Bacterial chemotaxis has been studied extensively. It serves as a model for responses to chemical stimuli in multicellular organisms and for the function of hormones and neurotransmitters. Chemotaxis is a virulence factor for bacteria. Understanding its underlying mechanisms may suggest ways to combat bacterial pathogens. Bacteria have two advantages for the study of signal reception and transmission. 1) They grow readily and are easily manipulated genetically and biochemically. 2) Their simple physiology permits direct observation of the phenotypic consequences of mutations. Furthermore, mutations that eliminate motility or taxis are rarely lethal. This proposal outlines research that extends work from the previous grant period. The study of interactions between the maltose-binding protein (MBP) and dipeptide-binding protein (DBP) and their cognate receptors in the cytoplasmic membrane (the Tar and Tap proteins) continues. MBP crosslinked between its N- and C-terminal domains is negatively dominant over wild-type MBP. The crosslinked MBP can be used in combination with mutations causing specific defects in maltose taxis to determine if these mutations interfere with binding of MBP to Tar or with subsequent initiation of a signal. Enough is known about how MBP binds to Tar to perform site-directed cysteine mutagenesis to create an intermolecular crossbridge between the two proteins in vivo. This achievement will usher in a series of experiments, including production of MBP-Tar co-crystals for X-ray analysis. The recently published crystal structure of DBP can be compared to that of MBP and used to direct mutational studies of DBP to determine if the DBP/Tap interaction is similar to the better-understood MBP/Tar interaction. Attention now turns to later steps in chemotactic signaling. Specific hypotheses about how small ligands and binding proteins initiate transmembrane signaling will be tested. Attractants inhibit receptor-mediated stimulation of CheA kinase and thus block synthesis of the tumble regulator phospho-CheY. How this inhibition accounts for behavioral responses remains obscure, since a change in net receptor occupancy of <1% generates a physiologically significant signal. Thus, amplification of an attractant signal remains a nettlesome problem. The observation that receptors cluster in the cell combined with new information about the transducers themselves suggests that amplification occurs via trans-inactivation . An attractant-bound receptor may propagate a signal that inhibits CheA kinase, or stimulates CheZ phosphatase within a receptor patch . Trans-inactivation may also explain signaling by the Tap and Trg receptors, which themselves have less ability to stimulate CheA in vivo.
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