Azorhizobium caulinodans is a nitrogen-fixing bacterium that forms root nodules on its host legume, Sesbania rostrata . This agriculturally significant symbiotic relationship is important in lowland rice cultivation, and allows for nitrogen fixation under flood conditions. Chemotaxis plays an important role in bacterial colonization of the rhizosphere. Plant roots release chemical compounds that are sensed by bacteria, triggering chemotaxis along a concentration gradient toward the roots. This gives motile bacteria a significant competitive advantage during root surface colonization. Although plant-associated bacterial genomes often encode multiple chemotaxis systems, A. caulinodans appears to encode only one. The che cluster on the A. caulinodans genome contains cheA , cheW , cheY2 , cheB , and cheR . Two other chemotaxis genes, cheY1 and cheZ , are located independently from the che operon. Both CheY1 and CheY2 are involved in chemotaxis, with CheY1 being the predominant signaling protein. A. caulinodans CheA contains an unusual set of C-terminal domains: a CheW-like/Receiver pair (termed W2-Rec), follows the more common single CheW-like domain. W2-Rec impacts both chemotaxis and CheA function. We found a preference for transfer of phosphoryl groups from CheA to CheY2, rather than to W2-Rec or CheY1, which appears to be involved in flagellar motor binding. Furthermore, we observed increased phosphoryl group stabilities on CheY1 compared to CheY2 or W2-Rec. Finally, CheZ enhanced dephosphorylation of CheY2 substantially more than CheY1, but had no effect on the dephosphorylation rate of W2-Rec. This network of phosphotransfer reactions highlights a previously uncharacterized scheme for regulation of chemotactic responses. IMPORTANCE Chemotaxis allows bacteria to move towards nutrients and away from toxins in their environment. Chemotactic movement provides a competitive advantage over non-specific motion. CheY is an essential mediator of the chemotactic response with phosphorylated and unphosphorylated forms of CheY differentially interacting with the flagellar motor to change swimming behavior. Previously established schemes of CheY dephosphorylation include action of a phosphatase and/or transfer of the phosphoryl group to another receiver domain that acts as a sink. Here, we propose A. caulinodans uses a concerted mechanism in which the Hpt domain of CheA, CheY2, and CheZ function together as a dual sink system to rapidly reset chemotactic signaling. To the best of our knowledge, this mechanism is unlike any that have previously been evaluated. Chemotaxis systems that utilize both receiver and Hpt domains as phosphate sinks likely occur in other bacterial species.
BackgroundMany bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners.ResultsWe report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1.ConclusionsPhosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator.Electronic supplementary materialThe online version of this article (10.1186/s12858-019-0104-5) contains supplementary material, which is available to authorized users.
Two-component signaling is a primary method by which microorganisms interact with their environments. A kinase detects stimuli and modulates autophosphorylation activity. The signal propagates by phosphotransfer from the kinase to a response regulator, eliciting a response. Response regulators operate over a range of time scales, corresponding to their related biological processes. Response regulator active site chemistry is highly conserved, but certain variable residues can influence phosphorylation kinetics. An Ala-to-Pro substitution (K+4, residue 113) in the Escherichia coli response regulator CheY triggers a constitutively active phenotype; however, the A113P substitution is too far from the active site to directly affect phosphochemistry. To better understand the activating mechanism(s) of the substitution, we analyzed receiver domain sequences to characterize the evolutionary role of the K+4 position. Although most featured Pro, Leu, Ile, and Val residues, chemotaxis-related proteins exhibited atypical Ala, Gly, Asp, and Glu residues at K+4. Structural and in silico analyses revealed that CheY A113P adopted a partially active configuration. Biochemical data showed that A113P shifted CheY toward a more activated state, enhancing autophosphorylation. By characterizing CheY variants, we determined that this functionality was transmitted through a hydrophobic network bounded by the β5α5 loop and the α1 helix of CheY. This region also interacts with the phosphodonor CheA P1 , suggesting that binding generates an activating perturbation similar to the A113P substitution. Atypical residues like Ala at the K+4 position likely serve two purposes. First, restricting autophosphorylation may minimize background noise generated by intracellular phosphodonors such as acetyl phosphate. Second, optimizing interactions with upstream partners may help prime the receiver domain for phosphorylation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.