A bipartite periplasmic receptor-diguanylate cyclase pair (XAC2383-XAC2382) in the bacterium Xanthomonas citri

Abstract

The second messenger cyclic diguanylate monophosphate (c-di-GMP) is a central regulator of bacterial lifestyle, controlling several behaviors, including the switch between sessile and motile states. The c-di-GMP levels are controlled by the interplay between diguanylate cyclases (DGCs) and phosphodiesterases, which synthesize and hydrolyze this second messenger, respectively. These enzymes often contain additional domains that regulate activity via binding of small molecules, covalent modification, or protein-protein interactions. A major challenge remains to understand how DGC activity is regulated by these additional domains or interaction partners in specific signaling pathways. Here, we identified a pair of co-transcribed genes (xac2382 and xac2383) in the phytopathogenic, Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), whose mutations resulted in opposing motility phenotypes. We show that the periplasmic cache domain of XAC2382, a membrane-associated DGC, interacts with XAC2383, a periplasmic binding protein, and we provide evidence that this interaction regulates XAC2382 DGC activity. Moreover, we solved the crystal structure of XAC2383 with different ligands, indicating a preference for negatively charged phosphate-containing compounds. We propose that XAC2383 acts as a periplasmic sensor that, upon binding its ligand, inhibits the DGC activity of XAC2382. Of note, we also found that this previously uncharacterized signal transduction system is present in several other bacterial phyla, including Gram-positive bacteria. Phylogenetic analysis of homologs of the XAC2382-XAC2383 pair supports several independent origins that created new combinations of XAC2382 homologs with a conserved periplasmic cache domain with different cytoplasmic output module architectures.

Keywords: Xanthomonas; bacterial signal transduction; cell motility; cyclic di-GMP (c-di-GMP); phytopathogen; plant pathogen; receptor; structural biology.

Figure 1.

xac2382 and xac2383 genes are co-transcribed in X. citri subsp. citri and the proteins interact in vitro. A, diagram of the locus containing the xac2383, xac2382, and xac2384 genes (shown as thick arrows). Black lines represent fragments (1, 2, and 3) amplified from the Xac cDNA preparation in C. B, domain architecture of XAC2382 and XAC2383. Black boxes show transmembrane regions of XAC2382 and the signal peptide of XAC2383. The lines above XAC2382 domain architecture indicate the constructs realized using pBRA vector. Asterisk indicates the sites of introducing a single amino acid modification in the heptad repeat of the HAMP domain (F259E) and the modification of the GGDEF motif to AAAEF. C, lanes 1, 4, and 7, PCRs using oligonucleotides that amplify a 200-bp fragment (fragment 1 in A) corresponding to the beginning of the xac2382 gene. Lanes 2, 5, and 8, PCRs using oligonucleotides that amplify a 200-bp fragment (fragment 2) corresponding to the end of the xac2383 gene. Lanes 3, 6, and 9, PCRs using oligonucleotides that amplify a 400-bp fragment (fragment 3) that overlaps genes xac2382 and xac2383. Lanes 1–3, PCR amplification using Xac cDNA produced from total mRNA as template. Lanes 4–6, negative control. PCR amplification using a mock cDNA sample (prepared by omitting reverse transcriptase) as template. Lanes 7–9, positive control. PCR amplification using genomic DNA as template. D, size-exclusion chromatography coupled to multiangle light scattering (SEC-MALS) analysis of the XAC2382^{Cache_37–192}–XAC2383^31–309 complex using a Superdex 200 10/300 column. Black line indicates the relative concentration of the proteins, and the gray line indicates the mass determined by the scattering analysis. E, SDS-PAGE of the purified complex.

Figure 2.

Crystal structure of XAC2383. A, XAC2383 topology and secondary structure. β-Strands are shown in red and α-helices in green. B, cartoon representation of XAC2383 crystal structure showing the two structurally similar lobes (PDB code 5UB3). C, comparison of the aperture between the lobes in-between XAC2383 (PDB code 5UB3), PhnD bound to 2AEP ligand (PDB code 3P7I), and apo-PhnD (PDB code 3S4U). Lobe 1, yellow; lobe 2, orange. The aperture between lobes in XAC2383 is smaller than that observed for apo-PhnD but larger than that observed for the PhnD–2AEP complex. The chloride ion bound to XAC2383 is shown as a green sphere. The 2AEP ligand bound to PhnD is completely buried and therefore not visible.

Figure 3.

XAC2383 ¹⁵²STS¹⁵⁴ motif in the ligand-binding site. A, 2F_o − F_c map contoured at 1.0σ of the conserved ¹⁵²STS¹⁵⁴ motif and a coordinated chloride ion (PDB code 5UB3). B, E. coli PhnD in complex with 2-aminoethylphosphonate (PDB code 3P7I). The numbers in A and B indicate distances in Å. C, alignment of PhnD from E. coli and P. aeruginosa and XAC2383 highlighting the residues in B responsible for 2-aminoethylphosphonate binding by PhnD. Black contour boxes show the residues responsible for the binding to the −PO₃²⁻ moiety. Gray contour boxes represent the residues responsible for the 2-aminoethyl R-group. The N-terminal signal peptides of the three sequences are highlighted.

Figure 4.

Ligand binding to the central groove of XAC2383. A, XAC2383 surface representation with the chloride ion (green) at the binding site (PDB code 5UB3). B–D, binding site with different ligands showing 2F_o − F_c map contoured at 1.0σ. B, phosphate (PDB code 5UB4). C, pyrophosphate (PDB code 5UB6). D, ATP (PDB code 5UB7). The 2F_o − F_c map for side chains in D was omitted for clarity. The numbers in B–D indicate distances in Å.

Figure 5.

XAC2382 and XAC2383 are involved in X. citri subsp. citri motility and have opposing phenotypes. A, sliding motility assay for the Xac WT strain containing the empty pBRA vector, the Δxac2382 containing the empty pBRA vector, and the Δxac2382 strain containing the pBRA vector expressing different XAC2382 fragments shown in Fig. 1B. B, Xac WT strain containing the empty pBRA vector, the Δxac2383 containing the empty pBRA vector, and the Δxac2383 strain containing the pBRA vector expressing XAC2383 WT and XAC2383 with the ¹⁵²STS¹⁵⁴ motif mutated to AAA. Pictures taken after 4 days growth at 30 °C on SB medium 0.5% agar plates.

Figure 6.

Common domain architectures found for XAC2382 homologs whose genes are predicted to be co-transcribed with xac2383 homologs. A, GGDEF domain-containing proteins. B, histidine kinases. C, σ54 with HTH motif domain, HD domain from phosphodiesterases, and guanylate cyclase.

Figure 7.

Phylogeny of XAC2382^Cache homologous domains found in association with XAC2383-like genes. The phylogeny tree was built by FastTree (Price et al. (77)), using default options, from a multiple sequence alignment generated by MUSCLE (75). Only the conserved columns of the alignment, as identified by TrimAL (76) using the heuristic similarity statistics, were used. Species names are colored after the domain effector type, as described in the legend in the bottom left corner. Branch color (when not black) refers to the domain architecture of an occasionally encountered third gene, located downstream to the effector GGDEF or histidine kinase XAC2382 homolog and unrelated to the XAC2383 homologs. Such genes also encode signal transduction proteins and contain effector domains (indicated by the colors in the legend). We note some instances of highly supported sibling branches that correspond to proteins with different effector domains but whose Cache domains are more closely to each other than to any other homologs with a similar architecture, thus pointing to novel and independent origins for these architectures.

Figure 8.

Model of the XAC2382–XAC2383 signal-transduction pathway. The periplasmic binding protein XAC2383 has two topologically similar (nonequivalent) lobes and can exist in two conformations whose equilibrium is determined by the binding of a specific ligand (L, unknown). It is not known whether XAC2383 binding to the XAC2382 Cache domain is affected by ligand binding, nor do we know the precise stoichiometry of the XAC2382–XAC2383 complex (here shown as 2:1 for simplicity). In the absence of the ligand (left), the XAC2383 interaction with the XAC2382 Cache domain permits XAC2382 to adopt a symmetric and catalytically active conformation. In the presence of ligand (right), the XAC2383–XAC2382 interaction changes, breaking the symmetry of the XAC2382 dimer, thereby inhibiting the latter's DGC activity.