Characterization of Opposing Responses to Phenol by Bacillus subtilis Chemoreceptors.

Bacillus subtilis employs 10 chemoreceptors to move in response to chemicals in its environment. While the sensing mechanisms have been determined for many attractants, little is known about the sensing mechanisms for repellents. In this work, we investigated phenol chemotaxis in B.

The Unconventional Cytoplasmic Sensing Mechanism for Ethanol Chemotaxis in Bacillus subtilis.

Motile bacteria sense chemical gradients using chemoreceptors, which consist of distinct sensing and signaling domains. The general model is that the sensing domain binds the chemical and the signaling domain induces the tactic response. Here, we investigated the unconventional sensing mechanism for ethanol taxis in Ethanol and other short-chain alcohols are attractants for Two chemoreceptors, McpB and HemAT, sense these alcohols.

The Mechanism of Bidirectional pH Taxis in Bacillus subtilis.

We investigated pH taxis in This bacterium was found to perform bidirectional taxis in response to external pH gradients, enabling it to preferentially migrate to neutral environments. We next investigated the chemoreceptors involved in sensing pH gradients. We identified four chemoreceptors involved in sensing pH: McpA and TlpA for sensing acidic environments and McpB and TlpB for sensing alkaline ones.

In Vitro Assay for Measuring Receptor-Kinase Activity in the Bacillus subtilis Chemotaxis Pathway.

The sensing apparatus of the Bacillus subtilis chemotaxis pathway involves a complex consisting of chemoreceptors, the CheA histidine kinase, and the CheV and CheW adaptor proteins. Attractants and repellents alter the rate of CheA autophosphorylation, either by directly binding the receptors or by indirectly interacting with them through intermediate binding proteins. We describe an in vitro assay for measuring receptor-kinase activity in B.

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

The Bacillus subtilis chemotaxis pathway employs three systems for sensory adaptation: the methylation system, the CheC/CheD/CheYp system, and the CheV system. Little is known in general about how these three adaptation systems contribute to chemotaxis in B. subtilis and whether they interact with one another.

The importance of the interaction of CheD with CheC and the chemoreceptors compared to its enzymatic activity during chemotaxis in Bacillus subtilis.

Bacillus subtilis use three systems for adaptation during chemotaxis. One of these systems involves two interacting proteins, CheC and CheD. CheD binds to the receptors and increases their ability to activate the CheA kinase.

The Bacillus subtilis chemoreceptor McpC senses multiple ligands using two discrete mechanisms.

Bacillus subtilis can perform chemotaxis toward all 20 L-amino acids normally found in proteins. Loss of a single chemoreceptor, McpC, was previously found to reduce chemotaxis to 19 of these amino acids. In this study, we investigated the amino acid-sensing mechanism of McpC.

Elucidation of the multiple roles of CheD in Bacillus subtilis chemotaxis.

Chemotaxis by Bacillus subtilis requires the CheD protein for proper function. In a cheD mutant when McpB was the sole chemoreceptor in B. subtilis, chemotaxis to asparagine was quite good.

Cellular stoichiometry of the chemotaxis proteins in Bacillus subtilis.

The chemoreceptor-CheA kinase-CheW coupling protein complex, with ancillary associated proteins, is at the heart of chemotactic signal transduction in bacteria. The goal of this work was to determine the cellular stoichiometry of the chemotaxis signaling proteins in Bacillus subtilis. Quantitative immunoblotting was used to determine the total number of chemotaxis proteins in a single cell of B.

Attractant binding induces distinct structural changes to the polar and lateral signaling clusters in Bacillus subtilis chemotaxis.

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.

Site-specific methylation in Bacillus subtilis chemotaxis: effect of covalent modifications to the chemotaxis receptor McpB.

The Bacillus subtilis chemotaxis pathway employs a receptor methylation system that functions differently from the one in the canonical Escherichia coli pathway. Previously, we hypothesized that B. subtilis employs a site-specific methylation system for adaptation where methyl groups are added and removed at different sites.

A PAS domain binds asparagine in the chemotaxis receptor McpB in Bacillus subtilis.

During chemotaxis toward asparagine by Bacillus subtilis, the ligand is thought to bind to the chemoreceptor McpB on the exterior of the cell and induce a conformational change. This change affects the degree of phosphorylation of the CheA kinase bound to the cytoplasmic region of the receptor. Until recently, the sensing domains of the B.

The molecular basis of excitation and adaptation during chemotactic sensory transduction in bacteria.

Chemotaxis is the process by which cells sense chemical gradients in their environment and then move towards more favorable conditions. In the case of Escherichia coli, the paradigm organism for chemotaxis, the pathway is now arguably the best characterized in all of biology. If one broadens their perspective to include other species of bacteria, then our knowledge of chemotaxis is far less developed.

The diverse CheC-type phosphatases: chemotaxis and beyond.

A new class of protein phosphatases has emerged in the study of bacterial/archaeal chemotaxis, the CheC-type phosphatases. These proteins are distinct and unrelated to the well-known CheY-P phosphatase CheZ, though they have convergently evolved to dephosphorylate the same target. The family contains a common consensus sequence D/S-X(3)-E-X(2)-N-X(22)-P that defines the phosphatase active site, of which there are often two per protein.

The three adaptation systems of Bacillus subtilis chemotaxis.

Adaptation has a crucial role in the gradient-sensing mechanism that underlies bacterial chemotaxis. The Escherichia coli chemotaxis pathway uses a single adaptation system involving reversible receptor methylation. In Bacillus subtilis, the chemotaxis pathway seems to use three adaptation systems.

The CheC phosphatase regulates chemotactic adaptation through CheD.

The bacterial chemotaxis system is one of the most extensively studied signal transduction systems in biology. The response regulator CheY controls flagellar rotation and is phosphorylated by the CheA histidine kinase to its active form. CheC is a CheY-P phosphatase, and this activity is enhanced in a CheC-CheD heterodimer.

CheX in the three-phosphatase system of bacterial chemotaxis.

Bacterial chemotaxis involves the regulation of motility by a modified two-component signal transduction system. In Escherichia coli, CheZ is the phosphatase of the response regulator CheY but many other bacteria, including Bacillus subtilis, use members of the CheC-FliY-CheX family for this purpose. While Bacillus subtilis has only CheC and FliY, many systems also have CheX.

Assays for CheC, FliY, and CheX as representatives of response regulator phosphatases.

Much study of two-component systems deals with the excitation of the histidine kinase, activation of the response regulator, and the ultimate target of the signal. Removal of the message is of great importance to these signaling systems. Many methods have evolved in two-component systems to this end.

A receptor-modifying deamidase in complex with a signaling phosphatase reveals reciprocal regulation.

Signal transduction underlying bacterial chemotaxis involves excitatory phosphorylation and feedback control through deamidation and methylation of sensory receptors. The structure of a complex between the signal-terminating phosphatase, CheC, and the receptor-modifying deamidase, CheD, reveals how CheC mimics receptor substrates to inhibit CheD and how CheD stimulates CheC phosphatase activity. CheD resembles other cysteine deamidases from bacterial pathogens that inactivate host Rho-GTPases.

Large increases in attractant concentration disrupt the polar localization of bacterial chemoreceptors.

In bacterial chemotaxis, the chemoreceptors [methyl-accepting chemotaxis proteins (MCPs)] transduce chemotactic signals through the two-component histidine kinase CheA. At low but not high attractant concentrations, chemotactic signals must be amplified. The MCPs are organized into a polar lattice, and this organization has been proposed to be critical for signal amplification.

Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo.

Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur.

Analysis of chimeric chemoreceptors in Bacillus subtilis reveals a role for CheD in the function of the McpC HAMP domain.

Motile prokaryotes use a sensory circuit for control of the motility apparatus in which ligand-responsive chemoreceptors regulate phosphoryl flux through a modified two-component signal transduction system. The chemoreceptors exhibit a modular architecture, comprising an N-terminal sensory module, a C-terminal output module, and a HAMP domain that connects the N- and C-terminal modules and transmits sensory information between them via an unknown mechanism. The sensory circuits mediated by two chemoreceptors of Bacillus subtilis have been studied in detail.

Diversity in chemotaxis mechanisms among the bacteria and archaea.

The study of chemotaxis describes the cellular processes that control the movement of organisms toward favorable environments. In bacteria and archaea, motility is controlled by a two-component system involving a histidine kinase that senses the environment and a response regulator, a very common type of signal transduction in prokaryotes. Most insights into the processes involved have come from studies of Escherichia coli over the last three decades.

Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis.

Bacillus subtilis has a more complex mechanism of chemotaxis than does the paradigm organism, Escherichia coli. In order to understand better the role of the novel chemotaxis proteins--CheC, CheD and CheV--mutants in which increasing numbers of the corresponding genes had been deleted were studied as tethered cells and their biases and sometimes durations of counterclockwise (CCW) and clockwise (CW) flagellar rotations in response to addition and removal of the attractant asparagine were observed. The cheC mutant was found to have considerably reduced switching frequency (that is, prolonged CCW and CW rotations) without a significantly different prestimulus CCW bias, compared with wild-type.

Bacillus subtilis CheC and FliY are members of a novel class of CheY-P-hydrolyzing proteins in the chemotactic signal transduction cascade.

Rapid restoration of prestimulus levels of the chemotactic response regulator, CheY-P, is important for preparing bacteria and archaea to respond sensitively to new stimuli. In an extension of previous work (Szurmant, H., Bunn, M.