Biochemical and molecular characterization of methyl-accepting chemotaxis proteins from Bacillus subtilis

Bacterial chemotaxis may be considered the archetype for the behavioral response by organisms to environmental stimuli. The movement of bacteria toward various chemical substances depends upon the ability of the organism to detect perturbations within the environment. A transmembrane signalling system mediates these behavioral changes by transducing the extracellular information received by these receptor proteins to the cytoplasm, such that the swimming behavior of the bacteria is altered appropriately. These transducers, or methyl-accepting chemotaxis proteins (MCPs) have been purified and reconstituted in a functionally active conformation from the gram positive bacterium, Bacillus subtilis. This forms the foundation for the establishment of an in vitro system using other purified chemotactic components. Four transducer genes were subsequently cloned and sequenced from a lambda DNA library using degenerate oligonucleotide probes which were designed based upon a conserved domain which is common to chemotactic receptors from enteric bacteria. It was initially hypothesized that this domain would also remain conserved among MCPs from B. subtilis. We speculate that this approach would be applicable for cloning transducer genes from other organisms. Inactivation of two of these transducer genes, designated mcpA and mcpB, reveals specific chemotactic defects towards sugars or amino acids. Despite strong homology, the two additional genes do not appear to be involved in chemotaxis. Based upon the morphological phenotype of these mutant bacteria, it is speculated that these transducer-like genes, tlpA and tlpB, may play a role in cell-to-cell communication. Furthermore, a previously unidentified methyl-accepting protein (MAP62) was characterized, but despite several characteristic MCP qualities, does not play an obvious role in the chemotactic process. Finally, a characterization of the cytoplasmic chemotaxis protein, CheW, reveals that this protein is necessary for efficient chemotaxis. In Escherichia coli, CheW is believed to couple events at the receptors with changes in the rate of CheA autophosphorylation and subsequent phosphoryl transfer to propagate the signal to the flagella. In B. subtilis, however, CheW is not required to mediate the effects of a chemoeffector binding at the MCPs with the biochemical changes associated with sensory transduction.U of I OnlyETDs are only available to UIUC Users without author permissio