A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Summary

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7_XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7_XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7_XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7_XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7_XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Introduction

Bacteria employ multiprotein secretion systems to translocate effector proteins or nucleoprotein complexes across the cell envelope where they modulate the bacterium's interactions with the environment. In Gram-negative bacteria, the secretion systems have been classified into 6 different types [1], [2]. The type IV secretion systems (T4SSs) are ancestrally related to bacterial conjugation machines [3] and are able to translocate proteins and/or protein-DNA complexes to the extracellular milieu or the host interior, in many cases contributing to the ability of the bacterial pathogen to colonize the host and evade its immune system [4]. T4SSs are important in the pathogenic mechanism of many microbes, including the animal pathogens Legionella pneumophila (Legionnaires' disease; [5]), Bordetella pertussis (whooping cough; [6]), Coxiella burnetii (Q fever; [7]), Bartonella henselae (cat scratch disease, [8]), Brucella spp. (brucellosis; [9]) and Helicobacter pylori (gastritis, ulcers, stomach cancer; [10]), as well as the plant pathogen Agrobacterium tumefaciens (crown gall disease; [11]) which has the prototype T4SS, composed of 12 proteins, VirB1-VirB11 and VirD4 [12].

Xanthomonas citri subsp. citri (formerly Xanthomonas axonopodis pv. citri) (Xac) is a gammaproteobacterial phytopathogen which causes citrus canker, a disease that affects all citrus plants and has significant economic impact worldwide [13]. The Xac genome was sequenced and many potential virulence-related genes were identified [14], including an 18 kb chromosomal vir locus that codes for a T4SS [13], [14], [15]. Orthologous T4SSs have been identified in the closely related species Xanthomonas campestris pv. campestris (strains ATCC 33913 [14], 8004 [16] and B100 [17]), Xanthomonas albilineans [18], Xanthomonas campestris pv. vasculorum NCPPB702 [19], Xanthomonas campestris pv. musacearum NCPPB4381 [19] and Stenotrophomonas maltophilia (strains K279A [20] and R551-3 [21]). The vir locus is incomplete in Xanthomonas campestris pv. vesicatoria [22] and is absent in four Xanthomonas oryzae strains (KACC10331 [23], MAFF311018 [24], PXO99A [25] and BLS256 (GenBank database AAQN00000000)) and in two Xanthomonas fuscans subsp. aurantifolii lineages (10535 and 11122 [26]). The unrelated Gram-negative bacteria Neisseria flavescens SK114 (GenBank database ACQV00000000) and Neisseria sp. oral taxon 014 str. F0314 (GenBank database ADEA00000000) also present a locus highly similar to the T4SS loci found in the Xanthomonadaceae family. Some Xanthomonas genomes, including Xac, carry megaplasmids that code for a second T4SS probably involved in plasmid mobilization and whose structural components exhibit only very low sequence identity to their counterparts in the chromosomally encoded T4SS under study [15], [27].

One particularly distinctive feature of the Xanthomonad T4SSs is related to the VirB7 component. In well-characterized T4SSs, VirB7 is a lipoprotein attached to the periplasmic side of the outer membrane [28]. It has been shown to interact with several T4SS subunits [29], including itself [30] and the VirB9 C-terminal region [31], [32]. The structure of a VirB7-VirB9-VirB10 complex (TraN-TraO-TraF) from the T4SS of the conjugative plasmid pKM101 demonstrated that these proteins form a hetero-tetradecameric outer membrane channel through which the substrates pass [33], [34]. Structures of the complex formed between the TraO C-terminal domain and TraN revealed that the 33-residue TraN forms an extended structure that winds around the TraO β-sandwich [34], [35].

Members of the VirB7 family are typically 45–65 residues long [36], becoming 15–20 residues shorter after removal of the N-terminal signal sequence and covalent attachment to lipid molecules. In Xac, several lines of evidence indicate that the gene xac2622 codes for a VirB7 equivalent; specifically, the gene position, the presence of signals for outer membrane localization and lipidation and the interaction of its product with VirB9 [15]. However, XAC2622 does not exhibit any sequence similarity with the VirB7 family and it is much larger than a typical VirB7 protein (139 and 118 residues before and after signal sequence removal, respectively).

In order to understand the unique structural features of Xanthomonad VirB7, we have solved the solution nuclear magnetic resonance (NMR) and X-ray crystal structures of XAC2622, which we now call VirB7_XAC2622, and studied its interaction with VirB9. VirB7_XAC2622 presents all of the characteristics of VirB7: predicted signal peptide and lipobox sequences followed by a short and extended region that contains the VirB9 binding site. However, unlike other VirB7 proteins, VirB7_XAC2622 has an extra domain whose topology is strikingly similar to that of N0 domains found in proteins from different multi-subunit complexes involved in transport across the bacterial outer membrane. We show that VirB7_XAC2622 oligomerizes through interactions involving conserved residues in the folded domain and the unfolded N-terminus and that oligomerization and the formation of the VirB7_XAC2622-VirB9 complex can occur simultaneously. We propose that these interactions are compatible with the tetradecameric core complex structure, resulting in the formation of an extra ring layer.

Results

Solution Structure of Xac VirB7 (VirB7_{XAC2622_24–139})

The construct used for NMR structure determination corresponds to residues 24–139 of VirB7_XAC2622 preceded by a Gly-Ser-His-Met sequence. Bioinformatics analysis predicts that residues 1–21 would be removed and Cys22 lipidated, leading to anchoring in the bacterial outer membrane [15]. Approximately 98% of main chain and aliphatic side chain and 93% of aromatic side chain ¹H, ¹³C and ¹⁵N resonances were assigned (Table 1 and Figure S1). A total of 2176 experimental NMR restraints, consisting of 2056 interproton distances and 120 torsion angles, were used in the structure calculation (Table 1). A small set of nuclear Overhauser effect (NOE) cross-peaks that were not assigned by CYANA [37] were subsequently identified as resulting from intermolecular contacts (see below).

The solution structure of VirB7_{XAC2622_24–139} consists of a globular domain (residues 52–133), flanked by a long disordered N-terminus (amino acids 24–51) and a short flexible C-terminus (residues 134–139) (Figure 1A). The globular domain is composed of two α-helices sandwiched between a mixed three stranded β-sheet on one side and an antiparallel two-stranded β-sheet plus a short 3₁₀ helix on the other (Figure 1B). The NMR structures were independently validated by ¹H-¹⁵N residual dipolar couplings (RDCs). The average Q factor obtained by fitting RDCs from secondary structure elements to the whole NMR ensemble is 0.271±0.019, consistent with a good quality structure [38], [39], [40].

High Resolution X-ray Crystal Structure of the VirB7_XAC2622 Globular Domain

To better characterize the globular domain, we cloned and purified a recombinant fragment encompassing residues 51–134 (VirB7_{XAC2622_51–134}) which was submitted to crystallization trials that resulted in large plates (Figure S2) which belong to space group C222₁ and diffracted up to 1.0 Å. Molecular replacement was performed using the solution structure of the VirB7_XAC2622 globular domain as the search model. Details of the data collection and structure refinement are listed in Table 2 and the model is presented in Figure 1C. There was clear electron density for all residues, with the exception of the first amino acid (threonine 51) and residues 122 and 123 in the loop between β4 and β5. As expected, the crystal structure is very similar to the NMR ensemble (Figure 1D), displaying an average root mean square deviation (RMSD) for the backbone heavy atoms and all heavy atoms of 1.05±0.05 Å and 1.61±0.06 Å for residues 53–130, and excellent agreement with the backbone ¹H-¹⁵N RDCs [Q factor of 0.187 (regular secondary structures only) and 0.272 (all residues)].

VirB7 Oligomerization

Changes in ¹⁵N heteronuclear single-quantum coherence (¹⁵N-HSQC) cross-peak positions as a function of protein concentration indicated that VirB7_XAC2622 oligomerizes in fast exchange on the NMR time scale (Figure 2A). Consistent with this observation, alterations in ¹⁵N T₁ and T₂ relaxation times indicated that the overall tumbling slows significantly at higher protein concentrations (see below). Chemical shift perturbation data showed that two regions are involved in VirB7_XAC2622 self-interactions: a region in the unfolded N-terminus (residues 42–49) and a patch on the surface of the globular domain made up of residues 63–65, 85–93, 111–119 and 131 (Figure 2B and C).

Two possible interaction schemes can be envisaged that would be compatible with the chemical shift perturbation data: i) a side-by-side arrangement in which the N-terminal unfolded segment and the globular domain of one molecule interact, respectively, with the same regions of a second molecule, or ii) a head-to-tail complex arrangement where the N-terminus of one molecule recognizes the folded domain of the other. In order to test the hypothesis of the formation of a head to tail complex, we performed a titration of the ¹⁵N-VirB7_{XAC2622_51–134} globular domain with an unlabeled peptide encompassing residues 38–52 from the N-terminal region (VirB7_{XAC2622_38–52}). During the titration, we observed changes in the same cross-peaks which were perturbed in the entire protein in a concentration dependent manner (Figure S3). This hypothesis was corroborated by the analysis of a set of 13 NOEs not assigned by CYANA [37], which showed that they could be accounted for by the formation of head-to-tail dimers. Indeed, those NOEs were observed between proton pairs derived from amino acids whose main-chain ¹H-¹⁵N chemical shifts are perturbed in a concentration dependent manner (the NOEs are listed in the Materials and Methods).

We then used the set of 13 intermolecular NOEs as geometric restraints to drive computational docking simulations of the VirB7_XAC2622 dimer using HADDOCK2.0 [41]. In one simulation, a fragment corresponding to the disordered N-terminal region (residues 24–50) was docked with a fragment corresponding to the globular domain (residues 51–139) while the second simulation involved the docking between two full length proteins (residues 24–139). The resulting models from both simulations were highly similar. In the second simulation three of the thirteen NOEs were violated in all solutions by approximately 1.4 to 3 Å. They correspond to weak peaks and could have contributions of spin diffusion or chemical exchange. These docking simulations were therefore able to determine the general nature of the oligomerization interface whose resolution is necessarily limited by the small number of restraints and by the absence of structural information for the N-terminal region. One cluster of docking solutions using full length VirB7_XAC2622 is shown in Figure 2D.

To investigate whether VirB7_{XAC2622_24–139} forms dimers or higher-order oligomers, glutaraldehyde cross-linking experiments were performed. These assays showed that VirB7_{XAC2622_24–139} forms dimers, trimers and higher order oligomers (Figure S4A). When the cross-linking experiment was performed in the presence of 1% SDS, no covalently cross-linked oligomers were observed, indicating that oligomerization requires the presence of a correctly folded protein (Figure S4A). As expected, no cross-links were observed when the experiment was performed using VirB7_{XAC2622_51–134} which lacks the disordered N-terminal region (Figure S4B).

VirB7_XAC2622 Interaction with VirB9_XAC2620

To analyze this interaction, VirB7_{XAC2622_24–139_His} was titrated into a solution of VirB9_{XAC2620_34–255} (full-length VirB9_XAC2620 minus the first 33 residues including a predicted signal peptide) and changes in intrinsic fluorescence were monitored. A monotonic increase in fluorescence emission was detected until saturation at a 1∶1 VirB7∶VirB9 stoichiometry (Figure S5). A dissociation constant of 4×10⁻⁸ M for the complex was estimated by fitting the observed fluorescence changes to a binding isotherm (equation 2, Materials and Methods).

To study this interaction by NMR, the ¹⁵N-HSQC spectrum of ¹⁵N-VirB7_{XAC2622_24–139} was acquired in the presence of unlabeled VirB9_XAC2620__34–255. At a 1∶1 molar ratio, essentially no cross peaks were detected, except for those derived from the flexible N- and C-terminal residues (amino acids 24–25 and 134–139; data not shown). This observation is consistent with the formation of a tight complex with long correlation time, with the majority of cross peaks beyond detection.

VirB7_XAC2622 was previously shown to interact with the C-terminal domain of VirB9_XAC2620 [15] as has been shown for other VirB7-VirB9 homologs [32], [35]. We therefore produced a fragment, VirB9_{XAC2620_154–255}, corresponding to the C-terminal domain of VirB9_XAC2620. Changes in the ¹⁵N-HSQC spectrum of VirB7_{XAC2622_24–139} upon adding VirB9_{XAC2620_154–255} showed that the binding occurs in slow exchange on the NMR time scale, consistent with the sub-micromolar affinity detected by fluorescence spectroscopy for the interaction with full length VirB9 (VirB9_{XAC2620_34–255}). In order to map the VirB9 binding site on the structure of VirB7, we assigned the backbone resonances of VirB7_{XAC2622_24–139} in complex with VirB9_{XAC2620_154–255} and analyzed the chemical shift differences (Figure 3A and B). This analysis showed that only residues 27–41, within the disordered VirB7 N-terminus, undergo significant chemical shift perturbations (Figure 3C and D). This region is adjacent to, but does not overlap, the N-terminal region involved in VirB7_XAC2622 oligomerization (residues 42–49). ¹H-¹⁵N RDCs and backbone chemical shifts for VirB7_{XAC2622_24–139} alone and in the presence of VirB9_{XAC2620_154–255} are essentially identical, with the exception of the N-terminal region that is involved in binding to VirB9 (Figure 3 and Figure S6). Thus, the folded domain does not participate in the recognition of the VirB9_XAC2620 C-terminal domain and the linker between the VirB9 interaction site and the VirB7 globular domain is flexible enough to permit the two regions to align independently in the alignment medium. Interestingly, VirB7_{XAC2622_24–139} is able to simultaneously oligomerize and interact with VirB9_{XAC2620_154–255}, as the cross-peaks related to the regions involved in VirB7 oligomerization shift even in the presence of VirB9_{XAC2620_154–255} (Figure S7).

VirB7_XAC2622-Induced Changes in the VirB9_XAC2620 C-terminal Domain

The ¹⁵N-HSQC spectrum of VirB9_XAC2620__154–255 showed characteristics of poor line shape and chemical shift dispersion (Figure 4; red) suggestive of conformational disorder and a probable lack of stable tertiary structure. However, when unlabeled VirB7_{XAC2622_24–139} was added to the solution containing ¹⁵N-VirB9_{XAC2620_154–255}, the ¹H-¹⁵N cross peaks became more uniform in shape and more highly resolved (Figure 4; green). These observations suggest that VirB9_{XAC2620_154–255} on its own is in a dynamic conformational equilibrium or not properly folded in the absence of VirB7_{XAC2622_24–139}. Nevertheless, it undergoes a significant conformational change which greatly reduces its conformational flexibility when bound to the latter protein.

In order to further investigate whether the interaction of VirB9_{XAC2620_154–255} with the N-terminal domain of VirB7_XAC2622 alone is sufficient to drive the formation of a specific VirB7-VirB9 complex, we studied the interaction of ¹⁵N-labeled VirB9 with an unlabeled peptide derived from the VirB7_XAC2622 N-terminus (residues 24–46), which includes the VirB7_XAC2622 N-terminal segment that interacts with VirB9_XAC2620 (amino acids 27–41). Indeed, the perturbations observed in the ¹⁵N-HSQC spectra of VirB9_{XAC2620_154–255} in the presence of the full-length VirB7_{XAC2622_24–139} or the VirB7_{XAC2622_24–46} peptide are essentially the same (Figure 4 and Figure S8), demonstrating that the VirB7 N-terminal region is sufficient to interact with the VirB9_XAC2620 C-terminal domain.

To investigate whether aggregation could be responsible for the line broadening observed in the ¹⁵N-HSQC spectrum of VirB9 C-terminal domain, we calculated the approximate overall rotational correlation time (τ_c) from estimates of ¹⁵N T₁ and T₂ obtained using 1D versions of the ¹⁵N relaxation experiments. The values of τ_c of VirB9_{XAC2610_154–255} alone or in complex with the VirB7_{XAC2622_24–46} peptide are approximately 6.5 ns (data not shown), which suggest that the VirB9_XAC2620 C-terminal domain is monomeric in both conditions.

Relaxation Studies of VirB7_{XAC2622_24–139}

Measurements of ¹⁵N relaxation times (T₁ and T₂) and heteronuclear {¹H}-¹⁵N NOE were performed for ¹⁵N-VirB7_{XAC2622_24–139} alone (at 100 and 800 µM) and in complex with unlabeled VirB9_{XAC2620_154–255}. The relaxation data are consistent with the presence of a flexible N-terminal tail and a more rigid globular domain (Figure 5). It is worth noting that the VirB9 binding surface of VirB7_{XAC2622_24–139} (residues 27–41) becomes less flexible upon binding to VirB9 (Figure 5A and C). In the absence of VirB9, residues 29–42 display {¹H}-¹⁵N heteronuclear NOE values close to zero, indicating that this segment is not fully flexible and may present transient structures poised to interact with VirB9. Changes in ¹⁵N-T₁ and ¹⁵N-T₂ observed upon raising the VirB7_{XAC2622_24–139} concentration from 100 to 800 µM indicate a significant decrease in the overall rotational correlation rate, consistent with the formation of oligomers (Figure 5B and C). Relaxation parameters for residues involved in oligomerization (43–47 and some between positions 86 and 93) were not obtained because they were not detectable in the ¹⁵N-HSQC spectrum at high protein concentration (800 µM). Although these cross peaks become visible upon diluting the sample, they display too low amplitude to allow precise measurements. From the ¹⁵N T₁ and T₂ data, the overall rotational correlation times of VirB7_XAC2622 at the concentrations of 100 and 800 µM were estimated to be 5.8 and 12.6 ns, respectively. These values are consistent with the predominance of a monomer at lower concentration and a dimer at higher concentration. This observation, however, does not exclude the possibility of the existence of an equilibrium which includes higher-order oligomers, as shown by glutaraldehyde cross-linking data.

VirB7_XAC2622 Contributes to VirB9_XAC2620 and VirB10_XAC2619 Stability in vivo

In Agrobacterium tumefaciens cells, the VirB7 protein contributes to VirB9 and VirB10 stability [42]. We therefore produced a xac2622 gene knockout strain and performed immunoblot experiments to assess the expression of VirB7_XAC2622, VirB9_XAC2620 and VirB10_XAC2619. The production of all three proteins could be detected in wild type cells but none could be detected in the ΔvirB7_XAC2622 strain (Figure 6A). To evaluate if the absence of VirB9_XAC2620 and VirB10_XAC2619 in the mutant strain is due to the lack of the VirB7_XAC2622 production or to a polar effect in the T4SS operon, ΔvirB7_XAC2622 cells were complemented with a plasmid encoding the VirB7_XAC2622 protein in trans. The expression of VirB7_XAC2622 from the pUFR-VirB7 plasmid restored VirB9_XAC2620 and VirB10_XAC2619 protein levels (Figure 6A). Furthermore, no significant differences in virB9_XAC2620 transcript levels were detected between the wild-type, ΔvirB7_XAC2622 and ΔvirB7_XAC2622+pUFR-VirB7 strains in quantitative RT-PCR experiments (data not shown). These results show that VirB7_XAC2622 is necessary for the stability of VirB9_XAC2620 and VirB10_XAC2619 proteins in vivo.

The VirB7, VirB9 and VirB10 orthologs TraN, TraO and TraF form a stable trimeric complex [33], [34]. In order to determine whether the VirB7_XAC2622, VirB9_XAC2620 and VirB10_XAC2619 form a stable complex in Xac cells, we performed immunoprecipitation experiments using antisera for each of the three proteins. Results described in Figure 6B show that VirB7_XAC2622, VirB9_XAC2620 and VirB10_XAC2619 are present in the material immunoprecipitated by each of the three antisera in wild-type cells. Furthermore, none of the three proteins were immunoprecipitated from ΔvirB7_XAC2622 cells while all three proteins were detected in immunoprecipitation experiments using ΔvirB7_XAC2622+pUFR-VirB7 cells (Figure 6B). Altogether, these results show that a complex between VirB7_XAC2622, VirB9_XAC2620 and VirB10_XAC2619 is formed in vivo in Xac cells.

In order to test the role of VirB7_XAC2622 in Xac virulence, sweet orange leaves were infiltrated with the wild type and ΔvirB7_XAC2622 strains. Both strains presented essentially the same infection phenotypes, including water-soaking, hyperplasia and necrosis (Figure 6C) [13]. In planta growth curves of the wild type and ΔvirB7_XAC2622 cells revealed that the two strains replicate at a similar rate (Figure 6D). These data indicate that the VirB7_XAC2622 protein does not participate in the Xac's ability to induce canker symptoms in orange leaves under the experimental conditions tested. Similarly, a T4SS knockout failed to affect the pathogenicity of X. campestris pv. campestris on several host plants [43]. Xac pathogenicity in citrus and canker symptoms are strongly dependent on the action of virulence factors secreted by the Type III secretion system [13]. The T4SS is probably involved in other, as yet unidentified, cellular functions. As noted in the Introduction, the Xac pXAC64 megaplasmid codes for a second T4SS probably involved in plasmid mobilization [15], [27] and whose structural components exhibit only very low sequence identity to their counterparts in the chromosomally encoded T4SS under study. For example, its VirB9 and VirB10 homologs (XACb0039 and XACb0038) are only 23–24% identical to VirB9_XAC2620 and VirB10_XAC2619 [15]. Furthermore, the pXAC64 plasmid does not code for any proteins with similarity to VirB7_XAC2622 or any other known VirB7 proteins [15]. Therefore, it is unlikely that the two Xac T4SSs exercise redundant functions.

Discussion

The structure of the outer membrane complex of the T4SS coded by the conjugative E. coli plasmid pKM101 has been determined by X-ray crystallography and consists of 14 repeats of the TraN – TraO_CT – TraF_CT trimer [34], homologs of VirB7, the C-terminal domain of VirB9 and the C-terminal domain of VirB10, respectively. This structure revealed that the inner lining of the channel is formed by the C-terminal domains of the 14 TraF subunits. The TraN-TraO subcomplexes surround the internal ring formed by TraF. The 33 residue long lipidated TraN subunit is the outermost subunit of the complex whose external diameter is 172 Å [34].

Xanthomonas VirB7 Structure and Interactions in the Context of the T4SS Core Complex

VirB7_XAC2622 is structurally distinct from all VirB7 homologs characterized to date. Its structural motifs are illustrated in Figure 7A. Like all other VirB7 proteins, it has a signal peptide, a conserved cysteine residue (Cys22) within a lipobox for covalent attachment to outer membrane lipids and a short extended region involved in interaction with VirB9. These motifs are all contained within the first 41 residues, which corresponds relatively well with the size of the majority of unprocessed VirB7 proteins (45–65 amino acids) [36].

VirB7 proteins are very poorly conserved though it has been observed that many proteins of this family have in common a PVNK motif that is involved in the interaction with VirB9 [35]. The VirB9 binding site in VirB7_XAC2622 includes a HVNH sequence (residues 36–39) found in almost all Xanthomonadaceae orthologs (PVNR in X. albilineans and Stenotrophomonas maltophilia) which aligns with a perfect PVNK motif in the Neisseria sp. oral taxon 014 str. F0314 ortholog (Figure 7B). The side chains of the two most conserved residues of this motif, Val and Asn, make intimate contacts with TraO/VirB9 in the pKM101 outer membrane complex structure [35]: the Val side chain is inserted between the two TraO β-sheets while the Asn amide Oδ1 and Nδ2 atoms makes a set of three H-bonds with the TraO main chain. Furthermore, the PVNK motif in TraN is preceded by an approximately 10 amino acid segment that makes contacts with TraO, including a 3 residue β-strand that adds to the edge of one of the TraO β-sheets [34]. The corresponding regions in VirB7_XAC2622 are precisely those that demonstrate the greatest chemical shift perturbations upon binding to VirB9_XAC2620 (Figure 3). This suggests that interactions between VirB7_XAC2622 and VirB9_XAC2620 are very similar to that observed in the TraN-TraO complex.

The VirB7-VirB9 interactions described are most likely responsible for the loss of conformational flexibility observed for the N-terminal tail of VirB7_XAC2622 and the C-terminal domain of VirB9_XAC2620. Furthermore, interaction with VirB7_XAC2622 is likely to be accompanied by a significant change in the structure of VirB9_{XAC2620_154–255}, as revealed by the very large chemical shift perturbations observed for the majority of the ¹H-¹⁵N cross-peaks (Figure 4). These changes in VirB9_XAC2620 structure and dynamics may be related to its instability in vivo in the absence of VirB7_XAC2622. Our immunoblot and coimmunoprecipitation data suggest that VirB7_XAC2622, VirB9_XAC2620 and VirB10_XAC2619 form a stable trimeric complex in vivo and that the stabilities of VirB9_XAC2620 and VirB10_XAC2619 are dependent on VirB7_XAC2622 (Figure 6). This may be a physiologically relevant mechanism by which excess VirB9 and VirB10 are degraded to maintain proper VirB7-VirB9-VirB10 stoichiometry.

VirB7_XAC2622 has a mosaic structure (Figure 7A). In addition to the canonical VirB7-like N-terminal region, VirB7_XAC2622 has a C-terminal region that includes a globular domain (residues 52–133). This globular domain does not interact with VirB9_{XAC2620_154–255} but does interact with an extended region (residues 42–49) that immediately precedes the globular domain in another VirB7_XAC2622 molecule (Figure 2D), leading to the formation of homo-oligomers. The unique structural features of VirB7_XAC2622 raise the question of how they may be incorporated into the model for the T4SS outer membrane complex tetradecamer previously solved for the pKM101 conjugation machine [34]. Specifically: can VirB7_XAC2622 oligomerization occur while maintaining VirB7-VirB9 interactions in the context of the pore? And how are the VirB7_XAC2622 globular domains oriented with respect to the pore? In order to investigate these questions, we used the VirB7_XAC2622-TraN alignment depicted in Figure 7B to manually place 14 different NMR models of residues 37–139 of VirB7_XAC2622 (VirB7_{XAC2622_37–139}) around a tetradecameric ring made up of TraO_CT subunits derived from the pKM101 TraN-TraO_CT-TraF_CT structure [34]. The VirB7_{XAC2622_37–139} subunits were placed so that Val37 and Asn38 occupied positions similar to Val33 and Asn34 of TraN. This (VirB7_{XAC2622_37–139}-TraO_CT)₁₄ complex was used as a starting model in a molecular dynamics simulation in which the following restraints were used: i) The 14 TraO_CT subunits were fixed in space, ii) VirB7_XAC2622 residues 37 and 38 were fixed in space and iii) intermolecular NOE derived distance restraints were placed on 5 hydrogen pairs between neighboring VirB7_XAC2622 subunits. During the initial cycles of the molecular dynamics simulation, the distance observed between the 13 intermolecular VirB7_XAC2622 hydrogen pairs for which NOEs were measured (Figure 2D and Results above) decreases from the 12–25 Å range observed in the starting model to a 4–7 Å range, consistent with observation of NOE signals. These short contact distances are then observed throughout the simulation for all 14 VirB7_XAC2622 pair interfaces made up of residues A43, T45, E46, I47, L49 in one VirB7_XAC2622 molecule and residues T63, S85, D86, Y87, T88, I90 in a neighboring VirB7_XAC2622. This is a consequence of the strong distance restraints included in the potential energy function. The final molecular dynamics configuration is shown in Figure 7C. Adding models of TraN residues 19–32 to represent VirB7_XAC2622 residues 22–36 (see alignment in Figure 7B) and TraF_CT (to represent VirB10_XAC2619) results in the structure shown in Figure 7D made up of 14 repeats of VirB7_{XAC2622_37–139}, TraN_19–32, TraO_CT and TraF_CT. This can be seen as putative model for the VirB7_XAC2622-VirB9_XAC2620-VirB10_XAC2619 outer membrane complex. Note that in Figures 7C and 7D, the VirB7 N0 domains (dark blue) adopt a variety of orientations with respect to the central ring and the membrane normal. This flexibility is derived from the conformational freedom of the VirB7_XAC2622 regions immediately before and after residues 42–49 (magenta) involved in VirB7-VirB7 interactions.

Our proposal of a physiological role for VirB7_XAC2622 oligomerization is supported by an analysis of conserved residues in the small group of full-length VirB7_XAC2622 homologs found in Xanthomonadaceae and the two Neisseria species Neisseria flavescens SK114 and Neisseria sp. oral taxon 014 str. F0314. An alignment of VirB7_XAC2622 and the two Neisseria proteins is shown in Figure 7B. The three proteins have only 21% sequence identity. However, 6 out of 11 of the residues involved in intermolecular NOEs at the B7-B7 interface (E46, I47, L49, T63, D86 and T88) are absolutely conserved in all Xanthomonadaceae, Neisseria str. F0314 and Neisseria flavescens and is either Phe or Tyr at position 87. Thus, the oligomerization interface is well conserved in an otherwise highly diverse protein family.

VirB7_XAC2622 oligomerization is different from the dimerization observed in the canonical VirB7 protein from A. tumefaciens [30] which occurs via the formation of disulfide bonds involving residue 24. The structure of the outer membrane complex [34] however, is not compatible with the maintenance of these disulfide bonds in a fully assembled T4SS. Furthermore, VirB7_XAC2622 does not have any cysteine residues other than at the N-terminal lipidation site.

The VirB7_XAC2622 Globular Domain is Structurally Similar to Proteins Associated with Transport Across Bacterial Outer Membranes

The VirB7_XAC2622 globular domain has no significant sequence similarity to proteins of known structure. We therefore used the Dali Server [44] to search for proteins with similar topology to this domain. This analysis revealed that the VirB7_XAC2622 folded region resembles domains found in the following proteins (Table 3 and Figure 8): i) the TonB-dependent receptors (periplasmic signaling domain; Figure 8A) [45], [46], ii) the outer membrane secretin channel GspD from the type II secretion system (T2SS; Figure 8B) [47], iii) the secretin EscC from the Type III secretion system (T3SS; Figure 8C) [48], iv) the needle-like cell-puncturing device components gp27 and gp44 from long-tailed phages like T4 and Mu (Figure 8D) [49], [50] and v) the Type VI secretion system (T6SS) protein VgrG (superposition not shown) [51]. These domains are found at the N-termini of T2SS and T3SS secretins and have been denominated N0 domains which have also been identified in the Type IV pilus secretin PilQ [47] and the filamentous phage secretin pIV [52]. It is striking that in spite of its small and compact nature, domains with the VirB7_XAC2622 topology are found only in a restricted number of proteins, all of which are involved in the transport of molecules across bacterial outer membranes. These observations suggest that all of these proteins may be distantly related and have evolved in the periplasm or outer membrane to adopt a variety of functions, from structural modules in outer membrane pores (secretins from type II and type III secretion systems, type IV pili and filamentous phages) to membrane-penetrating devices in T6SS and long-tailed bacteriophages, and signal-transduction modules in TonB-dependent receptors.

Nakano et al. [53] have recently described the crystal structure of the DotD lipoprotein of the Type IV-B secretion system from Legionella pneumophila. Type IV-B secretion systems have weak or no sequence similarity with the larger group of Type IV-A secretion systems [54] to which the Xanthomonad T4SS pertains. Interestingly, the DotD crystal structure presents a C-terminal region with an N0 domain topology similar to that of VirB7_XAC2622 structure (Figure 8E), though the proteins present no significant sequence similarity. An interesting difference between the two structures is that a 6 amino acid sequence (called the “lid”) of the otherwise disordered N-terminal region is visible in the crystal structure and makes β-strand addition to β1. This interaction is different from that observed in the VirB7_XAC2622 oligomer described here. Since no electron density for the 23 residues between the lid and the N0 domain were visible, it was not clear whether they come from the same molecule in the crystal lattice. We therefore predict that DotD will eventually be shown to exhibit the same characteristics as VirB7_XAC2622, namely to interact with both a VirB9-like protein and to form head-to-tail polymeric rings based on interactions between the flexible N-terminal region and globular N0 domains of neighboring subunits. This analysis points to previously unrecognized common structural features between the outer membrane complexes of Type IV-A and Type IV-B secretion systems.

The novel VirB7_XAC2622 structure, its oligomerization and its interaction with VirB9 described here point to a possible structural variation in the Xanthomonas T4SS that could result in the formation of an extra ring layer in the core complex. While the VirB7-VirB7 interactions observed in the NMR experiments were of relatively low affinity (fast exchange chemical shift changes were observed in the 7–850 µM range), these interactions would be strengthened by the increased effective concentration afforded by the fourteen neighboring VirB7_XAC2622 binding sites on the outer surface of the VirB9/VirB10 ring. Several hypotheses regarding the physiological role of this VirB7_XAC2622 ring can be proposed. One possibility is that the weak B7-B7 interactions break and reform again to accommodate different conformational states of the outer pore during substrate translocation or to permit allosteric communication between the outer and inner membrane core complexes. Another possibility is that the VirB7_XAC2622 N0 domains could act as a conduit for signal transmission between substrates or signaling molecules in the periplasm and the VirB9 and VirB10 subunits. Whether the VirB7_XAC2622 N0 domain carries out a purely structural function or is involved in substrate recognition or signal transduction will have to be tested in the future.

Materials and Methods

Cloning, Protein Expression and Purification

Plasmids, oligonucleotides and bacterial strains used in this study are listed in Table S1. The DNA fragments encoding residues 24–139 of VirB7_XAC2622 (GenBank AAM37471), 154–255 of VirB9_XAC2620 (GenBank AAM37469) and 85–389 of VirB10_XAC2619 (GenBank AAM37468) were amplified by PCR from Xanthomonas citri subsp. citri strain 306 genomic DNA and inserted into the pET28a expression vector (Novagen) using the NdeI and XhoI (VirB7_XAC2622) or NdeI and BamHI (VirB9_XAC2620 and VirB10_XAC2619) cloning sites, to express the recombinant proteins fused with an N-terminal His-tag. DNA fragments encoding residues 51–134 of VirB7_XAC2622 and 34–255 of VirB9_XAC2620 were cloned into the pET11a vector (Novagen) using NdeI and BamHI sites. All constructs were confirmed by DNA sequencing. E. coli BL21(DE3)RP and BL21(DE3)Star strains (Novagen) were transformed with the recombinant plasmids for expression of the protein products which are referred to as VirB7_{XAC2622_24–139_His}, VirB9_{XAC2620_154–255_His}, VirB7_{XAC2622_51–134}, VirB9_{XAC2620_34–255} and VirB10_{XAC2619_85–389_His}. Cells were grown in 2XTY for expression of unlabeled proteins or in M9 minimal media containing ¹⁵NH₄Cl and ¹²C-glucose or ¹³C-glucose (Cambridge Isotope Laboratories) for production of ¹⁵N or ¹⁵N and ¹³C isotopically labeled proteins. Recombinant protein expression was induced at midlog phase by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside and the cells were grown for four hours at 37°C. Cells were then harvested and lysed by French Press. In the case of VirB7_{XAC2622_24–139_His} and VirB9_{XAC2620_154–255_His}, the lysis supernatants were separated by passage through Q-Sepharose (VirB7_{XAC2622_24–139_His}) or SP-Sepharose (VirB9_{XAC2620_154–255_His}) ion exchange columns (GE Healthcare) followed by affinity chromatography using a nickel affinity column. The N-terminal His-tags were removed using the Thrombin CleanCleave kit (Sigma), creating the proteins VirB7_{XAC2622_24–139} and VirB9_{XAC2620_154–255}. A final purification step consisted of passage through a Superdex 75 26/60 size exclusion column (GE Healthcare). VirB7_{XAC2622_51–134} was purified using Q-sepharose anion exchange and Superdex 75 gel filtration chromatography. VirB9_{XAC2620_34–255} and VirB10_{XAC2619_85–389_His} are expressed as insoluble proteins present in inclusion bodies. After lysis, VirB9_{XAC2620_34–255} was solubilized in the presence of 8 M urea and purified by SP-Sepharose cation exchange and Superdex 75 size exclusion chromatographies using buffers containing 8 M urea. VirB10_{XAC2619_85–389_His} was purified by affinity chromatography using buffers containing 8 M urea. Purified proteins were refolded by successive dialysis against decreasing concentrations of urea in 10 mM sodium acetate (pH 5.0) for VirB9_{XAC2620_34–255} or 10 mM Tris-HCl (pH 8.0), 150 mM NaCl for VirB10_{XAC2619_85–389_His}. Synthetic peptides corresponding to VirB7_{XAC2622_24–46} and VirB7_{XAC2622_38–52} were purchased from Biomatik Corporation (Cambridge, Ontario, Canada) and EZBiolab (Carmel, Indiana, USA), respectively.

NMR Spectroscopy

NMR samples containing ¹⁵N or ¹⁵N/¹³C labeled protein were prepared in 10 mM ²H-sodium acetate (pH 5.0), containing 50 mM sodium chloride, 0.05% (w/v) sodium azide, 7% (v/v) ²H₂O and 1 mM trimethylsilyl-2,2,3,3-tetradeuteropropionic acid as internal standard for ¹H chemical shift referencing. NMR spectra were acquired on a 600 MHz Varian Unity-Inova spectrometer equipped with inverse triple resonance (¹H, ¹⁵N, ¹³C) cold probe, or on BRUKER 500 MHz DRX or 800 MHz Avance III spectrometers equipped with inverse triple resonance (¹H, ¹⁵N, ¹³C) room temperature probes. Unless otherwise mentioned, all NMR experiments were performed at 40°C. The backbone resonance assignment of ¹⁵N/¹³C-VirB7_{XAC2622_24–139} was achieved by analyzing the following three-dimensional spectra: HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB and CBCA(CO)NH [55]. Side-chain resonance assignments were determined by analyzing the following spectra: 2D ¹³C-HSQC, 2D TOCSY, 2D NOESY, 3D HNHA, 3D HBHA(CBCACO)NH, 3D ¹⁵N-TOCSY-HSQC, 3D H(CCO)NH-TOCSY, 3D (H)CC(CO)NH-TOCSY, 3D ¹⁵N-NOESY-HSQC, 3D H(C)CH-TOCSY and 3D ¹³C-NOESY-HSQC (specific for the aliphatic or the aromatic regions). The 3D H(C)CH-TOCSY and 3D ¹³C-NOESY-HSQC spectra were recorded in >99% (v/v) ²H₂O. NOESY experiments were carried out using 0.2–0.3 mM protein samples and all NOESY mixing times were 100 ms. Backbone resonance assignments for ¹⁵N/¹³C-VirB7_{XAC2622_24–139} in complex with unlabeled VirB9_{XAC2620_154–255} were obtained from the analysis of three-dimensional HNCA, HN(CO)CA, HNCO, HNCACB, CBCA(CO)NH, HBHA(CBCACO)NH and ¹⁵N-NOESY-HSQC experiments recorded on a sample of ¹⁵N/¹³C-VirB7_{XAC2622_24–139} in the presence of a 20% molar excess of unlabeled VirB9_{XAC2620_154–255}. The backbone resonance assignment (¹⁵N and ¹H) for VirB7_{XAC2622_51–134} was performed by comparison with the ¹⁵N-HSQC spectrum of the larger construct, VirB7_{XAC2622_24–139}. Residual ¹D_NH dipolar couplings for VirB7_{XAC2622_24–139} (0.25 mM), alone or in complex with VirB9_{XAC2620_154–255}, were determined from the differences in ¹H-¹⁵N splittings obtained in isotropic and anisotropic media. The ¹H-¹⁵N splittings were extracted from a series of J-modulated 2D ¹H-¹⁵N-HSQC experiments acquired at 30°C on the 500 MHz spectrometer, and analyzed essentially as described [56]. Residual alignment was induced by a liquid crystalline medium consisting of 5% penta-ethyleneglycol monododecyl ether (C12E5)/n-hexanol (r = 0.96) [57]. Fittings of RDCs to the NMR and X-ray structures were carried out using PALES [58] or MODULE [59]. All NMR spectra were processed with NMRPipe [60] and analyzed with CCPN Analysis [61]. Resonance assignments were deposited in the Biological Magnetic Resonance Data Bank (BMRB accession code 17257).

NMR Structure Calculation

Structure calculation and automated NOE assignment were performed using CYANA 2.1 [37]. Chemical shifts of ¹Hα, ¹³Cα, ¹³Cβ, ¹³C′ and ¹⁵N^H were used as input for TALOS [62] to predict φ and ψ dihedral angles that were subsequently used as restraints in the structure calculation. A total of 3471 NOE cross peaks were manually picked in the ¹⁵N-NOESY-HSQC and ¹³C-NOESY-HSQC (acquired specifically for the aliphatic or the aromatic regions) spectra and used as input for CYANA. The CYANA protocol consisted of 7 cycles of simulated annealing in torsion angle space with 10000 integration steps. In each cycle 300 conformers were calculated. The intra-residue, sequential and 10 unambiguous long range NOE cross peaks were assigned manually and imposed during all stages of the protocol. Chemical shift tolerances for automated NOE assignment were set to ±0.025 ppm in the direct ¹H dimension, ±0.030 ppm in the indirect ¹H dimension and ±0.25 ppm for the heteronuclei. The best 50 CYANA solutions were selected based on a target function criteria, and further refined in explicit water using CNS2.1 and HADDOCK2.0 [41]. The 1616 NOEs assigned by CYANA, the manually assigned NOEs, and the TALOS dihedral angle restraints were used as input during water refinement in HADDOCK. The best 20 lowest-energy conformers after water refinement were deposited in the Protein Data Bank (PDB code 2L4W). The quality of the final NMR structures was investigated by PROCHECK-NMR [63] and PSVS [64]. The structures were visualized using MOLMOL [65] or PYMOL (http://www.pymol.org).

¹⁵N Relaxation Measurements

Experiments for measuring backbone ¹⁵N longitudinal (T₁) and transverse (T₂) relaxation times, and the heteronuclear {¹H}-¹⁵N NOE, were recorded at 40°C on a Varian Inova 600 MHz spectrometer using standard Biopack pulse sequences (Varian, Inc.). The inversion recovery delays used for sampling T₁ relaxation were 50, 250, 450, 650, 850, 1050, 1250, 1450 and 1650 ms. The CPMG delays used for sampling T₂ relaxation were 10, 30, 50, 70, 90, 110, 130, 150, 170, 190 and 210 ms. The ¹H saturation period in the heteronuclear {¹H}-¹⁵N NOE experiment was 3 s. All 2D spectra were acquired sequentially as matrices of 512(¹H)×128(¹⁵N) complex points, and inter scan delay of 3 s. Relaxation rates were obtained by fitting the time decay of peak intensities to an exponential decay function with three fitted parameters, the relaxation rate, the intensity at time zero and an offset, using the CCPN Analysis software [61]. Overall rotational correlation times (τ_c) were obtained either from the mean ratios of ¹⁵N T₁/T₂ relaxation rates of ¹H-¹⁵N bond vectors located in regions of secondary structure using TENSOR2 [66], or from estimates of ¹⁵N T₁ and T₂ relaxation times obtained from 1D ¹⁵N-edited relaxation experiments, as previously described [67], [68].

NMR Titration Experiments

Unlabeled VirB9_{XAC2620_154–255} or VirB9_{XAC2620_34–255} were titrated into a solution of ¹⁵N-labeled VirB7_{XAC2622_24–139} and a ¹⁵N-HSQC spectrum was acquired after the addition of each aliquot until a molar excess of 1.5 of unlabeled protein with respect to ¹⁵N-VirB7_{XAC2622_24–139}. In analogous experiments ¹⁵N-labeled VirB9_{XAC2620_154–255} was titrated with unlabeled VirB7_{XAC2622_24–139} or VirB7_{XAC2622_24–46}. VirB7_{XAC2622_24–139} oligomerization was studied by monitoring changes in the ¹⁵N-HSQC spectra as a function of protein concentration in the range of 7 to 850 µM. ¹⁵N-labeled VirB7_{XAC2622_51–134} was also titrated with unlabeled VirB7_{XAC2622_38–52}. Chemical shift perturbations due to sample dilution or protein-protein interaction were obtained from the weighted chemical shift changes in the ¹⁵N-HSQC spectra calculated according to Eq. 1 [69]:(1)where ΔδH^N and ΔδN^H are the chemical shift changes (in ppm) of the amide proton and nitrogen resonances, respectively.

Protein-Protein Docking

Docking simulations were used to build a docking model for the dimer of VirB7_{XAC2622_24–139}. The calculations were performed with HADDOCK2.0 using CNS2.1 [41], and the first model of the NMR ensemble as starting structure. The docking was driven by unambiguous intermolecular NOEs and by ambiguous interaction restraints (AIR) between active residues and active plus passive residues. The following 13 NOE restraints were used: Ala43Hβ - Ile90Hδ1; Thr45Hα - T88Hγ2; Thr45Hβ - T88Hγ2; Glu46Hβ - Tyr87Hε; Ile47Hδ1 - Thr63Hβ; Ile47Hδ1 - Thr63Hγ2; Ile47Hδ1 - T88Hα; Ile47Hδ1 - T88Hγ2; Leu49Hγ - Ser85Hα; Leu49 Hδ1 - Asp86Hα; Leu49Hδ1 - Asp86Hβ1; Leu49Hδ1 - Asp86Hβ2 and Leu49Hδ2 - Asp86Hα. Active residues were identified as those showing chemical shift perturbations of at least one standard deviation above the mean, for ¹⁵N-VirB7_{XAC2622_24–139} as a function of protein concentration (Figure 2). Active residues were E42, A43, T45, E46 and I47 at the N-terminal tail of the first subunit and D86, T88, I90, G91, Q115 of the C-terminal globular domain of the second subunit. Passive residues were F40, D41, P44, P48 and L49 in the N-terminal segment, and P84, S85, Y87, P92, A113 and A114 in the C-terminal domain. The N-terminal tails (residues 24–50) were kept fully flexible during all stages of the docking, while C-terminal domain residues at the interface, 84–92, 61–63 and 113–115, were maintained semi-flexible, being allowed to move only during semi-flexible refinement. The rigid body stage consisted of the calculation of 1000 solutions, from which 100 with the lowest HADDOCK scores were selected for semi-rigid simulated annealing in torsion angle space including backbone and side chain flexibility, and final refinement in Cartesian space with explicit water. The refined solutions were then clustered based on the backbone RMSD at the interface. An analogous protocol was used for docking the N-terminal (residues 24–50) and C-terminal (residues 51–139) fragments of VirB7, except that in this calculation NOE restraints were not included and flexibility was introduced only at the interface.

Model Building and Molecular Dynamics Simulations

Models of residues 37–139 of VirB7_XAC2622 (VirB7_{XAC2622_37–139}) were positioned around a tetradecameric ring made up of TraO_CT subunits derived from the pKM101 T4SS TraN-TraO_CT-TraF_CT structure [34]. The VirB7_{XAC2622_37–139} subunits were placed so that Val37 and Asn38 occupied positions similar to Val33 and Asn34 of TraN. Molecular mechanics optimization and dynamics were then carried out from this initial model. The OPLS/AA force-field [70] for the protein and an implicit solvent representation of the generalized Born-formalism [71] were used. In addition to the molecular force-field, distance restraints with a flat-bottom harmonic functional form were included such that an energy penalty was added to the potential when the distance between specified pairs of atoms was less than a minimum threshold value of 3 Å or exceeded a maximum of 6 Å. A harmonic force constant of 5000 kJ⋅mol⁻¹⋅Å⁻² was used. The following 5 NOE proton pairs were specified for each VirB7_XAC2622 pair interface: Ala43Hβ - Ile90Hδ1, Thr45Hβ - T88Hγ2, Ile47Hδ1 - Thr63Hβ; Leu49Hγ - Ser85Hα; and Leu49Hδ2 - Asp86Hα. These pairs correspond to a subset of the pairs for which intermolecular NOEs were detected. Thus, a total of 70 (14×5) distance restrains were included in the potential. All ionizable residues besides histidine were treated in their charged forms. Molecular dynamics were carried out at a temperature of 300 K for a total time of 2 ns. All computations were carried out with the GROMACS 4.5.3 software [72].

Crystallization, Data Collection and Processing

A construct corresponding to the VirB7 C-terminal region, VirB7_{XAC2622_51–134}, at 14 mg/mL in 5 mM Tris-HCl pH 7.5 and 25 mM sodium chloride, was submitted to vapor diffusion sitting-drop crystallization trials at 18°C. Large plates appeared after one day over a reservoir solution comprising 1.4 M ammonium sulfate and 4% (v/v) isopropyl alcohol. Reservoir solution supplemented with 25% (v/v) glycerol was used as cryoprotectant. Crystals were flash frozen at 100 K and submitted to X-ray diffraction at beam line W01B-MX2 of the Brazilian Synchrotron Light Laboratory (LNLS) coupled to a Marmosaic-225 CCD detector (Mar, USA). The diffraction data were indexed, integrated and scaled using HKL2000 [73].

X-ray Structure Determination and Refinement

The crystal structure of VirB7_{XAC2622_51–134} was determined by molecular replacement by Phaser [74] using residues 51–134 of the lowest energy NMR structure as the search model. The model was iteratively refined using the graphics program Coot [75] with rounds of restrained refinement in Refmac5 [76] with individual anisotropic B-factor for all non-hydrogen atoms. All programs used for refinement were from the CCP4 suite [77]. The quality of the structure was analyzed by the programs Coot, Refmac5, Procheck [78], Rampage [79] and MolProbity [80]. The coordinates of the crystal structure were deposited in the PDB (PDB code 3OV5).

Fluorescence Spectroscopy

Fluorescence experiments were conducted in an AVIV ATF-105 spectrofluorimeter (AVIV Instruments). Fluorescence emission was collected after successive addition of 0.1 µM VirB7_{XAC2622_24–139_His} aliquots into a sample containing 1 µM VirB9_{XAC2620_34–255} in 5 mM sodium acetate (pH 5.0). The final VirB7_{XAC2622_24–139_His} concentration in the titration was 2.2 µM. Samples were pre-equilibrated for 2 min at 25°C, excited at 280 nm (2 nm bandwidth) and fluorescence emission was detected from 334–342 nm (7 nm bandwidth) at 2 nm intervals. The dissociation constant was calculated from a nonlinear regression fit of fluorescence titration data to Eq. 2 [81], [82], using the SigmaPlot program (SPSS, Inc.):(2)where F is the fluorescence intensity at any given point of the titration curve, v is the initial volume, V is volume at any given point of the titration, [X] and [Y] are the VirB9_{XAC2620_34–255} and VirB7_{XAC2622_24–139_His} concentrations at any given point of the titration, respectively, K_d is the dissociation constant and α is the ratio between the maximum fluorescence intensity and the initial fluorescence intensity.

Glutaraldehyde Cross-Linking Experiments

Samples with different concentrations of VirB7_{XAC2622_24–139} or VirB7_{XAC2622_51–134} were pre-equilibrated for 15 min, and then incubated with 0.01% (v/v) glutaraldehyde. After 20 min, the cross-linking reaction was stopped by the addition of SDS-PAGE sample buffer (100 mM Tris-HCl pH 6.8, 3.7% (w/v) SDS, 18.7% (v/v) glycerol, 140 mM 2-mercaptoethanol and 0.01% (w/v) bromophenol blue). The cross-linking products were analyzed by 16% Tricine SDS-PAGE [83].

virB7_XAC2622 Knockout and Complementation

Approximately 1 kb of the upstream and downstream regions of the virB7_XAC2622 gene were amplified by PCR from Xac genomic DNA and the two fragments were ligated to produce an in frame deletion, leaving only the region coding for the first three and last five codons. This sequence was then cloned into the BamHI restriction site of the pNPTS138 suicide vector (M. R. Alley, unpublished), thereby generating the plasmid pNPTS-Δxac2622. This vector was introduced into Xac by electroporation and replacement of the wild-type copy by the deleted version was obtained after two recombination events as described [84]. In order to complement the virB7_XAC2622 knockout, a fragment including the virB7_XAC2622 gene plus 1000 pb of upstream sequence was amplified by PCR from Xac genomic DNA and inserted into the pUFR047 vector [85] at the BamHI restriction site, creating the plasmid pUFR-VirB7. This plasmid was then transferred to the ΔvirB7_XAC2622 strain by electroporation and selection of gentamicin resistance.

In planta Studies

Virulence assays were performed by infiltration of the Xac strains into sweet orange leaves (Citrus sinensis L. Osbeck). The cell cultures were adjusted to an optical density of 0.1 at 600 nm and leaves were inoculated by syringe infiltration with needle. The plants were maintained at 28°C with a 12 h photoperiod and the development of the symptoms was regularly observed. Bacterial growth was measured by harvesting 10 mm² citrus leaf discs for each inoculated Xac strain, and the leaf discs were macerated in 150 mM sodium chloride. The solution was serially diluted and spread onto LBON plates [1% (w/v) tryptone, 0.5% (w/v) yeast extract and 1.5% (w/v) agar] containing ampicillin. The mean number of colony forming units per square centimeter (CFU/cm²) was calculated by counting individual colonies obtained from each dilution.

Antibody Production and Immunoblot

Rabbit polyclonal antibodies were raised against VirB7_{XAC2622_24–139}, VirB9_{XAC2620_34–255} and VirB10_{XAC2619_85–389_His}. To analyze protein levels in Xac, cells were grown in XVM2 medium [86] to an optical density (600 nm) of 1.0. Recombinant proteins and Xac cellular extracts were separated by 18% SDS-PAGE followed by immunoblot analysis. The rabbit sera were used at 1∶1000 dilutions and the antibodies were detected with staphylococcal protein A conjugated to horseradish peroxidase (Sigma). Immunoblots were developed with the ECL Advance Western Blotting system and exposed to Amersham Hyperfilm ECL film (GE Healthcare).

Immunoprecipitation

Strains of Xac were grown in XVM2 to an optical density (600 nm) of 1.0 and equal numbers of cells were harvested by centrifugation. Cells were resuspended in 500 µl of 50 mM Hepes (pH 8.0) containing 5 mM EDTA. Coimmunoprecipitation experiments were performed after solubilization of cells with deoxycholate (DOC) and N,N–Dimethyldodecylamine N-oxide (LDAO) detergents, as described [87]. Bacterial suspensions were treated with 200 µg/ml lysozyme for 1 h at 4°C. Cells were lysed by sonication in the presence of 100 mM NaCl, 1% (w/v) DOC and protease inhibitor cocktail (Sigma Aldrich). Volumes were adjusted to 1 mL with 50 mM Hepes (pH 8.0) and 2% (w/v) LDAO and lysates were solubilized by incubation for 16 h at 4°C with agitation. Solubilized material was isolated by centrifugation at 15,000×g for 20 min and incubated with Protein G agarose beads (Millipore) for 4 h at 4°C, removing nonspecifically bound proteins. For immunoprecipitation, supernatants from this pre-clearing step were incubated with protein G agarose beads in the presence of pre-immune serum (controls) or anti-VirB7, anti-VirB9 or anti-VirB10 antibody, for 16 h at 4°C, with agitation. The unbound material was discarded after centrifugation (400×g, 5 min) and beads were washed three times with decreasing concentrations of LDAO and DOC (final wash contained 0.1% DOC and 0.1% LDAO), each time for 10 min at 25°C, with agitation. Immunoprecipitates were eluted by addition of SDS sample buffer and boiling for 10 min. Samples were analyzed by SDS-PAGE followed by immunoblot.

Quantitative RT-PCR

RNAs from Xac cultures grown in XVM2 medium to an optical density (600 nm) of 1.0 were extracted using Illustra RNAspin Mini kit, according to manufacturer's instructions (GE Healthcare). Purified RNA was treated with 1 U/µg DNaseI, RNase-free (Fermentas) and successful removal of contaminating DNA was confirmed by PCR. Reverse transcription was performed using 1 µg of DNaseI-treated RNA and RevertAid H Minus First Strand cDNA Synthesis Kit, following manufacturer's protocol (Fermentas). Quantitative amplification of the resulting cDNA (40 ng) was performed using 0.3 µM of each primer (F_virB9_RT and R_virB9_RT; Table S1) and SYBR Green/ROX qPCR Master Mix in the ABI7300 Real-Time System (Applied Biosystems). Relative quantification of gene expression was performed using gene xac1631 (that codes for subunit A of DNA gyrase) as an endogenous control and the 2^−ΔΔCT method [88]. Primers were designed using the PrimerExpress Software (Applied Biosystems). Triplicates of two independent biological samples were used.

Accession Numbers

¹H, ¹³C and ¹⁵N resonance assignments of VirB7_XAC2622 were deposited in the BMRB: entry 17257. NMR and X-ray crystallography coordinates have been deposited in the PDB with accession codes 2L4W and 3OV5, respectively.

Supporting Information

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