PilZ interacts with the N-terminal domain of PilB

We have previously shown that PilZ interacts with the hexameric ATPase PilB, required for Type IV pilus biogenesis [23]. Fig 1A shows that the 578 residue PilB protein from X. citri can be divided into 3 domains: an N-terminal domain made up of sub-domains ND0 and ND1, a central ND2 domain and a C-terminal ATPase domain [36]. In order to determine which PilB domains are involved in the interaction with PilZ, we produced three preys consisting of fragments spanning sub domains ND0-ND1 (residues 1–190; PilB_1-190), domain ND2 (residues 159–306, PilB_159-306) and domains ND2-ATPase (residues 159–578, PilB_159-578) for use in yeast two-hybrid interaction assays with a PilZ bait. Fig 1B and 1C shows that an interaction was only detected using the PilB_1-190 prey. This result allows us to conclude that the PilB-PilZ interaction is mediated via the former´s N-terminal region.

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Fig 1. PilZ interacts with the N-terminal ND0/ND1 domain of PilB.

A) Schematic model of X. citri PilB domains. Fragments used as preys in the two-hybrid assays are indicated with coloured lines: PilB_1-190 (blue), PilB_159-306 (red), PilB_159-578 (green). B) Two hybrid assay showing yeasts cells grown on non-selective (SC-WL) and selective (SC-WHLA) media. Three different fragments of PilB were used as shown in A, only cells containing the PilZ-PilB_1-190 bait-prey combinations are able to grow on SC-WHLA media. C) Two-hybrid assay showing β-galactosidase activity of yeast cells containing only the PilB preys (left) and the PilZ-PilB bait-prey combinations. Only cells containing the PilZ-PilB_1-190 bait-prey combination exhibit significant β-galactosidase activity. D) Co-expressions and co-purification of PilB_1-190-PilZ- and PilB_12-163-PilZ complexes from E. coli cells. Complexes were first purified on a Ni²⁺-NTA column due to an N-terminal His-tag on PilB, the His tag removed by proteolytic cleavage, and the complex was further purified by passage through a Superdex 200 10/300 SEC column. E) ¹H-¹⁵N HSQC spectrum of ¹⁵N-labeled PilB_12-163 (lacking the 6xHis-tag) acquired at 25°C (red) shows that it has a partially disordered structure and that addition of unlabeled PilZ leads to folding of PilB_12-163 (blue). F) The fluorescence of PilZ_{W69_5OHW} was monitored during its titration with His-tagged PilB_1-190 (black boxes) or FimX_EAL (white boxes). 1 μM of PilZ_{W69_5OHW} was used. Each point is the average of at least two measurements. The fits (continuous lines) and the dissociation constants were calculated as previously described [82]. G) Ribbon representation of the PilB_12-163-PilZ complex with PilB colored green and PilZ colored yellow. The ND0 and ND1 sub-domains of PilB are indicated. H) Surface representation of the complex. I) Residues at the interaction interface between PilB (left) and PilZ (right). The PilZ MV motif makes important and extensive contacts with the ND1 sub-domain of PilB. MV motif residues (red) are indicated. J) Residue Met117 of PilZ fits into a hydrophobic pocket lined by conserved hydrophobic residues of PilZ and PilB.

https://doi.org/10.1371/journal.ppat.1009808.g001

Based on these results, we expressed and purified PilB_1-190 and a smaller PilB N-terminal fragment corresponding to residues 12–163 (PilB_12-163) that lacks regions predicted to be unstructured at the N- and C-terminal ends of PilB_1-190. Fig 1D shows that PilB_1-190-PilZ and PilB_12-163-PilZ complexes can be co-purified after co-expression in E. coli cells (in these experiments the PilB fragments were expressed with a cleavable N-terminal 6xHis tag). ¹⁵N-labelled PilB_12-163 has an ¹H-¹⁵N HSQC NMR spectrum characteristic of a partially disordered polypeptide. However, in the presence of unlabelled PilZ, the ¹H chemical shift dispersion increases which is indicative of a folded protein (Fig 1E). S1 Fig shows the results of typical SEC-MALS (size-exclusion chromatography coupled with multi-angle light scattering) experiments that show that PilB_1-190 and PilB_12-163 are predominantly monomeric on their own and each can form 1:1 binary complexes with PilZ.

In order to compare the affinities of PilZ for PilB and FimX, we expressed PilZ in a tryptophan auxotrophic strain of E. coli grown in minimal media containing 5-hydroxytryptophan (5OHW). The recombinant PilZ protein, PilZ_{W69_5OHW}, now contains this modified amino acid at position 69, normally occupied by tryptophan. The titration of PilZ_{W69_5OHW} by PilB_1-190 was accompanied by a 2.5-fold increase in 5OHW fluorescence and the data indicate that the dissociation constant is in the nanomolar or subnanomolar range (Fig 1F). The calculated K_D of 4.1 ± 0.9 nM should be taken as an upper limit since the PilZ protein concentration used in the experiment (1 μM) is between two and three orders of magnitude greater than the calculated dissociation constant. PilZ_{W69_5OHW} was also titrated by a fragment corresponding to the C-terminal EAL domain of FimX (FimX_EAL) [24], in which case a more moderate 25% increase in fluorescence was observed and the dissociation constant was calculated to be 0.2 μM ± 0.1 μM (Fig 1F). These results indicate that the PilZ interaction with PilB_1-190 is at least one or two orders of magnitude stronger than its interaction with FimX_EAL. We note that these experiments were performed in the absence of added c-di-GMP and that the W69_5OHW substitution may be affecting PilZ´s interactions with PilB and FimX to different degrees (see below).

The structure and interface of the PilB_12-163-PilZ complex

Native and selenomethionine PilB_12-163-PilZ complexes were crystallized as described in Materials and Methods, in space group P2₁. Two native crystal datasets were obtained, one with the wild type sequences and another with a spontaneous P70S mutation in the PilB_12-163 subunit. Data collection statistics for all three crystals are presented in S1 Table. Initial phases were estimated by single wavelength anomalous dispersion. The asymmetric unit contains two copies of the PilB_12-163-PilZ complex (chains AC and chains BD) (S2A Fig) and the overall structure of the heterodimeric complex is shown in Fig 1G and 1H. The PilB_12-163-PilZ interface buries 1026 Ų and 1018 Ų of surface area for the AC and BD PilB_12-163-PilZ dimers, respectively, as calculated by PISA [37]. A list of residues found at the interface is presented in S2 Table. Structural alignment of the two PilB_12-163 fragments (chains A and B) and the two PilZ molecules (chains C and D) show that they are very similar with a root mean square deviation (RMSD) of 0.6 Å and 0.4 Å for C^α atoms, respectively. No electron density was observed for the last 6 residues of PilB_12-163 and the first 8 residues of PilZ.

PilB_12-163 is made up of two sub-domains, a four helix ND0 (α1-α4) and an α/β ND1 (a β1-β3 antiparallel β-sheet surrounded by three helices (α5-α7) and a string of three consecutive short 3₁₀ helices (η1-η3)) (Figs 1G and S2B). The ND0 and ND1 sub-domains are topologically similar to the MshEN_N and MshEN_C domains, respectively, of the V. cholerae T4P ATPase MshE. It is noteworthy that X. citri PilB sub-domain ND0 lacks the specific amino acid sequence motifs required for c-di-GMP binding found in the MshEN_N domain.

Bioinformatics analysis indicates highly conserved PilB residues at the PilB-PilZ interface. S3 Table lists 51 bacterial species from 51 different genera whose genomes, all annotated in the KEGG database [38], code for all three PilB, PilZ and FimX homologs as well as a homolog of the conserved T4P component PilM. All except one of these genera are from the class Gammaproteobacteria but are distributed among different orders and families. The coincidence of genes coding for close PilB, PilZ and FimX homologs in different genomes could reflect a shared and conserved mechanism of T4P regulation in these species. We note that all of the PilB sequences in this list present both ND0 and ND1 sub-domains and none of the PilB sequences carry residues implicated in the binding of c-di-GMP by MshE and its closest homologs. Fig 2A presents the conservation profile of the N-terminal domains of these PilB proteins using the numbered positions of X. citri PilB as a reference. This conservation profile is mapped onto the PilB_12-163 structure in Fig 2B–2D. Fig 2B presents the PilB_12-163 surface involved in interactions with PilZ (predominantly through the former´s ND1 sub-domain; see also Fig 1G, 1H and S2 Table). Fig 2C and 2D presents the exposed PilB_12-163 surfaces after rotations of 120^o and 240^o about the vertical axis, respectively. Interestingly, in addition to the conservation of residues interacting with PilZ, the PilB_12-163 surface viewed by the 120^o rotation is also very well-conserved while the surface viewed by the 240^o rotation is relatively poorly conserved. Below, we will show that this second conserved PilB surface is most likely involved in interactions with PilM (Fig 2E).

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Fig 2. PilB sequence conservation and interactions.

A) Sequence logo generated from an alignment of 50 non-redundant PilB N-terminal sequences that contain ND0 and ND1 sub-domains. The X. citri PilB N-terminal (residues 1–190) sequence is shown below the alignment (highlighted in green). The secondary structure elements observed in the crystal structure of X. citri PilB (residues 12–163) are indicated above the alignment. B-D) Surface residues of PilB_12-163 colored according to degree of conservation. Panel B shows the PilB_12-163 surface oriented towards PilZ in the PilB_12-163-PilZ complex. * Indicates residues involved in direct contacts with PilZ. Panels C and D show PilB_12-163 after 120^o and 240^o rotations with respect to A. The surface in C presents residues proposed to be involved in the interaction with PilM. E) Possible mode of interaction between PilB_, PilZ and PilM. Shown is the superposition of the X. citri PilB_12-163-PilZ complex with homologous EpsE_ND1-EpsL complex (PDB ID: 2BH1) using the ND1 sub-domains as reference (see S12 and S13 Figs). PilZ and the EpsL cytoplasmic domain (PilM homolog) are shown in ribbon representation colored in yellow and blue, respectively. F) Proposed model for the PilB-PilZ-FimX ternary complex based on the superposition of the PilB_12-163-PilZ complex and the PilZ-FimX_EAL complex (interface 2). PilZ is the reference in the superposition. G) Above: SEC analysis of the mixture of full-length PilB-PilZ complex and full-length FimX shows that these proteins form a ternary complex (black continuous line). Addition of ATPγS and c-di-GMP results in a shift in the elution profile of the ternary complex (red continuous line). Also shown is the elution profile of the PilB_K343A/E407A-PilZ and FimX mixture in absence (dashed black line) and presence (dashed red line) of ATPγS and cdi-GMP. In all cases, a 1:1 molar ratio of PilB-PilZ complex to FimX was used. The elution profiles of full-length FimX (blue continuous line) and the PilB-PilZ complex (green continuous line) are also shown. To facilitate comparison, peak heights were normalized. Below: SDS-PAGE of the fractions of the major peak observed for the PilB-PilZ-FimX ternary complex in presence of ATPγS and cdi-GMP. H) ATPase activity of the PilB-PilZ complex in the absence and presence of full-length FimX. ATPase activity was determined by measuring the production of inorganic phosphate (Pi) at 30°C using 1 μM of PilB-PilZ complex, 1 mM de ATP in the absence and presence of 1 μM FimX. Where indicated, c-di-GMP was added to a final concentration of 20 μM. When wild-type PilB was substituted with the PilB_K343A/E407A mutant, no significant ATPase activity was observed. Each experiment was performed at least three times. Error bars: standard deviation.

https://doi.org/10.1371/journal.ppat.1009808.g002

The superposition of PilZ of the PilB_12-163-PilZ complex with the previously described crystallographic structures of X. citri PilZ [23] and the PilZ-FimX_EAL complex [24] shows that they are very similar, with a RMSD of 0.7 Å and 0.5 Å for C^α atoms, respectively (S2D Fig). The most significant difference between these three PilZ structures is that the electron density for the last 11 PilZ residues (residues 107–117) is well-defined in the PilB_12-163-PilZ complex (Figs 1I and S2E and S2F), while no density is observed for these residues for PilZ alone [23] and in the FimX_EAL-PilZ complex [24] (S2D Fig). These C-terminal residues (called motif V, or MV) are highly conserved in X. citri PilZ orthologs, including PilZ_PA2960 from P. aeruginosa, and are required for the interaction with PilB but not with FimX_EAL [23]. Specifically, in the PilB_12-163-PilZ complex, the carboxylate group of the C-terminal Met117 residue of PilZ makes a salt bridge with highly conserved Arg103 of PilB while the Met117 side chain fits into a hydrophobic pocket made up of PilB residues Leu72, Phe77, Leu100, Phe101, Phe108 and Ile134 and conserved PilZ residues Phe28 and Trp69 (Fig 1I and 1J and S2 Table). In addition to motif V, many other PilZ residues conserved in the PA2960/XAC1133 orthologous group [23] also participate in interactions with PilB (S2 Table).

Mutations at the PilB-PilZ interface destabilize the complex

We produced mutants in specific residues at the PilB-PilZ interface and tested the stability of these complexes using size-exclusion chromatography (SEC). S3A–S3F Fig shows the results using PilZ mutants in complex with PilB_1-190 (this larger PilB fragment was used due to its greater solubility in the absence of PilZ). Mutations in conserved PilZ residues Y22 and F28 did not affect binding to PilB_1-190 (S3A and S3B Fig). On the other hand, mutating PilZ residue W69 to alanine severely reduced the stability of the complex (S3C Fig). The PilZ_Δ107–117 mutant also failed to make a complex with PilB_1-190 (S3E Fig), consistent with previous observations [23]. Since the C-terminal residue M117 makes a number of contacts in the interface (Fig 1J), we produced two more PilZ mutants, one in which M117 was deleted (eliminating both carboxy-terminal and side chain interactions) and another in which it was substituted for a glycine residue (eliminating side-chain interactions). In both cases, interaction with PilB_1-190 was abolished (S3D and S3F Fig). We also mutated PilB residues found at the PilB-PilZ interface: F77A, F101A, F108A, R103A and E132A and the double mutant F101A/F108A. S3G–S3K Fig shows that all of the single mutants in PilB_1-190 retain the ability to interact with PilZ. However, the F101A/F108A double mutant no longer interacts (S3L Fig). Together these mutation studies point to the importance of hydrophobic interactions in the stabilization of the PilB-PilZ interface.

Determination of the relevant PilZ-FimX interface in the PilB-PilZ-FimX complex: PilZ interacts with FimX through a second interface distinct from that between PilZ and PilB

We also crystallized the complex between PilZ_Δ107–117 and a larger FimX fragment encompassing its GGDEF and EAL domains (FimX_GGDEF-EAL, residues 255–689) in the presence of c-di-GMP (S4 Fig). The PilZ_Δ107-117-FimX_GGDEF-EAL complex crystallized in space group P4₂2₁2 with one copy of each protein in the asymmetric unit (see S1 Table for data collection statistics). Very clear electron density for PilZ_Δ107–117 and the FimX EAL domain (beginning at residue 436) with bound c-di-GMP in the syn/anti conformation were observed (S4C and S4D Fig). However, no density for the FimX GGDEF domain could be detected (S4D and S4E Fig), a phenomenon similar to that observed for the crystal structure of the FimX_GGDEF-EAL fragment from P. aeruginosa [29] that is only 30% identical to X. citri FimX_GGDEF-EAL.

The PilZ_Δ107-117-FimX_GGDEF-EAL crystal presents the same two principal modes of crystal lattice contacts between PilZ and the FimX EAL domain observed in the previously published PilZ-FimX_EAL crystal structures from X. citri and X. campestris (Fig 3A–3C), in spite of the different fragments used and different space groups [24,30]. We name these two modes of contact interfaces 1 and 2. The most significant difference among the three structures has to do with the orientation of the first helix in FimX_EAL domains: in the X. citri PilZ-FimX_EAL and PilZ_Δ107-117-FimX_GGDEF-EAL complexes (residues 429–454 and 436–454, respectively), the helix is oriented so as to contribute significantly to the interaction with PilZ in interface 1 (362 or 308 Ų and 234 Ų of buried surface, respectively) while this helix is rotated approximately 140^o, pointing away from PilZ in the X. campestris PilZ-FimX_EAL structure [30] (Fig 3D and 3E). If the contribution of this helix is ignored, then the two interfaces bury similar amounts of surface area (Fig 3E). There are significant differences in the nature of the interfaces, however. While interface 1 involves a β-sheet extension and tenuous contacts between PilZ and the c-di-GMP ligand bound to FimX [24,30], interface 2 is dominated by hydrophobic interactions.

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Fig 3. Alternative modes of contact between PilZ and the FimX EAL domain observed in different crystal structures.

A-C) Left: The principal crystal contacts observed between PilZ and FimX subunits in the A) PilZ-FimX_EAL-c-di-GMP complex from X. citri (PDB: 4FOU; FimX_EAL colored in orange and PilZ colored in blue), B) PilZ-FimX_EAL-c-di-GMP complex from X. campestris pv. campestris (PDB: 4F48; FimX_EAL colored in green and PilZ colored in yellow) and C) PilZ_Δ107-117-FimX_GGDEF-EAL-c-di-GMP complex from X. citri (this study; FimX_EAL colored in magenta and PilZ colored in cyan). The dashed black circles and dashed red boxes delimit the two principal crystal contacts (interface 1 and 2, respectively). Right: Detailed view of interface 2 in the three structures. D) Superposition of one FimX_EAL domain with the two contacting PilZ subunits via interfaces 1 and 2 observed in the three structures shown in A-C with the same coloring scheme. Note the different orientations of the first alpha helix in the X. citri and X. campestris pv. campestris FimX EAL domains. This explains the reduced contact surface at interface 1 in the latter structure (see part E). E) Buried surface areas at the two interfaces in the three structures as calculated by the PISA server [37]. F) Size exclusion chromatography of PilZ-FimX_GGDEF-EAL binary complexes containing wild-type PilZ (PilZ_Wt) and its mutants (PilZ_F49E/L51E, PilZ_I10E and PilZ_D46A/E47A). In each chromatogram, the elution profile of the FimX_GGDEF-EAL−PilZ mixture (1:1.5 molar ratio) was performed in the absence (black line) and in the presence (red line) of a 2-fold excess of c-di-GMP to FimX_GGDEF-EAL. In these experiments, FimX_GGDEF-EAL has an N-terminal 6xHis-tag. Each experiment was performed at least three times and representative results are shown. These chromatograms are reproduced in S5A Fig with the respective SDS-PAGE analysis of the relevant fractions.

https://doi.org/10.1371/journal.ppat.1009808.g003

S5 Fig shows that the PilZ residues involved in contacts with FimX_EAL via interface 1 overlap significantly with PilZ residues involved in its interactions with PilB. Since we have previously reported that a ternary PilB-PilZ-FimX_GGDEF-EAL complex can be detected in far-western overlay assays, and that mutating conserved PilZ residue Trp69 to alanine (located at interface 1) reduces interactions with PilB but does not abolish PilZ-FimX_EAL interactions [23,24] we made a set of PilZ mutants to test the relevance of interface 2 for the stability of the PilZ-FimX interaction. Figs 3F and S6 show that mutations in PilZ residues Ile10, Phe49 and Leu51 (mutants I10E and F49E/L51E) significantly reduce the stability of the PilZ-FimX_GGDEF-EAL binary complex while the PilZ interaction with PilB_1-190 is maintained. These mutants have thermal stabilities (66 ^oC and 61 ^oC, respectively) very similar to that of wild-type PilZ (61 ^oC) (S7 Fig). On the other hand, simultaneously mutating non-interfacing PilZ residues D46 and E47 to alanine does not interfere with its interactions with FimX_GGDEF-EAL or with PilB_1-190 (Figs 3F and S6), even though it presents a lower Tm (48.8 ^oC) than wild-type PilZ (S7 Fig). We note that the binary complexes employing wild-type and D46A/E47A PilZ seem to be stabilized by the addition of c-di-GMP (Figs 3F and S5). Together, these observations raise the possibility that interface 2 between PilZ and FimX EAL domains may be physiologically relevant. Importantly, this second mode of contact would allow the simultaneous interaction of PilZ with both FimX and PilB as shown in Fig 2F. This hypothesis was tested in the experiments described below.

PilZ bridges PilB and FimX to form a ternary complex

When employing PilB_12-163 or PilB_1-190, ternary complexes can be observed in SEC experiments using PilZ and FimX_EAL or FimX_GGDEF-EAL fragments, but not when using full-length FimX or FimX_{PAS-GGDEF-EAL} (a FimX fragment (residues 153–689) lacking the N-terminal REC domain) (S8 Fig). We therefore determined if mutations in the PilZ-FimX and PilB-PilZ interfaces affect the stability of the PilB_1-190-PilZ-FimX_GGDEF-EAL complex. Indeed, mutations in PilZ residues participating in PilZ-FimX interface 2 (I10E, F49A/L51A, F49E/L51E) abolish ternary complex formation (Fig 4A). Furthermore, mutations in these PilZ residues (I10E, F49E, F49E/L51E, and F49A/L51A) compromised X. citri twitching motility and the infection of these cells by the bacteriophage ΦXacm4-11 (Fig 4B). On the other hand, simultaneously mutating PilZ residues Asp46 and Glu47 that are next to both PilZ-FimX_EAL interfaces but not directly participating in either one of them, did not compromise the stability of the PilB_1-190-PilZ-FimX_GGDEF-EAL complex (Fig 4A). Furthermore, mutants that destabilized the PilB_1-190-PilZ binary complex (W69A, M117G, ΔM117 and Δ107–117 in PilZ and the F101A/F108A mutant in PilB) also destabilize the ternary PilB_1-190-PilZ-FimX_GGDEF-EAL complex (Fig 4A). Finally, the W69A, ΔM117 and Δ107–117 mutations in PilZ that destabilize its interactions with PilB_1-190 and mutations in PilZ residues involved in interactions with FimX (I10E, F49E, F49E/L51E, and F49A/L49A) severely compromised X. citri twitching motility and the infection of these cells by the bacteriophage ΦXacm4-11 (Fig 4B and reference [4]). The above results clearly show that PilB, PilZ and FimX can form ternary complexes that involve the simultaneous interaction of PilZ with the N-terminal domain of PilB and the C-terminal EAL domain of FimX. A model for the PilB_12-163-PilZ-FimX_EAL complex, arrived at by using PilZ as a reference to superpose the PilB_12-163-PilZ structure and the PilZ-FimX_EAL structure based on interface 2, is shown in Fig 2F.

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Fig 4. Residues important for PilB_1-190-PilZ-FimX_GGDEF-EAL ternary complex stability and T4P function.

A) Size exclusion chromatography (Superdex 200 resin, 10/300 column) analysis of PilB_1-190-PilZ-FimX_GGDEF-EAL ternary complexes containing PilZ or PilB mutants. A set of mutants: PilZ_F28A, PilZ_D46A/E47A, PilZ_I10E, PilZ_W69A, PilZ_F49A/L51A, PilZ_F49E/L51E, PilZ_M117G, PilZ_ΔM117, PilZ_Δ107–117 and PilB_{1-190_F101A/F108A} (indicated in each chromatogram panel) were used to test their effect on ternary complex stability. In all the cases, a 1:1:1 PilB_1-190:PilZ:FimX_GGDEF-EAL molar ratio was used and the elution profile of the mixture is shown as a black line. All the mutants, except PilZ_F28A and PilZ_D46A/E47A, affect the stability of the FimX_GGDEF-EAL-PilZ-PilB_1-190 ternary complex. In all of the panels, the elution volume for the ternary complex is delimited by the vertical red broken lines. In the first panel, the elution profiles for wild type complex (black line) is shown together with the profiles for the three individual components FimX_GGDEF-EAL (red line), PilB_1-190 (blue line) and PilZ (green line). Each experiment was performed at least three times and representative results are shown. B) Top row: Fluorescence microscopy images of the edges of the twitching zones at the interstitial surface between the agar medium and the glass base of the microscopy chamber. For visualization, X. citri strains were transformed with the pBBR2-GFP plasmid. Wild-type cells, that are able to twitch, can separate from the main body of the colony and migrate on their own or in small groups, producing a rough, less organized boundary between the dense interior of the colony and the surrounding medium. Cells with mutations that compromise T4P function are not able to separate from the main body of the colony and so the colony border is much smoother and well-defined. Bottom row: Phage ΦXacm4-11 infection assays. Dark plaques are indicative of phage-induced bacterial lysis in a confluent culture background. For both twitching motility and bacteriophage infection assays: X. citri wild type (Xac_Wt), Xac_ΔpilZ_XAC1133 (ΔpilZ) and Xac_ΔpilZ_XAC1133 complemented with a plasmid (pURF047) directing the expression of the wild-type PilZ protein (ΔpilZc) or one of the following PilZ mutants: PilZ_I10E, PilZ_F49E, PilZ_F49E/L51E, PilZ_F49A/L51A,and PilZ_ΔM117. S14 Fig shows that PilZ_I10E, PilZ_F49E, PilZ_F49E/L51E, PilZ_F49A/L51A, PilZ_ΔM117 are all detected with anti-PilZ antibodies in these strains.

https://doi.org/10.1371/journal.ppat.1009808.g004

Full-length PilB-PilZ-FimX complexes

Recombinant full-length X. citri PilB containing an N-terminal polyhistidine tag (theoretical MW of 64.5 kDa) is insoluble when expressed on its own in E. coli (Fig 5A) but is soluble when co-expressed with PilZ (theoretical MW of 12.4 kDa) (Fig 5B). This full-length PilB-PilZ complex is stable and can be purified by affinity and size-exclusion chromatography (Figs 2G and 5C). The complex elutes as a broad peak with molecular weight, estimated by SEC-MALS analysis, that varies between 100 kDa and 140 kDa (Fig 5D). However, in the presence of ATPγS, the elution volume of this peak shifts slightly with an estimated molecular weight of 120–160 kDa (Fig 5D). These molecular weights are suggestive of a dynamic equilibrium between PilB-PilZ heterodimers (theoretical MW of 77.4 kDa) and (PilB-PilZ)₂ heterotetramers (theoretical MW of 154.8 kDa). Furthermore, the addition of ATPγS results in the appearance of a minor higher molecular weight peak with a large molecular weight distribution (between 350 and 600 kDa), indicative of larger aggregates (Fig 5D). These observations are consistent with previous reports on isolated PilB homologs from other systems that were observed as monomers, dimers and hexamers [12,39–45].

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Fig 5. Expression of a soluble form of full-length PilB requires the co-expression of PilZ.

A-B) SDS-PAGE analysis of E. coli cells expressing full-length PilB (A) and both PilB and PilZ (B). Lane labels: ‘M’: molecular weight markers, with the molecular weight for each band shown on the left; ‘NI’: total cell lysate before addition of IPTG; ‘I’: total cell lysates after induction with 0.2 mM IPTG; ‘S’: soluble fraction after cellular lysis; ‘P’: insoluble fraction after cellular lysis. C) Purification of the PilB-PilZ complex by affinity chromatography (Ni²⁺ HiTrap column; above) and size-exclusion chromatography (Superose 6 resin, 10/300 column; below). In these experiments, PilB contains an N-terminal polyhistidine tag. The elution profiles for each chromatography step are shown along with SDS-PAGE analysis of the relevant fraction. D) SEC-MALS analysis for the PilB-PilZ complex in the absence (continuous red line) and presence (continuous black line) of ATPγS (non-hydrolysable ATP analog). The red and black open circles show the calculated molecular mass distributions. E) SEC-MALS analysis for full-length FimX in the absence (continuous red line) and presence (continuous black line) of c-di-GMP. The red and black open circles show the calculated molecular mass distributions. In D and E, a silica-based WTC-050S5 (7.8/300) column (Wyatt Technology) was used. F) Left: SEC analysis of the PilB_K343A/E407A-PilZ complex in the absence (continuous red line) and presence (continuous black line) of ATPγS (non-hydrolysable ATP analog). Right: SDS-PAGE analysis of the relevant fractions containing the PilB_K343A/E407A-PilZ complex. In this experiment, a Superose 6 column (10/300, GE) was used.

https://doi.org/10.1371/journal.ppat.1009808.g005

Full-length X. citri FimX on its own has a molecular weight estimated by SEC-MALS of 145 and 147 kDa, in the absence and presence of its c-di-GMP ligand, respectively (Fig 5E). These molecular weights are consistent with dimer formation (theoretical MW of 152 kDa) as previously reported for the homologous protein in P. aeruginosa [29]. In contrast to results using PilB N-terminal fragments, when using full-length PilB, we are able to clearly observe ternary PilB-PilZ-FimX complexes containing full-length FimX (Fig 2G). These results indicate that the complete PilB protein is required for the stable incorporation of full-length FimX into the complex. SEC-MALS analysis of the PilB-PilZ-FimX complex again points to a heterogeneous mixture with molecular weight varying between 500 and 650 kDa (S9A Fig). The addition of ATPγS and c-di-GMP seems to stabilize this higher molecular weight complex (Figs 2G and S9A). We note that a PilB-PilZ-FimX stoichiometry of 6:6:2 corresponds to a theoretical molecular weight of approximately 620 kDa (see Discussion below).

FimX enhances PilB ATPase activity

PilB proteins from other bacterial species have been shown to exhibit ATPase activity in vitro [12,40,46]. Fig 2H shows that the X. citri PilB-PilZ complex can hydrolyze ATP in vitro with an intrinsic activity of approximately 8.5 nmol ATP hydrolyzed/min per mg PilB-PilZ complex, under the conditions tested. Upon addition of full-length FimX, an approximately 3-fold increase in ATPase activity was observed (22.5 nmol/min per mg PilB-PilZ complex; Fig 2H). As expected, no significant activity was observed for the PilB-PilZ and PilB-PilZ-FimX complexes containing PilB_K343A/E407A in which two active site residues, located within the ATPase domain, were mutated to alanine (Fig 2H). Fig 5F shows that this PilB mutant retains the ability to form a binary complex with PilZ but no mobility shift or evidence of higher order oligomers is observed upon addition of ATPγS. Finally, Figs 2G and S9B show that ternary complexes containing the PilB_K343A/E407A mutant, PilZ and FimX do form; but the higher-order oligomers seem to be less stable. These results show that i) FimX stimulates PilB ATPase activity, ii) the incorporation of FimX into the complex is not absolutely dependent on PilB ATPase activity and iii) PilB ATPase activity stabilizes higher order oligomeric forms of the PilB-PilZ binary and PilB-PilZ-FimX ternary complexes.

Unipolar colocalization of PilB, PilZ, FimX and PilQ at the leading edge of twitching X. citri cells

In order to study the localization of the X. citri Type IV pilus components, we produced strains in which the FimX, PilZ, PilB and PilQ genes were replaced with msfGFP-FimX, msfGFP-PilZ, msfGFP-PilB and PilQ-msfGFP fusions, respectively. Figs 6 and S10A show that FimX, PilZ and PilB all are localized predominantly at a single cell pole. The fraction of cells exhibiting polar foci was 54% for FimX, 32% for PilZ but only 6.5% for PilB under the conditions tested (Fig 6D). The outer membrane secretin subunit PilQ is also found mostly at one pole (79%) with a small fraction of cells showing foci at two poles simultaneously (Fig 6C and 6D). In the cases where bipolar PilQ localization was observed, one of the foci was almost always much more intense than the other (see S10A Fig for fluorescence intensity distributions along the major axes of all individual cells analyzed).

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Fig 6. Subcellular localization of FimX, PilZ, PilB and PilQ.

A) Representative images of fluorescent light microscopy of X. citri cells expressing chromosomal FimX, PilZ, PilB and PilQ fused with msfGFP (N-terminal fusion in FimX, PilZ and PilB and C-terminal fusion in PilQ) in wild type and mutants strains with deletion in indicated T4P regulatory components. The X. citri strains expressing the fluorescent fusion proteins were imaged by phase contrast and epifluorescence microscopy and analyzed at least five times independently with similar results. In these experiments, cells were grown on KB-agarose pads (1.5% w, using 0.2% casamino acids as nitrogen source) supplemented with 2 mM CaCl₂. Scale bar, 3 μm. B) Representative time lapse images showing that msfGFP-FimX, msfGFP-PilZ, msfGFP-PilB and PilQ-msfGFP localize to the leading pole in wild type X. citri cells undergoing twitching motility. The time lapse interval (h) is indicated for each frame. Images were taken by using an epi-fluorescence light microscope. Scale bar, 5 μm. See also S5–S8 Movies for time lapse images at shorter intervals. C) Graphical representation of the fluorescence intensity profile over the length of X. citri cells expressing msfGFP-FimX, msfGFP-PilZ, msfGFP-PilB and PilQ-msfGFP. The fluorescence intensity profile was obtained from 100 individual X. citri cells for each case (cell length was normalized). The profiles of all individual cells are shown in S10A Fig. Insets show the foci localizations. A and B show that msfGFP-FimX, msfGFP-PilZ and msfGFP-PilB foci exhibited unipolar localization. PilQ-msfGFP exhibits a mixture of both unipolar and bipolar localization; in the latter case one of the foci was almost always much more intense than the other (see S10A Fig). D) Comparison of the frequency of polar localization of msfGFP-FimX, msfGFP-PilZ, msfGFP-PilB and PilQ-msfGFP foci in wild-type, ΔfimX, ΔpilZ and ΔpilB backgrounds.

https://doi.org/10.1371/journal.ppat.1009808.g006

In order to determine if protein localization is influenced by whether the cells are grown in liquid (no twitching) or semisolid media (twitching) we grew mCherry-FimX and PilQ-msfGFP cells in liquid medium and observed them immediately after transfer to KB-agarose slabs (before they have a chance to begin twitching, time = 0 hours) and 6 hours after transfer during which time they are exhibiting twitching behaviour. S10B Fig shows that no mCherry-FimX foci are observed in cells immediately after transfer from liquid culture and that the foci appear only after growth for some time on semisolid media. In contrast, it was very common to observe PilQ-msfGFP foci at both cell poles (bipolar localization) immediately after growth in liquid medium; but after several hours growth on semi-solid media, most cells exhibited unipolar PilQ-msfGFP foci (S10B Fig). S11 Fig and S1–S4 Movies show that these X. citri strains all exhibit normal twitching motility and can all be infected with bacteriophage ΦXacm4-11, X. citri phenotypes that are dependent on a functional T4P [4].

Time-lapse fluorescence microscopy images of actively twitching X. citri msfGFP-FimX, msfGFP-PilZ, msfGFP-PilB and PilQ-msfGFP strains reveal that the most intense foci are found at the leading poles of the cells at the leading edge of the group of migrating cells (Fig 6B and S5–S8 Movies). These strains were then used to introduce deletions in specific Type IV pilus components. Fig 6A and 6D shows that deletion of pilZ or pilB genes results in a drastic reduction in the number of cells with fluorescent msfGFP-FimX foci. Likewise, deletion in the pilZ or pilB genes result in drastic reductions in the unipolar localization of msfGFP-PilB or msfGFP-PilZ, respectively (Fig 6A and 6D). In contrast, deletion of fimX, pilZ or pilB genes did not affect the polar localization PilQ-msfGFP (Fig 6A and 6D).

To confirm that FimX, PilZ, PilB and PilQ are in fact all localized at the same pole, mCherry-FimX fusions were used to substitute the FimX gene in the msfGFP-PilZ, msfGFP-PilB and PilQ-msfGFP strains to produce cells in which two T4P components could be simultaneously localized by fluorescence microscopy. These three strains are all able to be infected by ΦXacm4-11, indicative of normal T4P function (S11B Fig). Fig 7 shows fluorescence microscopy images of groups of X. citri cells in which most of the cells displaying mCherry-FimX foci also display msfGFP-PilB, msfGFP-PilZ or PilQ-msfGFP foci at the same pole. These results are consistent with studies on the co-localization of FimX, PilB and PilQ homologs in P. aeruginosa [25,26,34,47].

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Fig 7. Subcellular Colocalization of FimX with PilZ, PilB and PilQ.

Representative images of wild type X. citri cells co-expressing chromosomal msfGFP-PilZ and mCherry-FimX (A), msfGFP-PilB and mCherry-FimX (B) and PilQ-msfGFP and mCherry-FimX (C). X. citri expressing the fluorescent fusion proteins were imaged by phase contrast and epifluorescence microscopy at least three times with similar results. Scale bar, 3 μm. To the right, bar graphs present the fraction of cells presenting msfGFP, mCherry and both msfGFP and mCherry polar foci. the total number of cells analyzed (n) is given.

https://doi.org/10.1371/journal.ppat.1009808.g007

Bacterial strains, bacterial growth and cloning

S4 Table shows all strains used in this study. Primer sequences and plasmids are listed in S5 Table. Escherichia coli DH5α was used for DNA cloning. The coding sequence for the different constructions of FimX_XAC2398, PilZ_XAC1133 and PilB_XAC3398 were amplified by PCR from genomic DNA from X. citri pv. citri strain 306 genomic DNA and cloned into the expression vector pET28a or pET3a [66] in the NdeI and BamHI sites. The full-length PilB and PilZ were cloned into the co-expression vector pETDuet. The coding sequencing for PilB fragment (PilB_1-190, PilB_156-307 and PilB_156-578) was amplified by PCR from X. citri genomic DNA (primers listed in S3 Table) and cloned into the two-hybrid bait vector pOBD [67] in the NcoI and XhoI sites. A series of substitutions of amino-acid residues in the PilZ and PilB were carried out by via a single-step PCR protocol using the QuickChange Site-Directed Mutagenesis kit (STRATAGENE) (primers listed in S5 Table). The mutants were used for expression in E. coli or for complementation of X. citri strains. The PilZ wild type and F28A, W69A and Δ107–117 mutants were previously described [23,24]. All clones were confirmed by DNA sequencing. Antibiotics were used at the following final concentrations: kanamycin 50 μg/mL, ampicillin 100 μg/mL. In order to complement the ΔpilZXAC1133 knockouts, fragments coding for wild type PilZ (XAC1133) and its mutants M117G, ΔM117, I10E, F49E, F49E/L51E, F49A/L51A were amplified and cloned into pUFR047, as described previously [23].

Two-hybrid assays

Saccharomyces cerevisiae strain PJ694-a (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal80Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ)[68] was grown in SC medium (0.66% (w/v) nitrogen base without amino acids, 2% (w/v) glucose, 0.008% (w/v) adenine, 0.083% (w/v) amino acids mixture, pH 5.6). When necessary, SC medium was prepared lacking one or more of the components adenine, histidine, tryptophan, and leucine. Solid medium also contained 1.6% (w/v) Bacto-agar and 5 mM 3-aminotriazole. β-galactosidase activity was measured by selecting cells from SC-WL plates to inoculate SC-WL liquid cultures that were grown overnight at 30°C, collected by centrifugation, resuspended in 1 ml of YPD medium (20 g/L Tryptone, 10 g/L yeast extract, 20 g/L glucose, pH 5.8) and grown for another 4 h. Cells were collected, resuspended in the same volume of Z buffer (0.06 M Na₂HPO₄, 0.04 M NaH₂PO₄, 0.01 M KCl, 0.001 M MgSO₄, 0.05 M β-mercaptoethanol, pH 7.0) and the A_600nm of the resuspended cells was measured. After that, 100 μl of chloroform and 50 μl of 0.1% (w/v) SDS were added, vortex mixed and incubated for 5 min at 28°C. The reaction was initiated by adding 0.2 ml of 4 mg/mL o-nitrophenyl-β-d-galactoside (ONPG), vortex mixed, incubated at 28°C and time measured until the reaction was terminated by the addition of 0.5 ml of 1 M Na₂CO₃. The solution was centrifuged for 5 min at 13000 g and the A_420nm and at A_550nm were recorded. The Miller units were calculated as: Miller Unit = 1000 [(A_420nm− 1.75 A_550nm)]/(TVA_600nm) where T is reaction time (in minutes), and V is volume (in ml).

Protein expression and purification

X. citri PilZ (XAC1133), PilZ mutants (PilZI_10E, PilZ_Y22A, PilZ_F28A, PilZ_D46A/E47A, PilZ_F49E/L51E, PilZ_F49A/L51A, PilZ_W69A, PilZ_Δ107–117, PilZ_M117G and PilZ_ΔM117) and the FimX fragments (full-length FimX, FimX_EAL, FimX_GGDEF-EAL) were expressed and purified as described previously [23,24]. Conditions for protein expression (host E. coli strain and temperature) for FimX_GGDEF-EAL-PilZ_Δ107–117 complex, PilB, PilB fragments, their mutants (F77A, F101A, F108A, F101A/F108A, R103A e E132A) and PilB-PilZ complexes are summarized in S6 Table. Proteins were produced by growing E coli cells in 2×TY medium (16 g/L of bacto-tryptone, 10 g/L of yeast extract and 5 g/L of sodium chloride) supplemented with kanamycin (50 mg/mL) and/or ampicillin (100 mg/mL) under agitation of 200 r.p.m. to an OD_600nm of 0,6–0.8, at which point 0.4 mM isopropyl-β-D-1-thiogalactopyranoside was added. After induction, the cells were collected and resuspended in 25 mL of lysis buffer (50 mM Tris–HCl pH 8.0, 200 mM NaCl, 25% sucrose and 20 mM imidazole)/1L of culture and lysed by sonication. The lysate was centrifuged at 37,500 g, 4°C for 45 min. The soluble fraction was applied to Ni-HiTrap column (GE Healthcare) previously equilibrated in buffer A (50 mM Tris–HCl pH 8.0, 200 mM NaCl, 1mM MgCl₂, 20 mM imidazole and 5% glycerol). The resin was washed with 10–20 column volumes of buffer A and proteins were eluted with a gradient containing 20–500 mM imidazole in buffer A over 10 column volumes. The pooled protein fractions were concentrated, using centrifugal filter devices (Millipore), and further purified by SEC using a Superdex 200 26/600 column (GE Healthcare) equilibrated in gel-filtration buffer (20 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM MgCl₂). PilB-PilZ complex was purified using Superose 6 10/300 column equilibrated with 20 mM Tris–HCl pH 8.0, 50 mM NaCl and 5mM MgCl₂. Fractions containing the recombinant proteins were pooled and concentrated to 4–8 g/L using Amicon ultra concentrators (Millipore) with a 10 kDa membrane cutoff. Protein aliquots were stored at −20°C after the addition of 20% glycerol. Selenomethionine-containing protein was produced by growing a 500 ml culture in M9 medium to an OD_600nm of 0.8 at 37°C at which point 100 mg/L, lysine, 100 mg/L phenylalanine, 100 mg/L threonine, 50 mg/L isoleucine, 50 mg/L valine and 60 mg/L selenomethionine were added. After 15 min, 0.4 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added and the cells were grown for 16 h at 18°C. ¹⁵N-labeled PilB_12-163 was produced by growing transformed E. coli BL21(DE3) cells in M9 medium in the presence of 1 g/l of ¹⁵N-NH₄Cl and 4 g/l of unlabeled glucose. PilZ containing 5-hydroxytryptophan at the unique tryptophan position (PilZ_{W69_5OHW}) was expressed [69] and purified [23] as previously described.

Crystallization and structural determination of the PilB_12-163-PilZ complex

The crystallization of unlabeled and selenomethionine-labeled PilB_12-163-PilZ complexes was carried out in sitting-drop plates by mixing equal volumes (1 μL) of 4 mg/ml protein and well solution (0.1 M Tris pH 8.5; 2.0 M ammonium sulfate) and growing at 18°C. For the selenomethionine-labeled crystal, X-ray radiation at 0.97889 Å was used (corresponding to the peak of the fluorescence spectrum). The native crystal dataset was collected using 1.4587 Å radiation for the crystal containing the PilB P70S mutation and 1.5418 Å radiation for the crystal containing the wild-type PilB sequence (S1 Table). The data were indexed and integrated using the XDS program[70]. Selenium sites were found using AutoSol [71]. The resulting electron density map was used to construct a preliminary polyalanine model with ARP/wARP [72]. Interpretation of the electron density maps and construction of the missing residues was performed with COOT [73]. Structural refinement of the model was performed using REFMAC [74], Phenix [75] and COOT. Water molecules were added automatically using REFMAC and manually using COOT. Details of the refinement data statistics are shown in S1 Table. Structural alignments and figures were produced using Chimera [76]. The atomic models and experimentally determined structure factors have been deposited in the protein data bank with pdb codes 7LKM, 7LKN and 7LKO.

Crystallization and structural determination of the PilZ_Δ101-117-FimX_GGDEF-EAL complex

The crystallization of native PilZ_Δ101-117-FimX_GGDEF-EAL complex was performed in sitting-drop plates by mixing equal volumes (1 μL) of 10 mg/ml protein and well solution (0.1 M MES pH 6.5; 1.6 M Mg₂SO₄) and growing at 18°C. The data were indexed and integrated using the HKL2000 software [77]. Phases were calculated by molecular replacement using the model of the X. citri PilZ-FimX_EAL complex (PDB ID 4FOU; [24]). Structural refinement was performed as described above using REFMAC, Phenix and COOT. Details of the refinement data statistics are shown in S1 Table. Structural alignments and figures were produced using Chimera. The atomic model and experimentally determined structure factors have been deposited in the protein data bank with pdb code 7LKQ.

Size-exclusion chromatography (SEC)

A Superdex S200 10/300 (GE Healthcare) column was used to investigate protein–protein interactions between the PilB_12-163 or PilB_1-190, PilZ and the different constructions of FimX. The column was previously equilibrated with 50 mM Tris-HCl pH 8,0, 50 mM NaCl, 5mM MgCl₂. Protein samples, for binary complex (PilB_1-190 and wild type PilZ or their mutants) and ternary complex (PilB_12-163-PilZ and PilB_1-190-PilZ complex with FimX fragments), were mixed at an equimolar ratio (60–100 μM) in a final volume of 100 μL and applied to the SEC column. In SEC experiments studying the binary complex formed between FimX_GGDEF-EAL and wild type or mutant PilZ, a 1:1.5 molar ratio was employed (100 μM FimX_GGDEF-EAL and 150 μM PilZ). Where indicated, a 2-fold excess of c-di-GMP to FimX was added. In SEC experiments studying complexes containing full-length PilB, PilZ and FimX, a Superose S6 10/300 (GE Healthcare) column was used, equilibrated in the same buffer. In this case, protein samples were mixed in equimolar ratio (30–50 μM) in a final volume of 100 μL and applied to the SEC column. Where indicated, two-fold molar excess of ATPγS and/or c-di-GMP was added to the sample.

Multi-angle laser light scattering coupled with size-exclusion chromatography (SEC-MALS)

SEC-MALS analysis was used to determine the molar mass of PilB_1-190, PilB_12-163, PilB_1-190-PilZ complex and PilB_12-163-PilZ complex at two concentrations (3.0 mg/ml to 4.0 mg/ml) in 50 mM Tris–HCl (pH 8.0) and 50 mM NaCl, 1 mM MgCl₂, 1 mM β-mercaptoethanol. Proteins samples (100 μL injection volume) were separated using a Superdex 200 10/300 column coupled to a miniDAWN TREOS multi-angle light scattering system and an Optilab rEX refractive index detector. For FimX, a silica-based column 7.8/300 was used (WTC-050S5, Wyatt Technology). For PilB-PilZ complex and PilB-PilZ-FimX ternary complex, a silica-based column 7.8/300 was used (WTC-015S5, Wyatt Technology). In these cases, the column was equilibrated with 50 mM Tris–HCl (pH 8.0) and 50 mM NaCl, 5mM MgCl₂. The ligands (ATPγS and /or c-di-GMP) were added at molar ratio 1:2 protein to ligand when indicated. Data analysis was performed using the Astra Software package, version 7.1 (Wyatt Technology Corp). Molecular mass was calculated assuming a refractive index increment dn/dc = 0.185 ml/g [78].

NMR spectroscopy

NMR sample of ¹⁵N-labeled PilB_12-163 was prepared in 20 mM Tris-HCl pH 7.0, 50 mM NaCl and 1 mM MgCl₂ at 150 μM concentration. The ¹⁵N-HSQC experiments were acquired in the absence and presence of unlabeled PilZ at molar ratio of 1:1.5, at 298 K on a Bruker Avance III spectrometer operating at 800 MHz (¹H frequency) and equipped with a cryogenic TCI probe. NMR spectra were processed with NMRPipe [79] and analyzed using CCPNMR Analysis 2.4.1 [80].

ATPase assay

The ATPase activity of PilB-PilZ complex was analyzed by malachite green assay as previously described [81]. Briefly, 1 μM of PilB-PilZ complex was used for each reaction in the absence and presence of 1 μM FimX. The assay was carried out in reaction buffer containing 25 mM Tris-HCl pH 7,0, 50 mM NaCl, 2 mM MgCl₂, 2 mM β-mercaptoethanol and 1 mM ATP (and 20 μM c-di-GMP where indicated) and incubated for 12 h at 30°C. An aliquot (50 μL) of each reaction was transferred to a 96-well plate and the reaction was stopped by the addition of 200 μl of the phosphate assay reagent. Phosphate assay reagent was freshly prepared using 3 volumes of 0.045% malachite green hydrochloride (Sigma-Aldrich), 1 volume of ammonium molybdate (4.2% in 4 N HCl) and 1/100 volume of 1% Triton X-100. After 10 min of incubation at room temperature, the optical density was measured at 650 nm using a 96-well microplate reader (Spectramax–Molecular Devices). Reactions lacking PilB-PilZ complex or ATP were included as negative controls. The phosphate released during the reaction was measured using a standard curve of 1 to 150 μM KH₂PO₄.

Fluorescence titration

The formation of the PilB_1-190-PilZ_{W69_5OHW} and FimX_EAL-PilZ_{W69_5OHW} complexes were monitored by changes in the 5-hydroxytryptophan fluorescence emission spectra of PilZ_{W69_5OHW} upon addition of different amounts PilB_1-190 or FimX_EAL. The assay was carried out in buffer containing 50 mM Tris-HCl pH 8,0, 50 mM NaCl, 1 mM MgCl₂, 1 mM β-mercaptoethanol and the initial concentrations of PilZ_{W69_5OHW} was 1 μM. The samples were equilibrated for 2 min before each measurement. Titration experiments were performed using a RF-6000 fluorescence spectrophotometer (SHIMADZU). The excitation wavelength was 310 nm (bandwidth: 5 nm), and the emission spectra were recorded between 325–445 nm (bandwidth: 5 nm). Dissociation constants were calculated assuming a simple 1:1 bonding model, as previously described [82] using the SigmaPlot 11 software (Systat Software Inc.).

Cloning of constructs for genomic deletions and insertions

Primers, plasmid and strains used for cloning and PCR verifications are listed in S4 and S5 Tables. Genomic deletion and insertion in the X. citri (strain 306) genome were introduced by two-step allelic exchange method [83] with small modifications as described [84]. Briefly, for gene deletions, two fragments of approximately 1000 bp corresponding to up- and downstream from the region of interest were amplified by PCR (Phusion polymerase, Thermo Scientific) using primers containing either homology region to the pNPTS138 vector backbone or the up or down-fragment and cloned into the pNPTS138 suicide vector by Gibson assembly (NEB). For the gene insertion of fluorescent reporter, the msfgfp or mcherry genes was amplified from pDHL1029 [85] and pDHL-mCherry [86], respectively, introducing a flexible N- or C-terminal liker (Ser-Gly-Gly-Gly-Gly). Separately, ~1,000 bp fragments of the upstream and downstream regions from the star (N-terminal fusion) or stop (C-terminal fusion) codon were amplified by PCR (Phusion polymerase, Thermo Scientific) from X. citri genomic DNA, using primers containing either homology to the pNPTS138 vector backbone or the msfgfp or mcherry gene (S1 Table). The three fragments were cloned into the pNPTS138 vector using Gibson Assembly (NEB). The resulting plasmid was used to transform the appropriate X. citri strain by electroporation (2.0 kV, 200 Ω, 25 μF, 0.2 cm cuvettes; Bio-Rad). A first recombination event was selected for on LB plates containing 50 μg/ml kanamycin. Transformants were streaked for single colonies on kanamycin plates after which several single colonies of the merodiploids (Kan^R, Suc^S) were streaked on NaCl-free LB-agar supplemented with sucrose (10 gl⁻¹ tryptone, 5 gl⁻¹ yeast extract, 60 gl⁻¹ sucrose and 15 gl⁻¹ agar), selecting for a second recombination event creating either a wild-type (reversion) or mutant allele. After confirmation of the loss of the kanamycin resistance cassette together with sacB, a PCR was performed using primers that hybridize outside of the homology regions to identify the genomic deletions or insertion and confirmed by Sanger sequencing of the PCR product.

Twitching motility assay

Twitching motility was assayed by the stab-inoculation method in KB medium in a microscopy chamber covered with glass slides [4]. Briefly, X. citri strains were grown on LB agar (1.5% wt/vol) supplemented with the appropriate antibiotic at 28°C for 48 hours. Using a sterile toothpick, X. citri cells were collected from an isolated colony and stabbed through KB-agar (1%wt/vol) supplemented with 2 mM CaCl₂ to the plastic surface of microscopy chamber (Nu155411; Lab-Tek, NUNC). Chambers were statically incubated in a humidified chamber at 28°C for 48 hours. Twitching zone was visualized by an inverted fluorescence microscope (Nikon Eclipse Ti-E) with specific excitation and emission filters for GFP. The images obtained were analyzed with Fiji (ImageJ) software [87]. For time-lapse experiments, overnight cultures of X. citri strains were grown in 2xTY medium at 28°C with shaking at 200 rpm. After a first overnight growth period, cells were transferred at a 100-fold dilution into 2xTY media for a second overnight growth to synchronize growth. Cells were then diluted 100-fold in fresh media and 1 μL of cell suspension was spotted on a thin slab of KB-agar (1% w/v) supplemented with 2 mM CaCl₂, after 4- to 6-hour growth, phase contrast images were obtained with a Leica DMi-8 epifluorescent microscope. The KB-agar slabs were constructed as described [88].

Fluorescence microscopy

Overnight cultures of X. citri strains were grown as described above. Cells were then diluted 100-fold in KB fresh media (using 0,2% casamino acids as nitrogen source) and 1 μL of cell suspension was spotted on KB-agarose (1,5% w/v, using 0,2% casamino acids as nitrogen source) supplemented with 2 mM CaCl₂ and incubated at 30°C for 6 hours before imaging. The KB-agar slabs were constructed as described [88]. Phase contrast and msfGFP and/or mCherry emission images were obtained with a Leica DMi-8 epifluorescent microscope. Fluorescence emission of msfGFP and mCherry were captured using 2000 and 3000 ms exposure times at maximum excitation light intensities. The microscope was equipped with a DFC365 FX camera (Leica), a HC PL APO 100x/1.4 Oil ph3 objective (Leica) and excitation-emission band-pass filter cubes for GFP (Ex.: 470/40, DC: 495, Em.: 525/50; Leica) and mCherry (Ex.: 540/80, DC: 585, Em.: 592–668; Leica) foci. To determine the polar localization of foci and quantify the polar fluorescence intensities, the images were analyzed using the MicrobeJ software package [89].

Bacteriophage plaque assay

Overnight cultures X. citri wild-type and mutant strains were grown in 2×TY medium, collected by centrifugation, and resuspended in fresh medium at an OD_600nm of 0.3. X. citri strains were mixed with warm liquefied KB agar (0.7% wt/vol) supplemented with 2 mM CaCl₂ to form the top agar layer poured into petri dishes carrying previously solidified KB agar (1% wt/vol) + 2 mM CaCl₂. Plates were dried for 5 min at room temperature and then spotted with 5 μL of dilutions (from 10⁰ to 10¹⁰) of the bacteriophage ΦXacm4-11 stock solution (10¹² PFU/mL) and incubated at 28°C for 24 h as described [4].

Western blot assay

Western blot assays were performed using total protein extract of X. citri wild type or ΔpilZ strains carrying the pUFR047 vector directing the expression of wld-type or mutant PilZ proteins (I10E, F49E, F49E/L51E, F49A/L51A and ΔM117). X. citri cells were grown in petri dishes on KB-agar 1% supplemented with 2 mM CaCl₂ and 10 μg mL^-1 of gentamicin and incubated at 28°C for 3 days. Bacteria were collected using a plastic spatula and resuspended in 15 mL of 1xPBS and the OD_600nm for all strains was measured and adjusted to 1.5. Bacterial cells were collected by centrifugation and the bacterial pellet washed three times with 10 mL of 1xPBS. After this, the bacterial pellet was resuspended in 400 μL of 1xPBS and 100 μL of denaturing sample buffer (5x) and incubated for 5 min at 95°C. Samples (20 μl) were resolved in 10-well Tricine–SDS–PAGE gels, transferred to nitrocellulose membrane (Bio Rad), and blocked for 12 h using 5% skimmed milk in 1×PBS. Primary antibodies produced in rabbit against PilZ (AbPilZ; 1:5000) [23] and VirB8 (AbVirB8; 1:8000) [90] were used. Secondary goat anti-rabbit IgG-IRDye 800CW (Li-Cor 32211; 1:8000) were used for AbPilZ and visualizing using a ChemiDoc MP Imaging System (Bio Rad), and secondary goat anti-rabbit IgG-AP conjugate (Bio Rad 1706518; 1:8,000) was used for and AbVirB8 with BCIP (VWR 0885) and NBT (Sigma-Aldrich N6876) for detection.

Thermal denaturation of PilZ and mutants

The thermal denaturation of wild-type PilZ and its mutants (I10E, D46A/E47A and F49E/L51E) was accompanied by differential scanning fluorescence (DSF) as previously described [91] using a QuantStudio 3 Real-Time PCR (Thermo Fisher Scientific) instrument. Briefly, 50 μL aliquots of purified protein dissolved in 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 5 mM MgCl₂, 125x concentrated SYPRO Orange dye (Thermo Fisher Scientific) were distributed into optical 96-well plates (Life Technologies). The final protein concentrations were: PilZ_Wt: 240 μM; PilZ_F49E/L51E: 240 μM; PilZ_I10E: 240 μM; and PilZ_D46A/E47A: 60 μM. The plates were sealed with real-time compatible adhesive film (Life Technologies). Fluorescence data were collected using the built-in ROX filter while the temperature was raised from 25°C to 95°C at a rate of 3°C/minute. Data were subsequently processed using the Protein Thermal Shift software (Thermo Fisher Scientific), and the Tm values were determined based on the maxima of the first derivative of the fluorescence versus temperature plot. The reported Tm values represent the mean and standard error of three different experiments.

Construction of homology models

Homology models were built using the I-Tasser [92] and Phyre2 [93] servers. The homology model for the X. citri PilB hexameric core (ND2 and ATPase domains, residues 202–568) was based on G. metallireducens PilB ND2-ATPase structure (PDB code 5TSH) [36]. The homology model of the cytosolic portion of the X. citri PilM-PilN complex (PilM residues 15–359 and PilN residues 1–8) was built based on the P. aeruginosa PilM-PilN structure (PDB: 5EOU) [55]. The homology model for X. citri PilC dimer (residues 74–418) was built based on the low resolution cryo-EM tomography model of M. xanthus PilC (PDB: 3JC8) [9].