The genetic expression of the phosphoglycerate transport system of S. typhimurium is induced by external inducers but not by internal inducers. The long-term objectives of the proposed research are to gentically and biochemically characterize the regulatory machinery involved in gene expression of this transport system and to understand the biochemical and molecular mechanism underlying this novel gene regulation. During the current grant period we dissected the genetic regulatory machinery, determined the DNA sequence, identified gene products and determined the cellular locations of these products.
The specific aims of the present proposal are: to isolate, purify and characterize the pgt regulatory proteins; to study the regulatory mechanism in in vitro transcription-translation coupled system in order to determine the functional role of each regulatory proteins with respect to inducer binding, DNA binding and signal transmission; to analyze pgt transcripts and determine pgtP transcription initiation start and DNA binding regions, in order to distinguish two models of regulation and to define the control regions; to isolate constitutive regulatory mutants by genetic selection and site-directed mutagenesis and to characterize the mutant proteins; to study processing of regulatoryp proteins and determine the cleavage sites; and to perform chemical cross-linking experiments to investigate the interaction of the regulatory proteins.
