Attractant Binding Induces Distinct Structural Changes to the Polar and Lateral Signaling Clusters in Bacillus subtilis Chemotaxis

Abstract

Bacteria employ a modified two-component system for chemotaxis, where the receptors form ternary complexes with CheA histidine kinases and CheW adaptor proteins. These complexes are arranged in semi-ordered arrays clustered predominantly at the cell poles. The prevailing models assume that these arrays are static and reorganize only locally in response to attractant binding. Recent studies have shown, however, that these structures may in fact be much more fluid. We investigated the localization of the chemotaxis signaling arrays in Bacillus subtilis using immunofluorescence and live cell fluorescence microscopy. We found that the receptors were localized in clusters at the poles in most cells. However, when the cells were exposed to attractant, the number exhibiting polar clusters was reduced roughly 2-fold, whereas the number exhibiting lateral clusters distinct from the poles increased significantly. These changes in receptor clustering were reversible as polar localization was reestablished in adapted cells. We also investigated the dynamic localization of CheV, a hybrid protein consisting of an N-terminal CheW-like adaptor domain and a C-terminal response regulator domain that is known to be phosphorylated by CheA, using immunofluorescence. Interestingly, we found that CheV was localized predominantly at lateral clusters in unstimulated cells. However, upon exposure to attractant, CheV was found to be predominantly localized to the cell poles. Moreover, changes in CheV localization are phosphorylation-dependent. Collectively, these results suggest that the chemotaxis signaling arrays in B. subtilis are dynamic structures and that feedback loops involving phosphorylation may regulate the positioning of individual proteins.Many motile bacteria employ for chemotaxis a modified two-component system to sense and respond to chemicals, where the receptors form ternary complexes with the CheA histidine kinase and the CheW adaptor protein (1, 2). The clustering of these ternary complexes into semi-ordered hexagonal lattices has been documented in multiple species (3) and is presumably conserved in all chemotactic bacteria where the three proteins are found. These arrays are thought to amplify the response to attractant binding (4,5). A number of models have specifically proposed that cooperative interactions between the receptors within these arrays enable bacteria to sense small differences in the number of attractant-bound receptors over a wide range of concentrations (see Ref. 6).Multiple studies have investigated the structure and molecular determinants of these clusters (e.g. Refs. 7 and 8) along with their role in signal transduction. In Escherichia coli, the receptors form mixed trimers of receptor homodimers. These trimers are believed to form the basic building blocks for the larger clusters, which range in size from tens to thousands of receptors (9). These clusters are found predominantly at the cell poles, although they are also found along the lateral length of the cell. Attractant binding, which inhibi...

The protein interaction network of a taxis signal transduction system in a Halophilic Archaeon

“…In several prokaryotic organisms taxis receptors assemble into large, mixed clusters [74-81] which facilitate signal integration, large signal amplification and high sensitivity [76,82-85]. Due to this cluster formation it is not possible to deduce whether certain Htrs directly interact with a Che protein from copurification experiments.…”

“…Dynamic changes in the signaling clusters have recently been directly observed in B.subtilis [81]. Immunofluorescence microscopy showed that attractant binding caused a decrease in the number of observable polar receptor clusters and an increase in the lateral receptor clusters.…”

“…Immunofluorescence microscopy showed that attractant binding caused a decrease in the number of observable polar receptor clusters and an increase in the lateral receptor clusters. The disappearance or appearance of receptor clusters is probably caused by an altered degree of receptor packing [81]. At the same time, the localization of CheV changed from primarily lateral to primarily polar.…”

Interactions among the three adaptation systems of Bacillus subtilis chemotaxis as revealed by an in vitro receptor‐kinase assay

“…An additional facet of CheV worth noting concerns mobility. In a previous study investigating the spatial localization of the chemotaxis proteins in B. subtilis, we observed changes in the localization of CheV following the addition and subsequent adaption to asparagine (Wu et al ., ). Specifically, CheV was found to associate more strongly with the receptors and CheA at the cell poles following the addition of asparagine than with the lateral receptors, where CheA and CheW are scarce.…”

The Importance of the Interaction of CheD with CheC and the Chemoreceptors Compared to Its Enzymatic Activity during Chemotaxis in Bacillus subtilis

“…B. subtilis has three systems that foster adaptation: a system involving receptor methylation [5], [9], a system involving phosphorylation of CheV [10], [11], and a system involving CheC, CheD, and CheYp [12], [13]. CheV is homologous to the coupling protein CheW except that it also has an N-terminal response regulator domain [14], [15].…”