Transcription of the Escherichia coli uhpT gene, which encodes an active transport system for sugar phosphates, is induced by extracellular, but not by intracellularly generated glucose-6- phosphate. The uhpT gene and the three contiguous regulatory genes have been cloned and their nucleotide sequence determined. All three regulatory genes are required for proper regulation. UhpA appears to be an activator of uhpT transcription, whereas UhpB and UhpC appear to act to prevent UhpA action in the absence of external inducer. Projects in this proposal address the regulation of this system through the isolation of the three regulatory proteins, and the exploration of their interactions with inducer, the membrane, each other, and the uhpT promoter region. Both biochemical and genetic approaches will be employed to investigate these interactions. Deletion and point mutations in the uhpT promoter region will be combined with footprinting analyses to determine the sites of binding of UhpA, RNA polymerase, and the cAMP receptor protein. Mutants that overcome the loss of one regulatory protein will be isolated in hopes that they will provide further information on the interaction of the regulatory components. Ultimately, attempts will be made to establish an in vitro transcription-translation system that reproduces proper regulation. Since the UhpT transporter has recently been shown to number function as a phosphate antiport, point mutations and fusions to the structural genes for alkaline phosphatase and beta-galactosidase will be isolated, with the goal of defining the transmembrane orientation of the protein. Studies will be addressed to the use of site- directed mutagenesis for identification of residues involved in transport function, substrate recognition, or energy coupling. It is anticipated that these studies will provide information about both the novel regulatory system and its transmembrane signalling, as well as about this novel mechanism of energy coupling.
