Construction and expression of hybrid plasmids containing the structural gene of the Escherichia coli K-12 3-deoxy-2-oxo-D-gluconate transport system

Abstract

The kdgT gene of Escherichia coli, which encodes for the 3-deoxy-2-oxo-D-gluconate transport system, was isolated as a ColE1-kdgT hybrid plasmid from the Clarke and Carbon bank. A restriction and genetic map of the min 88 region of the chromosome was established; by subcloning the restriction fragments into the plasmid vector pBR322, the kdgT gene was localized on a 1.4-megadalton PstI DNA fragment, and the direction of transcription of the gene was determined by making use of an in vitro gene fusion between kdgT and lacZ genes. Amplification of the gene product of kdgT was up to 14-fold the level found in a haploid strain. When plasmids bearing kdgT were expressed in an in vivo maxicell system, a specific polypeptide of 28,000 daltons appeared that was found to be associated with the membrane fraction.