Analysis of HubP-dependent cell pole protein targeting in Vibrio cholerae uncovers novel motility regulators

Abstract

In rod-shaped bacteria, the emergence and maintenance of long-axis cell polarity is involved in key cellular processes such as cell cycle, division, environmental sensing and flagellar motility among others. Many bacteria achieve cell pole differentiation through the use of polar landmark proteins acting as scaffolds for the recruitment of functional macromolecular assemblies. In Vibrio cholerae a large membrane-tethered protein, HubP, specifically interacts with proteins involved in chromosome segregation, chemotaxis and flagellar biosynthesis. Here we used comparative proteomics, genetic and imaging approaches to identify additional HubP partners and demonstrate that at least six more proteins are subject to HubP-dependent polar localization. These include a cell-wall remodeling enzyme (DacB), a likely chemotaxis sensory protein (HlyB), two presumably cytosolic proteins of unknown function (VC1210 and VC1380) and two membrane-bound proteins, named here MotV and MotW, that exhibit distinct effects on chemotactic motility. We show that while both ΔmotW and ΔmotV mutants retain monotrichous flagellation, they present significant to severe motility defects when grown in soft agar. Video-tracking experiments further reveal that ΔmotV cells can swim in liquid environments but are unable to tumble or penetrate a semisolid matrix, whereas a motW deletion affects both tumbling frequency and swimming speed. Motility suppressors and gene co-occurrence analyses reveal co-evolutionary linkages between MotV, a subset of non-canonical CheV proteins and flagellar C-ring components FliG and FliM, whereas MotW regulatory inputs appear to intersect with specific c-di-GMP signaling pathways. Together, these results reveal an ever more versatile role for the landmark cell pole organizer HubP and identify novel mechanisms of motility regulation.

Fig 1. Comparative proteomics of minicells in search of polar proteins.

(A) Representative fluorescent microscopy image of minicells purified from YBB2143 (ΔminD ΔparA1 hubP::hubP-yfp). YFP signals are pseudocolored in green and merged with the corresponding phase contrast image. (B) Summary plot of iTRAQ results. Horizontal axis: proteins’ locus tag on each chromosome; Y-axis, log2 of HubP^-/HubP⁺ value from two independent experiments. A blue square indicates HubP (VC0998); known proteins related to chemotaxis are indicated by orange diamonds. (C) Representative fluorescence microscopy images of C-terminal GFP fusions of indicated candidate proteins in hubP⁺ and ΔhubP V. cholerae cells. Bars = 2 μm.

Fig 2. Bipolarity of polar proteins.

(A-D) Representative fluorescent microscopy images of cells expressing indicated fluorescent protein fusions expressed from either plasmid (_p) or native chromosomal locus (referred as _c). (E) Fraction of cells (%) exhibiting uni- (1) or bi- (2) polar foci. Average and standard deviations of 2 independent analyses (each counted >1000 cells) are shown.

Fig 3. Motility of V. cholerae mutants.

(A) Motility defects examined by swimming in soft agar plates. Average diameter relative to WT, along with standard deviations from at least three experiments are shown. Only limited number of strains are shown and the exhaustive results can be found in S5 Fig. * motV⁺ complemented in the suppressor strain. (B) Representative trajectories of V. cholerae cells in liquid (left) and 0.25% agarose (right) environments. Bars = 10 μm. Tumbling frequency (C) and velocity (D) of V. cholerae cells from video-tracking in liquid. Numbers of cells analyzed (n) are shown on the left in parenthesis. † denotes no tumbling. Standard deviations from at least three replicates are shown for (D). (E) Mean square displacement (MSD) of V. cholerae cells in 0.25% agarose. Average (thick lines), standard error of the mean (vertical bars), and standard deviations (shades) of projected individual trajectories are shown. n is indicated in parentheses.

Fig 4. Multiple sequence alignments of FliG and FliM.

(A) Presence of C-ring (FliG/M), HubP, MotV and MotW in 132 γ-proteobacteria species shown in Venn diagram. (B) Alignments of FliG and FliM from different γ-proteobacterial species. For each organism, presence of HubP, MotW and CheV homologs are indicated. (§) In addition to total number of CheV proteins, number of CheV variants with non-canonical ‘switch’ residues are indicated in round (A substitution) and square [T substitution] brackets (see text and Fig 5). Asterisk (*), colon (:), and period (.) indicate amino acids with full conservation, strongly similar properties, and weakly similar properties, respectively. Key residues differentially conserved in MotV⁺ versus MotV^- bacteria are labeled in yellow.

Fig 5. Motility regulators with identified ΔmotV and/or ΔmotW suppressor mutations.

(A) Domain architecture of conserved C-ring component FliG (Left) and FliM (right). _N, N-terminal domain; _M, middle domain; _C, C-terminal domain; NM, FliG_N-FliG_M domain linker; Arm, armadillo repeat-like motifs; MC, FliG_M-FliG_C domain linker; CheY-P, phosphorylated CheY. (B) Left, schematic of the V. cholerae flagellum with C-ring components in color. Right, cartoon representation of a homology model of a single FliG-FliM-FliN-CheY unit with color coding as in (A) and based on structural information from [45]. FliG and FliM amino acids mutated in the suppressor strains are highlighted as red spheres. The thumbnail representation of the flagellum is adapted with modifications from [45], under the license CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/legalcode). (C) A three-dimensional homology model of the assembled multi-copy FliG-FliM-FliN C-ring with indicated positions (red spheres) of the suppressor mutations are as in (B). (D) VCA0954/CheV4 domain architecture. REC, receiver domain. (E) Key residues involved in receiver domain phosphorylation as compared to canonical CheY proteins and CheV homologs in MotV⁻and MotV⁺ species. (F) Representative fluorescence microscopy images of cells expressing full length (top rows) or truncated (bottom rows) CheV4*-GFP fusions from the native cheV chromosomal locus in different genetic backgrounds, as indicated. Non-polar fluorescence foci are indicated with white arrowheads. Bar = 2 μm; (G) Predicted VC1653/VieS domain architecture. PBPb, bacterial periplasmic substrate-binding protein domain; TM, transmembrane region; CC, coiled-coil; DHp, dimerization histidine phosphotransfer domain; ATPase, ATP binding and hydrolysis domain; HPt, histidine phosphotransfer domain; PDE, phosphodiesterase; c-di-GMP, cyclic diguanylate; pGpG, linear diguanylate.

Fig 6. HubP-dependent cell pole organization.

Proposed integrated model for HubP-dependent cell pole organization and functional linkages among HubP, identified partners, flagellar motility, chemotaxis signaling and c-di-GMP signaling pathways. The thumbnail representation of the flagellum is adapted with modifications from [45], under the license CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/legalcode).