Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Bacterial chemoreceptors, the histidine kinase CheA, and the coupling protein CheW form transmembrane molecular arrays with remarkable sensing properties. The receptors inhibit or stimulate CheA kinase activity depending on the presence of attractants or repellants, respectively. We engineered chemoreceptor cytoplasmic regions to assume a trimer of receptor dimers configuration that formed well-defined complexes with CheA and CheW and promoted a CheA kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar electron-spin resonance spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo–electron microscopy. The kinase-off state was especially stable, had relatively low domain mobility, and associated the histidine substrate and docking domains with the kinase core, thus preventing catalytic activity. Together, these data provide an experimentally restrained model for the inhibited state of the core signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

Bacterial chemoreceptors, the CheA histidine kinase, and the coupling protein CheW comprise transmembrane molecular arrays with remarkable sensing properties. An unanswered question concerns how receptors turn off CheA kinase activity. Chemoreceptor cytoplasmic regions engineered to assume a trimer-of-receptor-dimers configuration form well-defined complexes with CheA and CheW and promote a kinase-off state. These mimics of core signaling units were assembled to homogeneity and investigated by site-directed spin-labeling with pulse-dipolar ESR spectroscopy (PDS), small-angle x-ray scattering, targeted protein cross-linking, and cryo-electron microscopy. The kinase-off state is especially stable, has relatively low domain mobility and associates the histidine substrate domain P1 and docking domain P2 with the kinase core. Distances measured between spin-labeled ADP molecules bound to the P4 kinase domain provide evidence for a “dipped conformation” that has been previously proposed from molecular dynamics simulations. Taken together, the data provide an experimentally restrained model for the inhibited state of the core-signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.

Chemoperception of Specific Amino Acids Controls Phytopathogenicity in Pseudomonas syringae pv. tomato

ABSTRACT Chemotaxis has been associated with the pathogenicity of bacteria in plants and was found to facilitate bacterial entry through stomata and wounds. However, knowledge regarding the plant signals involved in this process is scarce. We have addressed this issue using Pseudomonas syringae pv. tomato, which is a foliar pathogen that causes bacterial speck in tomato. We show that the chemoreceptor P. syringae pv. tomato PscA (PsPto-PscA) recognizes specifically and with high affinity l-Asp, l-Glu, and d-Asp. The mutation of the chemoreceptor gene largely reduced chemotaxis to these ligands but also altered cyclic di-GMP (c-di-GMP) levels, biofilm formation, and motility, pointing to cross talk between different chemosensory pathways. Furthermore, the PsPto-PscA mutant strain showed reduced virulence in tomato. Asp and Glu are the most abundant amino acids in plants and in particular in tomato apoplasts, and we hypothesize that this receptor may have evolved to specifically recognize these compounds to facilitate bacterial entry into the plant. Infection assays with the wild-type strain showed that the presence of saturating concentrations of d-Asp also reduced bacterial virulence. IMPORTANCE There is substantive evidence that chemotaxis is a key requisite for efficient pathogenesis in plant pathogens. However, information regarding particular bacterial chemoreceptors and the specific plant signal that they sense is scarce. Our work shows that the phytopathogenic bacterium Pseudomonas syringae pv. tomato mediates not only chemotaxis but also the control of pathogenicity through the perception of the plant abundant amino acids Asp and Glu. We describe the specificity of the perception of l- and d-Asp and l-Glu by the PsPto-PscA chemoreceptor and the involvement of this perception in the regulation of pathogenicity-related traits. Moreover, a saturating concentration of d-Asp reduces bacterial virulence, and we therefore propose that ligand-mediated interference of key chemoreceptors may be an alternative strategy to control virulence.

Flexible Hinges in Bacterial Chemoreceptors

ABSTRACTTransmembrane bacterial chemoreceptors are extended, rod-shaped homodimers with ligand-binding sites at one end and interaction sites for signaling complex formation and histidine kinase control at the other. There are atomic-resolution structures of chemoreceptor fragments but not of intact, membrane-inserted receptors. Electron tomography ofin vivosignaling complex arrays lack distinct densities for chemoreceptor rods away from the well-ordered base plate region, implying structural heterogeneity. We used negative staining, transmission electron microscopy, and image analysis to characterize the molecular shapes of intact homodimers of theEscherichia coliaspartate receptor Tar rendered functional by insertion into nanodisc-providedE. colilipid bilayers. Single-particle analysis plus tomography of particles in a three-dimensional matrix revealed two bend loci in the chemoreceptor cytoplasmic domain, (i) a short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil and (ii) aligned glycines in the extended, four-helix coiled coil, the position of a bend noted in the previous X-ray structure of a receptor fragment. Our images showed HAMP bends from 0° to ∼13° and glycine bends from 0° to ∼20°, suggesting that the loci are flexible hinges. Variable hinge bending explains indistinct densities for receptor rods outside the base plate region in subvolume averages of chemotaxis arrays. Bending at flexible hinges was not correlated with the chemoreceptor signaling state. However, our analyses showed that chemoreceptor bending avoided what would otherwise be steric clashes between neighboring receptors that would block the formation of core signaling complexes and chemoreceptor arrays.IMPORTANCEThis work provides new information about the shape of transmembrane bacterial chemoreceptors, crucial components in the molecular machinery of bacterial chemotaxis. We found that intact, lipid-bilayer-inserted, and thus functional homodimers of theEscherichia colichemoreceptor Tar exhibited bends at two flexible hinges along their ∼200-Å, rod-like, cytoplasmic domains. One hinge was at the short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil. The other hinge was at aligned glycines in the extended, four-helix coiled coil, where a bend had been identified in the X-ray structure of a chemoreceptor fragment. Our analyses showed that flexible hinge bending avoided structural clashes in chemotaxis core complexes and their arrays.

Sensory Repertoire of Bacterial Chemoreceptors

SUMMARY Chemoreceptors in bacteria detect a variety of signals and feed this information into chemosensory pathways that represent a major mode of signal transduction. The five chemoreceptors from Escherichia coli have served as traditional models in the study of this protein family. Genome analyses revealed that many bacteria contain much larger numbers of chemoreceptors with broader sensory capabilities. Chemoreceptors differ in topology, sensing mode, cellular location, and, above all, the type of ligand binding domain (LBD). Here, we highlight LBD diversity using well-established and emerging model organisms as well as genomic surveys. Nearly a hundred different types of protein domains that are found in chemoreceptor sequences are known or predicted LBDs, but only a few of them are ubiquitous. LBDs of the same class recognize different ligands, and conversely, the same ligand can be recognized by structurally different LBDs; however, recent studies began to reveal common characteristics in signal-LBD relationships. Although signals can stimulate chemoreceptors in a variety of different ways, diverse LBDs appear to employ a universal transmembrane signaling mechanism. Current and future studies aim to establish relationships between LBD types, the nature of signals that they recognize, and the mechanisms of signal recognition and transduction.

Identification of a Chemoreceptor for C2and C3Carboxylic Acids

ABSTRACTChemoreceptors are at the beginnings of chemosensory signaling cascades that mediate chemotaxis. Most bacterial chemoreceptors are functionally unannotated and are characterized by a diversity in the structure of their ligand binding domains (LBDs). The data available indicate that there are two major chemoreceptor families at the functional level, namely, those that respond to amino acids or to Krebs cycle intermediates. Since pseudomonads show chemotaxis to many different compounds and possess different types of chemoreceptors, they are model organisms to establish relationships between chemoreceptor structure and function. Here, we identify PP2861 (termed McpP) ofPseudomonas putidaKT2440 as a chemoreceptor with a novel ligand profile. We show that the recombinant McpP LBD recognizes acetate, pyruvate, propionate, andl-lactate, withKD(equilibrium dissociation constant) values ranging from 34 to 107 μM. Deletion of themcpPgene resulted in a dramatic reduction in chemotaxis toward these ligands, and complementation restored a native-like phenotype, indicating that McpP is the major chemoreceptor for these compounds. McpP has a CACHE-type LBD, and we present data indicating that CACHE-containing chemoreceptors of other species also mediate taxis to C2and C3carboxylic acids. In addition, the LBD of NbaY ofPseudomonas fluorescens, an McpP homologue mediating chemotaxis to 2-nitrobenzoate, bound neither nitrobenzoates nor the McpP ligands. This work provides further insight into receptor structure-function relationships and will be helpful to annotate chemoreceptors of other bacteria.