Cloning and expression of the glycerol-3-phosphate transport genes of Escherichia coli

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Abstract
The 2000 E. coli:Col E1 hybrid plasmid strains of the Clarke and Carbon colony bank were screened by conjugation for those that correct the deficiency of a mutant unable to transport glycerol-3-phosphate. Six strains harboring recombinant plasmids carrying the glpT region were identified and characterized with respect to plasmid size and transport properties. The initial rate of glycerol-3-phosphate transport in both whole cells and membrane vesicles prepared from such strains was elevated 3- to 10-fold over strains carrying random DNA inserts, but Km of glycerol-3-phosphate transport was near 12 .mu.M in both experimental and control strains. Four of the 6 glpT carrying plasmid strains demonstrated elevated levels of the anaerobic glycerol-3-phosphate dehydrogenase coded for by the neighboring glpA gene. The glpT hybrid plasmids were transferred into a minicell-producing strain of E. coli X1197 and the minicells were used for specific in vitro synthesis of plasmid-coded proteins. The glpT plasmids code for a 40,000 polypeptide which is localized in the periplasmic space. The also code for a membrane-associated protein of 26,000 which may be the carrier polypeptide.