In chemotaxis by Bacillus subtilis, the information that attractant has bound to chemoreceptors is transduced along a pathway, which includes loss of methyl groups from the methyl-accepting chemotaxis proteins (MCPs), and ultimately results in a transient increase of counterclockwise rotation by the flagella. It is very important to trace this pathway, to discover what proteins are involved and what their functions are. B. subtilis is perhaps the best organism for this work since we have, using purified methyltransferase and methylesterase, shown that attractants retard methylation and strongly enhance demethylation of MCPs in vitro. Only in B. subtilis have these enzymes been purified to homogeneity. We have also developed two complementation systems and have obtained evidence for over twenty new genes. Thus, the stage is now set to investigate proteins that modulate methylation reactions. Using this system, we will explore the mechanism of several novel events induced by attractant: transfer of methyl groups between MCPs, turnover of methyl groups on MCPs, and transfer to an intermediate acceptor before formation of methanol. The last step is blocked by repellents and is the first biochemical handle on their mechanism of action. Isolation and characterization of mutants, including those containing a transposon that may express Beta-galactosidase, and cloning the chemotaxis genes will greatly assist us. Once the genes are cloned, we will carry out a restriction analysis of chromosomal segments and correlate the genetic and physical maps. We will express the genes in E. coli minicells as radioactive proteins so that we can ascertain their size and hydrophobicity and search for protein/protein interactions, especially with the switch, which controls direction of flagellar rotation. By second-site reversion studies, we will also seek evidence for such interactions. Our understanding of the MCPs, which represent our major biochemical handle on chemotaxis, is hampered by lack of mutants and insufficient gene expression and, using the techniques of molecular biology, we exprect to remedy this. Our knowledge, not only of the MCPs, but of over twenty other chemotaxis proteins, will be enhanced by mapping and complementing the mutants and by varying levels of genes expression. Armed with a uniquely suitable in vitro system for evaluation the effects of chemotaxis proteins, we believe that a renewed emphasis on genetics and molecular biology will prove especially fruitful.
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