Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM

Abstract

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array.chemotaxis | chemoreceptor arrays | Vibrio cholerae | electron cryotomography | microscopy M ost motile bacteria move toward favorable environments through a process called chemotaxis. The molecular basis of this behavior is best understood in Escherichia coli, where transmembrane methyl-accepting chemotaxis proteins (MCPs, or chemoreceptors) form large arrays at the cell pole. The chemoreceptors bind attractants or repellents in the periplasm (1-3) and relay signals to histidine kinases (CheA) in the cytoplasm (4). When activated, CheA first autophosphorylates and then transfers the phosphoryl group to the response regulators CheY and CheB. Phosphorylated CheY binds to the flagellar motor, changing the direction of flagellar rotation. This allows the cells to switch from swimming forward smoothly (so-called "runs") to tumbling randomly. Changes in the duration and frequency of run and tumble phases drive a biased random walk that moves the cells toward favorable environments (5). The other response regulator, CheB, is a methylesterase, which, in conjunction with the constitutively active methyltransferase CheR, tunes the sensitivity of the system by changing the methylation state of the chemoreceptors (6-8).Although there is only one chemotaxis system in E. coli, most chemotactic bacterial and archaeal species have multiple systems (9). For instance, Rhodobacter sphaeroides has three chemotaxis systems encoded in three distinct clusters of genes, one of which (CheOp2) encodes two receptors without predicted transmembrane domains, TlpT and TlpC (10). Analysis of fluorescently tagged TlpT and TlpC revealed that they localize in foci around midcell. The foci split before cell division and are segregated to the midcell position within the future daughter cells. The chromosome-associated ParA-like ATPase PpfA controls the localization and segregation of the foci through an interaction with the N terminus of TlpT in a ParB-like manner (11).Using electron cryotomography (ECT), we discovered that these cytoplasmic foci in R. sphaerodes are ordered arrays of chemore...

Structural and proteomic changes in viable but non-culturableVibrio cholerae

“…V. cholerae encodes three distinct chemotaxis systems (cluster I, II, III) and 44-45 different MCPs [48]. The MCPs that are known to interact with cluster II all belong to the 40H class [22,49]. Cluster II arrays sense a plethora of cues and are so far the only chemosensory system that controls the chemotactic swimming behavior of V. cholerae [50].…”

Differential Localization of Chemotactic Signaling Arrays during the Lifecycle of Vibrio parahaemolyticus

“…Preparation of V. parahaemolyticus swimmer cells for fluorescence microscopy was carried out essentially as described (Ringgaard et al, 2015; Briegel et al, 2016). A volume of 10 mL of LB was inoculated with a bacterial colony of V. parahaemolyticus from an over-night LB agar plate grown at 37°C and relevant antibiotic.…”

Repurposing a macromolecular machine: Architecture and evolution of the F7 chemosensory system

“…The genome of V. cholerae encodes three chemosensory systems: F6, F7 and F9 (Table S2) (9). Having already identified the F6 and F9 systems (11,16), we hypothesized that the novel arrays were formed by the F7 system. The genome of P. aeruginosa encodes four chemosensory systems: F6, F7, ACF and TFP (Table S2) (9).…”

“…showed that this array is formed by proteins of the F6 chemosensory system (16) (known to control flagellar rotation). In late stationary phase, we found that cells contain another, purely cytoplasmic array consisting of two CheA/CheW baseplates 35 nm apart sandwiching a double layer of chemoreceptors (16). This array is formed by proteins of the F9 chemosensory system, but its function is unknown.…”