Azospirillum brasilense AerC and Tlp4b Cytoplasmic Chemoreceptors Are Promiscuous and Interact with the Two Membrane-Bound Chemotaxis Signaling Clusters Mediating Chemotaxis Responses.

Chemotaxis in Bacteria and Archaea depends on the presence of hexagonal polar arrays composed of membrane-bound chemoreceptors that interact with rings of baseplate signaling proteins. In the alphaproteobacterium Azospirillum brasilense, chemotaxis is controlled by two chemotaxis signaling systems (Che1 and Che4) that mix at the baseplates of two spatially distinct membrane-bound chemoreceptor arrays. The subcellular localization and organization of transmembrane chemoreceptors in chemotaxis signaling clusters have been well characterized but those of soluble chemoreceptors remain relatively underexplored. By combining mutagenesis, microscopy, and biochemical assays, we show that the cytoplasmic chemoreceptors AerC and Tlp4b function in chemotaxis and localize to and interact with membrane-bound chemoreceptors and chemotaxis signaling proteins from both polar arrays, indicating that soluble chemoreceptors are promiscuous. The interactions of AerC and Tlp4b with polar chemotaxis signaling clusters are not equivalent and suggest distinct functions. Tlp4b, but not AerC, modulates the abundance of chemoreceptors within the signaling clusters through an unknown mechanism. The AerC chemoreceptor, but not Tlp4b, is able to traffic in and out of chemotaxis signaling clusters depending on its level of expression. We also identify a role of the chemoreceptor composition of chemotaxis signaling clusters in regulating their polar subcellular organization. The organization of chemotaxis signaling proteins as large membrane-bound arrays underlies chemotaxis sensitivity. Our findings suggest that the composition of chemoreceptors may fine-tune chemotaxis signaling not only through their chemosensory specificity but also through their role in the organization of polar chemotaxis signaling clusters. IMPORTANCE Cytoplasmic chemoreceptors represent about 14% of all chemoreceptors encoded in bacterial and archaeal genomes, but little is known about how they interact with and function in large polar assemblies of membrane-bound chemotaxis signaling clusters. Here, we show that two soluble chemoreceptors with a role in chemotaxis are promiscuous and interact with two distinct membrane-bound chemotaxis signaling clusters that control all chemotaxis responses in Azospirillum brasilense. We also found that any change in the chemoreceptor composition of chemotaxis signaling clusters alters their polar organization, suggesting a dynamic interplay between the sensory specificity of chemotaxis signaling clusters and their polar membrane organization.

Distinct Chemotaxis Protein Paralogs Assemble into Chemoreceptor Signaling Arrays To Coordinate Signaling Output.

Most chemotactic motile bacteria possess multiple chemotaxis signaling systems, the functions of which are not well characterized. Chemotaxis signaling is initiated by chemoreceptors that assemble as large arrays, together with chemotaxis coupling proteins (CheW) and histidine kinase proteins (CheA), which form a baseplate with the cytoplasmic tips of receptors. These cell pole-localized arrays mediate sensing, signaling, and signal amplification during chemotaxis responses. Membrane-bound chemoreceptors with different cytoplasmic domain lengths segregate into distinct arrays. Here, we show that a bacterium, Azospirillum brasilense, which utilizes two chemotaxis signaling systems controlling distinct motility parameters, coordinates its chemotactic responses through the production of two separate membrane-bound chemoreceptor arrays by mixing paralogs within chemotaxis baseplates. The polar localization of chemoreceptors of different length classes is maintained in strains that had baseplate signaling proteins from either chemotaxis system but was lost when both systems were deleted. Chemotaxis proteins (CheA and CheW) from each of the chemotaxis signaling systems (Che1 and Che4) could physically interact with one another, and chemoreceptors from both classes present in A. brasilense could interact with Che1 and Che4 proteins. The assembly of paralogs from distinct chemotaxis pathways into baseplates provides a straightforward mechanism for coordinating signaling from distinct pathways, which we predict is not unique to this system given the propensity of chemotaxis systems for horizontal gene transfer.IMPORTANCE The assembly of chemotaxis receptors and signaling proteins into polar arrays is universal in motile chemotactic bacteria. Comparative genome analyses indicate that most motile bacteria possess multiple chemotaxis signaling systems, and experimental evidence suggests that signaling from distinct chemotaxis systems is integrated. Here, we identify one such mechanism. We show that paralogs from two chemotaxis systems assemble together into chemoreceptor arrays, forming baseplates comprised of proteins from both chemotaxis systems. These mixed arrays provide a straightforward mechanism for signal integration and coordinated response output from distinct chemotaxis systems. Given that most chemotactic bacteria encode multiple chemotaxis systems and the propensity for these systems to be laterally transferred, this mechanism may be common to ensure chemotaxis signal integration occurs.

Metabolic adaptations of Azospirillum brasilense to oxygen stress by cell-to-cell clumping and flocculation.

The ability of bacteria to monitor their metabolism and adjust their behavior accordingly is critical to maintain competitiveness in the environment. The motile microaerophilic bacterium Azospirillum brasilense navigates oxygen gradients by aerotaxis in order to locate low oxygen concentrations that can support metabolism. When cells are exposed to elevated levels of oxygen in their surroundings, motile A. brasilense cells implement an alternative response to aerotaxis and form transient clumps by cell-to-cell interactions. Clumping was suggested to represent a behavior protecting motile cells from transiently elevated levels of aeration. Using the proteomics of wild-type and mutant strains affected in the extent of their clumping abilities, we show that cell-to-cell clumping represents a metabolic scavenging strategy that likely prepares the cells for further metabolic stresses. Analysis of mutants affected in carbon or nitrogen metabolism confirmed this assumption. The metabolic changes experienced as clumping progresses prime cells for flocculation, a morphological and metabolic shift of cells triggered under elevated-aeration conditions and nitrogen limitation. The analysis of various mutants during clumping and flocculation characterized an ordered set of changes in cell envelope properties accompanying the metabolic changes. These data also identify clumping and early flocculation to be behaviors compatible with the expression of nitrogen fixation genes, despite the elevated-aeration conditions. Cell-to-cell clumping may thus license diazotrophy to microaerophilic A. brasilense cells under elevated oxygen conditions and prime them for long-term survival via flocculation if metabolic stress persists.