Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri

Abstract

Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 Å-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein-protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution.

Figure 1. Biochemistry and electron microscopy map and model of the X. citri T4S system core complex.

a, SDS-PAGE of the VirB7-VirB9-VirB10 core complex. Left lane labeled “Marker”: molecular weight markers, with the molecular weight for each band shown at left. Right lane labeled “CC”: purified core complex, with the three components labeled at right. Purification assays were repeated more than ten times with similar results (see also Supplementary Figure 9a). b, Electron micrograph of the X. citri core complex, with some particles highlighted in blue circles. Scale bar: 50 nm. Experiments were repeated at least 8 times showing similar results. c, Representative of top, tilt, and side view 2D class averages obtained using RELION 2.0 (see the Methods section for more information). d, Overview of the 3.3-Å electron density, contoured at 4 σ level. e, Two representative regions of the electron density of the X. citri core complex. Electron density map contoured at a 4 σ level is shown in chicken wire representation, color-coded in grey-blue. The final model built into the map is shown in a ribbon and stick representation color-coded dark blue, red, yellow, and light blue for nitrogen, oxygen, sulfur and carbon atoms, respectively. Secondary structures are labeled. The regions depicted are both from the VirB10_CTD (left panel for α-helices and right panel for β-strands). f, Topology secondary structure diagrams of VirB7 (red), VirB9 (green), and VirB10 (blue). β-strands and α-helices are represented as arrows and cylinders, respectively. Regions for which no electron density was observed are indicated by dashed lines. Note that the 182 N-terminal residues of VirB10 were disordered and could not be traced except for a small helix corresponding to residues 150-161. This is not unexpected considering that the 101 amino acid stretch (residues 84-184) between the inner membrane spanning helix and the globular VirB10_CTD is rich in proline residues (Supplementary Fig. 2).

Figure 2. Structure of the X. citri T4S system core complex.

a, Top view of the structure in ribbon representation with one VirB7-VirB9-VirB10 heterotrimer shown in red, green and blue, respectively (coloring maintained in parts b-e). The heterotrimer numbering used in this study is indicated, with the colored heterotrimer serving as reference and therefore numbered 0. b and c, Top view (b) and side view (c, upper panel) of the structure in surface representation. c, lower panel: cut-away side view of the model. External and internal dimensions of specific structural features are indicated in b and c. The 80-Å opening at the bottom of the I-chamber is similar to that observed for the 8.5-Å cryo-EM structure of the elastase-digested pKM101 core complex. d and e, Side view of the structure in surface representation as in c but rotated 60 degrees counter-clockwise to show heterotrimer 0 more clearly. e is a zoomed in view of the region delimited by the dashed line in d.

Figure 3. The heterotrimer of the X. citri T4S system core complex structure and comparison with that of pKM101 from E. coli.

The X. citri VirB7-VirB9-VirB10 heterotrimer. a, Proteins are shown in ribbon representation and color-coded as in Figure 2. Specific domains are labeled, as also are the secondary structures in each protein. The linkers between VirB9 NTD and CTD, the two α-helices of the VirB10 “antennae” and the CTD of VirB10 and VirB10_{NTD_150-161} helix are shown in dashed green and blue lines, respectively, as they are disordered in the structure. The orientation is as in Figure 2a. b, Same as in a, but rotated 90 degrees to correspond to the side view shown in Figure 2c. The box delimits the central compact core. c, Superposition of the structure of the X. citri heterotrimer (in red, green and blue) with that of the pKM101 complex (grey) composed of TraN/VirB7, TraO_CTD/VirB9_CTD, and TraF_CTD/VirB10_CTD.

Figure 4. Interactions between heterotrimers in the X. citri T4S system core complex.

a, Overall structure of the core complex with 3 heterotrimers highlighted. These are labeled 0, -1, and -2 and color-coded as in Figure 2 with different shades of the respective colors for each heterotrimer. b, Interactions between the VirB10_CTDs of heterotrimer 0 (0VirB10_CTD) and -1 (-1VirB10_CTD). The β1 strand is labeled “Lever arm” due to its correspondence to part of the same structural feature observed in pKM101 core complex O-layer. c, Interactions between the VirB9_CTD of heterotrimer 0 (0VirB9_CTD) and VirB7 of heterotrimer 0 (0VirB7) and -1 (-1VirB7). d, Interactions between adjacent VirB9_NTDs. The two VirB9_NTDs shown are from heterotrimer 0 (0VirB9_NTD) and -1 (-1VirB9_NTD). e, Interactions between the VirB9_NTD of heterotrimer 0 (0VirB9_NTD) and VirB10_CTD of heterotrimer -1 (-1VirB10_CTD) and -2 (-2VirB10_CTD). f, Interactions of a helical structure belonging to VirB10_NTD (VirB10_{NTD_150-161}) with two adjacent VirB9_NTDs. Left panel: surface representation of the lumen of the X. citri core complex structure, color-coded by proteins with VirB7, VirB9, and VirB10 in red, green and blue, respectively. Upper-right panel: zoomed view of the region contained in the black box shown at left. VirB9_NTDs and the VirB10_{NTD_150-161} helix are shown in ribbon representation. The electron density for the VirB10_{NTD_150-161} is also shown (grey) contoured at 4 σ level. Lower-right panel: stereo figure of residue specific interactions between the VirB10_{NTD_150-161} helix and two adjacent VirB9_NTDs. The side chains of interfacing residues are in stick representation with nitrogen and oxygen atoms colored in blue and red, respectively. Carbon atoms are in the color of the ribbon they emanate from. Two shades of green were used to distinguish residues from two different VirB9 chains.

Figure 5. Effect of specific mutations in the core complex on T4S system-mediated cell lysis of neighboring E. coli cells and VirB10 localization.

a, Left panel: Bacterial killing assay measuring the ability of X. citri to use the T4S system to lyse neighboring E. coli cells. The results obtained for wild-type X. citri cells (dashed grey line) are compared to the assay performed using the X. citri virB10-msfGFP strain (solid black line). The dashed black horizontal line corresponds to the no lysis baseline after subtracting the E. coli only background signal from the data. Data are mean ± s.d. (n=9 for X. citri virB10-msfGFP and n=27 for wild-type X. citri). Central panel: Representative image of the X. citri virB10-msfGFP strain as obtained by epifluorescence microscopy displaying discrete msfGFP foci that indicate the presence of T4S systems. Image shows msfGFP intensity levels of a 0.5-µm region containing the focal plane of the cells. The inset shows the superposition of the locations of fluorescent VirB10-msfGFP foci obtained from 100 individual X. citri virB10-msfGFP cells. Right panel: An enhanced image obtained by deconvolution of the obtained Z-slices (not used for quantification) more clearly showing discrete foci (see Methods). Scale bar: 5 μm. The X. citri virB10-msfGFP strain was imaged and analyzed at least 6 times independently with similar results. b, Representative epifluorescence microscopy images (as described in a) for a selected series of X. citri virB10-msfGFP mutant strains in VirB7, VirB9 or VirB10. Note that ΔVirB7 and VirB10_C206S cells are mostly devoid of fluorescence. Other strains, such as VirB7_W34A, present more diffuse fluorescence and lack clear foci. Also note that the few foci shown in the cell contour insets of mutants severely deficient in killing (see below) are due to occasional background detection. Scale bar: 5 μm. All X. citri virB10-msfGFP mutant strains were imaged and analyzed together on two separate occasions independently with similar results. c, Bacterial killing assays of the X. citri virB10-msfGFP strains shown in b. Each X. citri virB10-msfGFP mutant was compared to the X. citri virB10-msfGFP strain (solid black line). Data are mean ± s.d. (n=9 for X. citri virB10-msfGFP and n=4 for each mutant).

Figure 6. Killing efficiency is correlated with T4S system assembly in X. citri.

a, Top panel: The relative number of VirB10-msfGFP foci per cell is plotted for each strain. The distribution of cells according to the number of foci per cell is represented by the randomly placed shaded circles in each bin. Tukey box and whisker plots depict the data: black central line (median), box (first and third quartiles), whiskers (data within 1.5 IQR), green triangles (mean) (see Supplementary Table 4). The sample size (n) of cells of each strain analyzed by fluorescence microscopy (from two independent experiments) is listed at the top. Bottom panel: The mean (± s.d.) relative capacity of each mutant to lyse E. coli cells in a X. citri/E. coli co-culture is shown (as in Figure 5 and Supplementary Table 4). Red dots represent mutants produced in the VirB10-msfGFP background and black dots represent mutants produced in the non-GFP background. In competitions using GFP background strains the n=4 (except for VirB9_A142-S147 (n=3), VirB10_T325-T335 (n=3), ΔVirB7 (n=6), and VirB10-msfGFP (n=9). In competitions using non-GFP background strains the n=3. b, Western blot assays using polyclonal antibodies (Ab) against specific T4S system components or against msfGFP in different X. citri virB10-msfGFP mutant strains (see Methods). The first lane contains total protein extract from E. coli BL21(DE3) expressing the X. citri core complex with normal length VirB10 as does that for the VirB9_V29D mutant (red asterisks), which was obtained only in non-GFP genomic background. Experiments were repeated 3 times with similar results. † indicates mutations in the VirB9 Linker and ‡ indicates mutations in the VirB10 linker for VirB10_ΔQ175-D182 or the VirB10 antenna for VirB10_ΔT325-T335. Colored diamonds denote the values of the relative killing efficiency according to the code: Less than 0.3 (red), 0.3 to 0.7 (yellow), greater than 0.7 (green) (see a and Supplementary Table 4). WT=wild-type. Theoretical molecular weight (in kDa) for each mature protein is shown at right. c, Western blot detection of VirB10 in mutant strains produced in the non-GFP genomic background. Annotations are the same as in b. Experiments were repeated twice with similar results. Full western blots are presented in Supplementary Figure 9b-g.