CLONING AND EXPRESSION OF THE GENE ENCODING GLUCOSE PERMEASE OF THE PHOSPHOTRANSFERASE SYSTEM FROM BREVIBACTERIUM-FLAVUM IN ESCHERICHIA-COLI

A Brevibacterium flavum gene coding for glucose permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned by complementing the Eschenichia coli ZSC113 mutations affecting a ptsG gene with the B. flavum genomic library. From the E. coli clone grown as red colony on a Mac-Conkey plate supplemented with glucose as an additional carbon source, a recombinant plasmid was isolated and named pBFT93. The plasmid pBFT93 was identified as carrying a 3.6-kb fragment of B. flavum chromosomal DNA which enables the E. coli transformant to use glucose or mannose as a sale carbon source in an M9 minimal medium. The non-metabolizable sugar analogues, 2-deoxy-D-glucose (2-DG) and methyl-alpha-D-glucopyranoside (MeGlc) affected the growth of ZSC113 cells carrying the plasmid pBFT93 on minimal medium supplemented with non-PTS carbohydrate, glycerol, as a sole cabon source, while the analogues did not repress the growth of ZSC113 cells without pBFT93. It was also found that both 2-deoxy-D-[U-C-14]glucose and methyl-alpha-D-[U-C-14]glucopyranoside could be effectively transported into ZSC113 cells transformed with plasmid pBFT93. Several in vivo complementation studies suggested that the B. flavum DNA in pBFT93 encodes a glucose permease specific for glucose and mannose.