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Abstract Sinorhizobium melilotisenses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novelS. melilotichemotaxis protein CheT. While CheT and theEscherichia coliphosphatase CheZ share little sequence homology, CheT is predicted to possess an α‐helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate‐sink response regulator, CheY1~P by enhancing its decay two‐fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a K_Dof 2.9 μM, which is 25‐fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheTmutant andcheTDXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a K_Dof 19 μM, and a SEC‐MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation. 

ABSTRACT Chemotaxis and motility are important traits that support bacterial survival in various ecological niches and in pathogenic and symbiotic host interaction. Chemotactic stimuli are sensed by chemoreceptors or m ethyl-accepting c hemotaxis p roteins (MCPs), which direct the swimming behavior of the bacterial cell. In this study, we present evidence that the cellular abundance of chemoreceptors in the plant symbiont Sinorhizobium meliloti can be altered by the addition of several to as few as one amino acid residues and by including common epitope tags such as 3×FLAG and 6×His at their C termini. To further dissect this phenomenon and its underlying molecular mechanism, we focused on a detailed analysis of the amino acid sensor McpU. Controlled proteolysis is important for the maintenance of an appropriate stoichiometry of chemoreceptors and between chemoreceptors and chemotactic signaling proteins, which is essential for an optimal chemotactic response. We hypothesized that enhanced stability is due to interference with protease binding, thus affecting proteolytic efficacy. Location of the protease recognition site was defined through McpU stability measurements in a series of deletion and amino acid substitution mutants. Deletions in the putative protease recognition site had similar effects on McpU abundance, as did extensions at the C terminus. Our results provide evidence that the programmed proteolysis of chemotaxis proteins in S. meliloti is cell cycle regulated. This posttranslational control, together with regulatory pathways on the transcriptional level, limits the chemotaxis machinery to the early exponential growth phase. Our study identified parallels to cell cycle-dependent processes during asymmetric cell division in Caulobacter crescentus . IMPORTANCE The symbiotic bacterium Sinorhizobium meliloti contributes greatly to growth of the agriculturally valuable host plant alfalfa by fixing atmospheric nitrogen. Chemotaxis of S. meliloti cells toward alfalfa roots mediates this symbiosis. The present study establishes programmed proteolysis as a factor in the maintenance of the S. meliloti chemotaxis system. Knowledge about cell cycle-dependent, targeted, and selective proteolysis in S. meliloti is important to understand the molecular mechanisms of maintaining a suitable chemotaxis response. While the role of regulated protein turnover in the cell cycle progression of Caulobacter crescentus is well understood, these pathways are just beginning to be characterized in S. meliloti . In addition, our study should alert about the cautionary use of epitope tags for protein quantification. 

Chemotaxis systems enable microbes to sense their immediate environment, moving towards beneficial stimuli and away from those that are harmful. In an effort to better understand the chemotaxis system of Sinorhizobium meliloti , a symbiont of the legume alfalfa, the cellular stoichiometries of all ten chemotaxis proteins in S. meliloti were determined. A combination of quantitative immunoblot and mass spectrometry revealed that the protein stoichiometries in S. meliloti varied greatly from those in Escherichia coli and Bacillus subtilis . To compare protein ratios to other systems, values were normalized to the central kinase CheA. All S. meliloti chemotaxis proteins exhibited increased ratios to varying degrees. The ten-fold higher molar ratio of adaptor proteins CheW1 and CheW2 to CheA might result in the formation of rings in the chemotaxis array that only consist of CheW instead of CheA and CheW in a 1:1 ratio. We hypothesize that the higher ratio of CheA to the main response regulator CheY2 is a consequence of the speed-variable motor in S. meliloti , instead of a switch-type motor. Similarly, proteins involved in signal termination are far more abundant in S. meliloti , which utilizes a phosphate-sink mechanism based on CheA retro-phosphorylation to inactivate the motor response regulator versus CheZ-catalyzed dephosphorylation as in E. coli and B. subtilis . Finally, the abundance of CheB and CheR, which regulate chemoreceptor methylation, was increased when compared to CheA, indicative of variations in the adaptation system of S. meliloti . Collectively, these results mark significant differences in the composition of bacterial chemotaxis systems. IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti contributes greatly to host-plant growth by fixing atmospheric nitrogen. The provision of nitrogen as ammonium by S. meliloti leads to increased biomass production of its legume host alfalfa and diminishes the use of environmentally harmful chemical fertilizers. To better understand the role of chemotaxis in host-microbe interaction, a comprehensive catalogue of the bacterial chemotaxis system is vital, including its composition, function, and regulation. The stoichiometry of chemotaxis proteins in S. meliloti has very few similarities to the systems in E. coli and B. subtilis . In addition, total amounts of proteins are significantly lower. S. meliloti exhibits a chemotaxis system distinct from known models by incorporating new proteins as exemplified by the phosphate sink mechanism.