liquid (Ecolite, ICN) and counted with a Beckman Coulter. The entire TrIP protocol was repeated three times in triplicate and the average value from a single experiment was reported.

Membrane solubilization and immunoprecipitation

To identify possible membrane protein–protein complexes by immunoprecipitation, A. tumefaciens cultures (500 ml) were induced for vir gene expression in ABIM for 18 h at 22°C. Cells were harvested, lysed by French-Press treatment, and total membranes recovered as previously described (Rashkova et al., 2000). Membranes were resuspended in buffer A (50 mM Tris-HCl buffer, pH 7.4, 20% sucrose, 5 mM EDTA, protease inhibitor cocktail; Roche Molecular Biochemicals). Approximately 3 mg of total membrane was diluted to 1.3 ml with the buffer A containing 3% DDAO and solubilized by gentle rocking at 4°C for 12 h. The solubilized material was centrifuged at 90 000 g for 1 h at 4°C and the supernatant was used for immunoprecipitation as previously described (Atmakuri et al., 2003).

Protein analyses

Proteins recovered by immunoprecipitation and suspended in Laemmli’s buffer were subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) using glycine- or tricine-buffer systems as previously described (Jakubowski et al., 2003). For detection by immunostaining, proteins were transferred onto nitrocellulose membranes, and immunoblots were developed with antibodies to the VirD4 and VirB proteins from our laboratory collection (Atmakuri et al., 2003; Jakubowski et al., 2003; Cascales and Christie, 2004) and goat anti-rabbit antibodies conjugated to alkaline phosphatase (Bio-Rad). Membrane proteins were loaded on SDS-polyacrylamide gels on a per cell equivalent basis for comparisons of steady-state levels of T4SS subunits between strains.

Acknowledgements

We thank members of the laboratory for helpful discussions, and Yanjie Liu for plasmid constructions. We thank Joe Vogel for helpful discussions and the ‘ping-pong’ terminology for our model. We gratefully acknowledge the financial support of the NIH (GM48746) for this study.

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