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

Chemotaxis Methyltransferase Ii From Bacillus Subtilis

156 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1983.Chemotaxis is the process by which motile cells sense and respond to their chemical environment. Following a period of time during which chemotactic bacteria respond to chemical stimulation, they revert to their pre-stimulus behavior, or adapt. Biochemically, adaptation is correlated with the methylation or demethylation of certain integral membrane proteins, the methyl-accepting chemotaxis proteins (MCPs). We have purified and characterized a chemotaxis methyltransferase (methyltransferase II) from Bacillus subtilis. By using a combination of DEAE Bio-Gel A chromatography, CM Bio-Gel A chromatography, ammonium sulfate fractionation, and S-adenosylhomocysteine affinity chromatography, a virtually homogeneous product was obtained.The enzyme was shown to be a monomer with a native molecular weight of 30,000, a K(,m) for S-adenosylmethionine of 5 (mu)M, and a K(,i) for S-adenosylhomocysteine of 0.2 (mu)M. The MCP methylation reaction in vitro was activated by divalent cation and inhibited by NaCl or KCl. It had a pH optimum of 7 and a temperature optimum of 20-25(DEGREES)C. The linkage between the MCP and the transferred methyl group was preliminarily characterized as gammaglutamyl methyl ester.Methyltransferase II appeared to have the ability to gain access to the intracellular space in permeabilization assays. It was also determined that a 68,000 molecular weight cytoplasmic protein (with two sub-units) could serve as a methyltransferase II substrate. The cytoplasmic protein was partially purified and characterized.Finally, functional homology between Bacillus subtilis methyl-transferase II and Escherichia coli cheR protein was established.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Simplified testing for antibodies to human immunodeficiency virus.

Test strips for the detection of antibodies to human immunodeficiency virus type 1 were investigated using specimens from risk groups in Thailand (141 reactive; 445 nonreactive) in a local Thai laboratory. The diagnostic sensitivity and specificity were both 100%. Using a set of seroconversion panels, the sensitivity of the test strips was within the range of sensitivities obtained with enzyme immunoassays. The test was developed for performance at decentralized settings under nonlaboratory conditions