BINDING-PROTEIN DEPENDENT TRANSPORT OF GLYCINE BETAINE AND ITS OSMOTIC REGULATION IN ESCHERICHIA-COLI-K12

Glycine betaine, which functions as an osmoprotectant, is accumulated to high intracellular concentrations in Escherichia coli at high osmolarity. We demonstrate the presence of a high-affinity, binding protein dependent transport system for glycine betaine, which is encoded by the proU region. We show the osmotically regulated synthesis of a 32 kDa periplasmic protein that is a glycine betaine binding protein with a KD of 1.4 .mu.M. ProU-mediated glycine betaine transport is osmotically stimulated at the level of gene expression. The osmolarity of the medium also regulates the activity of the transport system, while binding of glycine betaine to its binding protein is independent of the osmolarity. We also find a second glycine betaine transport system that is dependent on proP and exhibits a lower substrate affinity. Like ProU, this system is regulated at two levels; both gene expression and the activity of the transport system are osmotically stimulated. Using .lambda.placMu-generated lacZ operon gene fusions, we find that expression of the proU regions is osmotically regulated at the level of transcription. We cloned a part of the proU region together with the .PHI.(proU-lacZ)hyb2 gene fusion into a multicopy plasmid and show that the DNA sequences required in cis for osmotic regulation are present on the plasmid.