Cloning and Expression of the Gene Encoding Glucose Permease of the Phosphotransferase System from Brevibacterium flavum in Escherichia coli

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 Escherichia coli ZSCl13 mutations affecting a ptsG gene with the B. flavum genomic library. From the E. coli clone grown as red colony on a MacConkey 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 man nose as a sole 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 ZSCl13 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 ZSCl13 cells without pBFT93. It was also found that both $2-deoxy-D-[U-^{14}C]glucose{\;}and{\;}methyl-{\alpha}-D-[U-^{14}C]glucopyranoside$ could be effectively transported into ZSCl13 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.