The structural analysis of the periplasmic domain of Sinorhizobium meliloti chemoreceptor McpZ reveals a novel fold and suggests a complex mechanism of transmembrane signaling.

Chemotaxis is a fundamental process whereby bacteria seek out nutrient sources and avoid harmful chemicals. For the symbiotic soil bacterium Sinorhizobium meliloti, the chemotaxis system also plays an essential role in the interaction with its legume host. The chemotactic signaling cascade is initiated through interactions of an attractant or repellent compound with chemoreceptors or methyl-accepting chemotaxis proteins (MCPs). S. meliloti possesses eight chemoreceptors to mediate chemotaxis. Six of these receptors are transmembrane proteins with periplasmic ligand-binding domains (LBDs). The specific functions of McpW and McpZ are still unknown. Here, we report the crystal structure of the periplasmic domain of McpZ (McpZPD) at 2.7 Å resolution. McpZPD assumes a novel fold consisting of three concatenated four-helix bundle modules. Through phylogenetic analyses, we discovered that this helical tri-modular domain fold arose within the Rhizobiaceae family and is still evolving rapidly. The structure, offering a rare view of a ligand-free dimeric MCP-LBD, reveals a novel dimerization interface. Molecular dynamics calculations suggest ligand binding will induce conformational changes that result in large horizontal helix movements within the membrane-proximal domains of the McpZPD dimer that are accompanied by a 5 Å vertical shift of the terminal helix toward the inner cell membrane. These results suggest a mechanism of transmembrane signaling for this family of MCPs that entails both piston-type and scissoring movements. The predicted movements terminate in a conformation that closely mirrors those observed in related ligand-bound MCP-LBDs.

McpT, a Broad-Range Carboxylate Chemoreceptor in Sinorhizobium meliloti.

Chemoreceptors enable the legume symbiont Sinorhizobium meliloti to detect and respond to specific chemicals released from their host plant alfalfa, which allows the establishment of a nitrogen-fixing symbiosis. The periplasmic region (PR) of transmembrane chemoreceptors act as the sensory input module for chemotaxis systems via binding of specific ligands, either directly or indirectly. S. meliloti has six transmembrane and two cytosolic chemoreceptors. However, the function of only three of the transmembrane receptors have been characterized so far, with McpU, McpV, and McpX serving as general amino acid, short-chain carboxylate, and quaternary ammonium compound sensors, respectively. In the present study, we analyzed the S. meliloti chemoreceptor McpT. High-throughput differential scanning fluorimetry assays, using Biolog phenotype microarray plates, identified 15 potential ligands for McpTPR, with the majority classified as mono-, di-, and tricarboxylates. S. meliloti exhibited positive chemotaxis toward seven selected carboxylates, namely, α-ketobutyrate, citrate, glyoxylate, malate, malonate, oxalate, and succinate. These carboxylates were detected in seed exudates of the alfalfa host. Deletion of mcpT resulted in a significant decrease of chemotaxis to all carboxylates except for citrate. Isothermal titration calorimetry revealed that McpTPR bound preferentially to the monocarboxylate glyoxylate and with lower affinity to the dicarboxylates malate, malonate, and oxalate. However, no direct binding was detected for the remaining three carboxylates that elicited an McpT-dependent chemotaxis response. Taken together, these results demonstrate that McpT is a broad-range carboxylate chemoreceptor that mediates chemotactic response via direct ligand binding and an indirect mechanism that needs to be identified. IMPORTANCE Nitrate pollution is one of the most widespread and challenging environmental problems that is mainly caused by the agricultural overapplication of nitrogen fertilizers. Biological nitrogen fixation by the endosymbiont Sinorhizobium meliloti enhances the growth of its host Medicago sativa (alfalfa), which also efficiently supplies the soil with nitrogen. Establishment of the S. meliloti-alfalfa symbiosis relies on the early exchange and recognition of chemical signals. The present study contributes to the disclosure of this complex molecular dialogue by investigating the underlying mechanisms of carboxylate sensing in S. meliloti. Understanding individual steps that govern the S. meliloti-alfalfa molecular cross talk helps in the development of efficient, commercial bacterial inoculants that promote the growth of alfalfa, which is the most cultivated forage legume in the world, and improves soil fertility.

Sinorhizobium meliloti Chemoreceptor McpV Senses Short-Chain Carboxylates via Direct Binding.

Sinorhizobium meliloti is a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors have been characterized, with McpU and McpX serving as general amino acid and quaternary ammonium compound sensors, respectively. Both receptors use a dual Cache (calcium channels and chemotaxis receptors) domain for ligand binding. We identified that the ligand-binding periplasmic region (PR) of McpV contains a single Cache domain. Homology modeling revealed that McpVPR is structurally similar to a sensor domain of a chemoreceptor with unknown function from Anaeromyxobacter dehalogenans, which crystallized with acetate in its binding pocket. We therefore assayed McpV for carboxylate binding and S. meliloti for carboxylate sensing. Differential scanning fluorimetry identified 10 potential ligands for McpVPR Nine of these are monocarboxylates with chain lengths between two and four carbons. We selected seven compounds for capillary assay analysis, which established positive chemotaxis of the S. meliloti wild type, with concentrations of peak attraction at 1 mM for acetate, propionate, pyruvate, and glycolate, and at 100 mM for formate and acetoacetate. Deletion of mcpV or mutation of residues essential for ligand coordination abolished positive chemotaxis to carboxylates. Using microcalorimetry, we determined that dissociation constants of the seven ligands with McpVPR were in the micromolar range. An McpVPR variant with a mutation in the ligand coordination site displayed no binding to isobutyrate or propionate. Of all the carboxylates tested as attractants, only glycolate was detected in alfalfa seed exudates. This work examines the relevance of carboxylates and their sensor to the rhizobium-legume interaction.IMPORTANCE Legumes share a unique association with certain soil-dwelling bacteria known broadly as rhizobia. Through concerted interorganismal communication, a legume allows intracellular infection by its cognate rhizobial species. The plant then forms an organ, the root nodule, dedicated to housing and supplying fixed carbon and nutrients to the bacteria. In return, the engulfed rhizobia, differentiated into bacteroids, fix atmospheric N2 into ammonium for the plant host. This interplay is of great benefit to the cultivation of legumes, such as alfalfa and soybeans, and is initiated by chemotaxis to the host plant. This study on carboxylate chemotaxis contributes to the understanding of rhizobial survival and competition in the rhizosphere and aids the development of commercial inoculants.

Sinorhizobium meliloti Chemotaxis to Multiple Amino Acids Is Mediated by the Chemoreceptor McpU.

The legume symbiont Sinorhizobium meliloti is chemoattracted to compounds exuded by germinating seeds of its host alfalfa. This response is mainly mediated by the S. meliloti chemoreceptor McpU. McpU also has a prominent contribution in sensing a synthetic amino acid (aa) mixture mimicking the amounts and composition observed in seed exudate. Here, we used the hydrogel capillary assay to quantify chemotactic responses of S. meliloti to individual aa exuded by germinating alfalfa seeds and to define the role of McpU in this behavior. S. meliloti exhibited positive chemotaxis responses to all proteinogenic aa, except for aspartate, and to citrulline, cystine, gamma-aminobutyric acid, and ornithine. Wild-type responses were diverse in intensity, while a strain lacking mcpU displayed strongly diminished responses. Differential scanning fluorimetry demonstrated interaction of the purified periplasmic region of McpU (McpU-PR) with the aa, except glutamate and aspartate. We additionally tested organic acids and sugars, but there were no significant interactions with the McpU ligand-binding domain, except for citrate. Using ligand displacement, we confirmed the interaction of McpU-PR with aa representing strong and weak attractants. Our results show that S. meliloti McpU is a broad-range aa receptor mediating differential responses to individual attractants, which does not bind negatively charged aa.

Complete Genome Sequence of Curtobacterium sp. Isolated from Surface-Sterilized Germinating Alfalfa Seeds.

We reported the complete genome sequence of a member of the pathogenic Curtobacterium genus. The sample includes a circular 3,955-kb chromosome, a 164-kb megaplasmid and a 42-kb plasmid. This strain was isolated from surface-sterilized alfalfa seeds.

Rhizobial Chemoattractants, the Taste and Preferences of Legume Symbionts.

The development of host-microbe interactions between legumes and their cognate rhizobia requires localization of the bacteria to productive sites of initiation on the plant roots. This end is achieved by the motility apparatus that propels the bacterium and the chemotaxis system that guides it. Motility and chemotaxis aid rhizobia in their competitiveness for space, resources, and nodulation opportunities. Here, we examine studies on chemotaxis of three major model rhizobia, namely Sinorhizobium meliloti, Rhizobium leguminosarum, and Bradyrhizobium japonicum, cataloging their range of attractant molecules and correlating this in the context of root and seed exudate compositions. Current research areas will be summarized, gaps in knowledge discussed, and future directions described.

An Updated Perspective on Sinorhizobium meliloti Chemotaxis to Alfalfa Flavonoids.

The symbiotic interaction between leguminous plants and their cognate rhizobia allows for the fixation of gaseous dinitrogen into bioavailable ammonia. The perception of host-derived flavonoids is a key initial step for the signaling events that must occur preceding the formation of the nitrogen-fixing organ. Past work investigating chemotaxis - the directed movement of bacteria through chemical gradients - of Bradyrhizobium japonicum, Rhizobium leguminosarum, and Rhizobium meliloti discovered chemotaxis to various organic compounds, but focused on chemotaxis to flavonoids because of their relevance to the symbiosis biochemistry. The current work sought to replicate and further examine Sinorhizobium (Ensifer) meliloti chemotaxis to the flavonoids previously thought to act as the principal attractant molecules prior to the initial signaling stage. Exudate from germinating alfalfa seedlings was analyzed for composition and quantities of different flavonoid compounds using mass spectrometry. The abundance of four prevalent flavonoids in germinating alfalfa seed exudates (SEs) was at a ratio of 200:5:5:1 for hyperoside, luteolin, luteolin-7-glucoside, and chrysoeriol. Using quantitative chemotaxis capillary assays, we did not detect chemotaxis of motile S. meliloti cells to these, and two other flavonoids identified in seed exudates. In support of these findings, the flavonoid fraction of seed exudates was found to be an insignificant attractant relative to the more hydrophilic fraction. Additionally, we observed that cosolvents commonly used to dissolve flavonoids confound the results. We propose that the role flavonoids play in S. meliloti chemotaxis is insignificant relative to other components released by alfalfa seeds.