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.

Genome-wide peptidoglycan profiling of Vibrio cholerae

“…Similar changes in the PG were observed for the vc0377 and vc0378 mutants (Fig. 3e), encoding a CheX-like protein proposed to be involved in motility regulation (Altinoglu et al, 2022) and the zinc uptake regulator Zur (Sheng et al, 2015) respectively, which influences PG endopeptidase activity in V. cholerae (Murphy et al, 2019).…”

Impact of Gene Repression on Biofilm Formation of Vibrio cholerae

“…All of these randomly selected candidates were identified at least two times in the screen ( Table 3 ). Notably, VC0998 (HubP) is also linked to chemotactic cascades, being a landmark protein in cell pole differentiation which targets several other proteins related to chemotaxis, motility and cell wall homeostasis to the cell poles via their cytosolic or periplasmic modules ( Yamaichi et al, 2012 ; Altinoglu et al, 2022 ).…”

“…Vice versa, constitutive (over)-expression of VC0998 increased VPS expression and static biofilm formation. VC0998 (HubP) is a large transmembrane protein involved in localizing and organizing several other proteins in the cell pole, namely proteins involved in cell division, cell wall remodeling, chemotaxis and motility ( Yamaichi et al, 2012 ; Altinoglu et al, 2022 ). One of the recently identified proteins requiring HubP for proper membrane localization is MotW, which has been linked to c-di-GMP degradation ( Altinoglu et al, 2022 ).…”

“…VC0998 (HubP) is a large transmembrane protein involved in localizing and organizing several other proteins in the cell pole, namely proteins involved in cell division, cell wall remodeling, chemotaxis and motility ( Yamaichi et al, 2012 ; Altinoglu et al, 2022 ). One of the recently identified proteins requiring HubP for proper membrane localization is MotW, which has been linked to c-di-GMP degradation ( Altinoglu et al, 2022 ). Thus, altered expression of HubP could interfere via MotW in c-di-GMP signaling, which in turn influences VPS expression and biofilm formation.…”

“…Thus, altered expression of HubP could interfere via MotW in c-di-GMP signaling, which in turn influences VPS expression and biofilm formation. Differential expression of HubP also impaired motility, which might also be linked to disturbances in c-di-GMP levels or other chemotaxis and motility proteins, whose polar localization is HubP-dependent ( Altinoglu et al, 2022 ).…”