Site-specific methylation in Bacillus subtilis chemotaxis: effect of covalent modifications to the chemotaxis receptor McpB

The Bacillus subtilis chemotaxis pathway employs a receptor methylation system that functions differently from the one in the canonical Escherichia coli pathway. Previously, we hypothesized that B. subtilis employs a site-specific methylation system for adaptation where methyl groups are added and removed at different sites. This study investigated how covalent modifications to the adaptation region of the chemotaxis receptor McpB altered its apparent affinity for its cognate ligand, asparagine, and also its ability to activate the CheA kinase. This receptor has three closely spaced adaptation sites located at residues Gln371, Glu630 and Glu637. We found that amidation, a putative methylation mimic, of site 371 increased the receptor's apparent affinity for asparagine and its ability to activate the CheA kinase. Conversely, amidation of sites 630 and 637 reduced the receptor's ability to activate the kinase but did not affect the apparent affinity for asparagine, suggesting that activity and sensitivity are independently controlled in B. subtilis. We also examined how electrostatic interactions may underlie this behaviour, using homology models. These findings further our understanding of the site-specific methylation system in B. subtilis by demonstrating how the modification of specific sites can have varying effects on receptor function

Characterization of the Chemoreceptors in the Bacillus Subtilis Chemotaxis System

146 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.Physical characterization of the chemoreceptors was probed by identification of a zinc ion binding area, using isothermal titration calorimetry. In addition, in vivo chemotactic assays showed that altering the charges of the methylation sites on the chemoreceptors can have a significant effect on the activity of the receptor. Furthermore, individual methylation sites were shown to be responsible for both changes in the affinity of the chemoreceptor for attractant and receptor activation. These findings suggest a novel, more complicated role for methylation than previously suggested, and lead to a clearer picture of the coordination of the three adaptational systems in the B. subtilis chemotactic sensory transduction.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Characterization of the Chemoreceptors in the Bacillus Subtilis Chemotaxis System

146 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.Physical characterization of the chemoreceptors was probed by identification of a zinc ion binding area, using isothermal titration calorimetry. In addition, in vivo chemotactic assays showed that altering the charges of the methylation sites on the chemoreceptors can have a significant effect on the activity of the receptor. Furthermore, individual methylation sites were shown to be responsible for both changes in the affinity of the chemoreceptor for attractant and receptor activation. These findings suggest a novel, more complicated role for methylation than previously suggested, and lead to a clearer picture of the coordination of the three adaptational systems in the B. subtilis chemotactic sensory transduction.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

The importance of the interaction of CheD with CheC and the chemoreceptors compared to its enzymatic activity during chemotaxis in Bacillus subtilis.

Bacillus subtilis use three systems for adaptation during chemotaxis. One of these systems involves two interacting proteins, CheC and CheD. CheD binds to the receptors and increases their ability to activate the CheA kinase. CheD also binds CheC, and the strength of this interaction is increased by phosphorylated CheY. CheC is believed to control the binding of CheD to the receptors in response to the levels of phosphorylated CheY. In addition to their role in adaptation, CheC and CheD also have separate enzymatic functions. CheC is a CheY phosphatase and CheD is a receptor deamidase. Previously, we demonstrated that CheC's phosphatase activity plays a minor role in chemotaxis whereas its ability to bind CheD plays a major one. In the present study, we demonstrate that CheD's deamidase activity also plays a minor role in chemotaxis whereas its ability to bind CheC plays a major one. In addition, we quantified the interaction between CheC and CheD using surface plasmon resonance. These results suggest that the most important features of CheC and CheD are not their enzymatic activities but rather their roles in adaptation

GST pulldowns with CheD’s two binding partners (CheC and McpAc) show reduced binding by GST-CheD-F102E.

<p>Lanes 1 (GST-CheD only control), 2 (+CheC), 3 (+McpAc) and 4 (+McpCc) contain GST-CheD bound to glutathione beads. Lanes 5 (GST-CheD-F102E only control), 6 (+CheC), 7 (+McpAc) and 8 (+McpCc) contains GST-CheD-F102E bound to glutathione beads. Multiple bands of the Mcps correspond to receptor deamidation.</p

Western blots of <i>in vitro</i> deamidation assays of McpAc show CheD-mediated receptor modifications.

<p>Deamidation of the receptor fragment results in higher-migrating bands on a polyacrylamide gel. CheC is added, as indicated, and inhibits deamidation. Lanes 2 and 3 show McpAc incubated with wild-type GST-CheD, and lanes 4–11 show McpAc incubated with various GST-CheD mutants.</p

Kinetic parameters of CheC-CheD binding from SPR experiments.

<p>Kinetic parameters of CheC-CheD binding from SPR experiments.</p

A CheC/CheD structural model based on the <i>T. maritima</i> crystal structure (PDB ID 2F9Z) shows residues at the binding interface [<b>21</b>].

<p>Residues M101 and F102 on CheD are highlighted in cyan, and residue D149 on CheC is highlighted in yellow.</p