Molecular cloning of the uhp region and evidence for a positive activator for expression of the hexose phosphate transport system of Escherichia coli.

Abstract

The uhp locus of Escherichia coli contains genes for the sugar phosphate transport system (uhpT) and the regulatory system which allows its induction by external glucose 6-phosphate (uhpRA). The uhp region was cloned onto high-copy-number plasmids, both from Uhp(+) plasmids of the Clarke-Carbon collection and from genetically characterized specialized transducing phages carrying uhpT-lac operon fusions. Two Clarke-Carbon plasmids and their Uhp(+) subclones in pBR322 shared restriction sites defining the uhp region, but exhibited different regulation of Uhp expression and dependence on chromosomal uhp genotype. Plasmid pLC17-47 and derivatives conferred constitutive glucose 6-phosphate uptake activity in all strains, even those with complete deletions of uhp. These plasmids also rendered constitutive the expression of a chromosomal uhpT-lac operon fusion. Plasmid pLC40-33 conferred inducible Uhp expression, which required the presence of the uhpA(+) gene on the chromosome. The induced transport levels in all strains carrying these plasmids were not appreciably amplified over haploid levels. Similar behavior was seen with the cloned operon fusions. A fusion-bearing plasmid that carried an intact regulatory system (uhpR(+)A(+)) exhibited trans-dominant constitutive expression of beta-galactosidase, regardless of the chromosomal uhp genotype. In contrast, the cloned fusion carrying only uhpR(+) gave glucose 6-phosphate-inducible production of beta-galactosidase that was dependent on the presence of chromosomal uhpA(+). Expression of both fusions in the haploid state was inducible. From these results, it was concluded that the uhpA product is necessary for uhpT transcription and that elevated dosage of uhpA results in at least partially constitutive expression of uhpT. A tentative model for uhp regulation is presented.