Search for: All records

Abstract Sensory adaptation in bacterial chemotaxis is mediated by posttranslational modifications of methyl‐accepting chemotaxis proteins (MCPs). InEscherichia coli, the adaptation proteins CheR and CheB tether to a conserved C‐terminal receptor pentapeptide. Here,we investigated the function of the pentapeptide motif (N/D)WE(E/N)F inSinorhizobium melilotichemotaxis. Isothermal titration calorimetry revealed stronger affinity of the pentapeptides to CheR and activated CheB relative to unmodified CheB. Strains with mutations of the conserved tryptophan in one or all four MCP pentapeptides resulted in a significant decrease or loss of chemotaxis to glycine betaine, lysine, and acetate, chemoattractants sensed by pentapeptide‐bearing McpX and pentapeptide‐lacking McpU and McpV, respectively. Importantly, we discovered that the pentapeptide mediates chemotaxis when fused to the C‐terminus of pentapeptide‐lacking chemoreceptors via a flexible linker. We propose that adaptational assistance and a threshold number of available sites enable the efficient docking of adaptation proteins to the chemosensory array. Altogether, these results demonstrate thatS. melilotieffectively utilizes a pentapeptide‐dependent adaptation system with a minimal number of tethering units to assist pentapeptide‐lacking chemoreceptors and hypothesize that the higher abundance of CheR and CheB inS. meliloticompared toE. coliallows for ample recruitment of adaptation proteins to the chemosensory array. 

Abstract The flagellar motor is a powerful macromolecular machine used to propel bacteria through various environments. We determined that flagellar motility of the alpha‐proteobacteriumSinorhizobium melilotiis nearly abolished in the absence of the transcriptional regulator LdtR, known to influence peptidoglycan remodeling and stress response. LdtR does not regulate motility gene transcription. Remarkably, the motility defects of the ΔldtRmutant can be restored by secondary mutations in the motility genemotAor a previously uncharacterized gene in the flagellar regulon, which we namedmotS. MotS is not essential forS. melilotimotility and may serve an accessory role in flagellar motor function. Structural modeling predicts that MotS comprised an N‐terminal transmembrane segment, a long‐disordered region, and a conserved β‐sandwich domain. The C terminus of MotS is localized in the periplasm. Genetics based substitution of MotA with MotA_G12Salso restored the ΔldtRmotility defect. The MotA_G12Svariant protein features a local polarity shift at the periphery of the MotAB stator units. We propose that MotS may be required for optimal alignment of stators in wild‐type flagellar motors but becomes detrimental in cells with altered peptidoglycan. Similarly, the polarity shift in stator units composed of MotB/MotA_G12Smight stabilize its interaction with altered peptidoglycan. 

Abstract Chemotaxis is a fundamental process whereby bacteria seek out nutrient sources and avoid harmful chemicals. For the symbiotic soil bacteriumSinorhizobium 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. melilotipossesses 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. 

The bacterial flagellum is a complex macromolecular machine that drives bacteria through diverse fluid environments. Although many components of the flagellar motor are conserved across species, the roles of FliL are numerous and species-specific. Here, we have characterized an additional player required for flagellar motor function in Sinorhizobium meliloti, MotF, which we have identified as a FliL paralog. We performed a comparative analysis of MotF and FliL, identified interaction partners through bacterial two-hybrid and pull-down assays, and investigated their roles in motility and motor rotation. Both proteins form homooligomers, and interact with each other, and with the stator proteins MotA and MotB. The ∆motF mutant exhibits normal flagellation but its swimming behavior and flagellar motor activity are severely impaired and erratic. In contrast, the ∆fliL mutant is mostly aflagellate and nonmotile. Amino acid substitutions in cytoplasmic regions of MotA or disruption of the proton channel plug of MotB partially restored motor activity to the ∆motF but not the ∆fliL mutant. Altogether, our findings indicate that both, MotF and FliL, are essential for flagellar motor torque generation in S. meliloti. FliL may serve as a scaffold for stator integration into the motor, and MotF is required for proton channel modulation. 

ABSTRACT 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 McpT PR , 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 McpT PR 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. 

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.