Cloning, expression, and nucleotide sequencing of the gene encoding glucose permease of phosphotransferase system from Brevibacterium ammoniagenes

A Brevibacterium ammoniagenes gene coding for glucose/mannose-specific enzyme II (EIIGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned by complementing an Escherichia coli mutation affecting a ptsG gene, and the complete DNA nucleotide sequence was determined. The cloned gene was identified to be a ptsG, which enables the E. coli transformant to use glucose more efficiently than mannose as the sole carbon source in an M9 minimal medium. The ptsG gene of B. ammoniagenes consists of an open reading frame of 1,983 nucleotides putatively encoding a polypeptide of 661 amino acid residues and a TAA stop codon. The deduced amino acid sequence of the B. ammoniagenes EIIGlc shows, at 46%, the highest degree of sequence similarity with the Corynebacterium glutamicum EII specific for both glucose and mannose. In addition, the EIIGlc shares approximately 30% sequence similarities with sucrose-specific and beta-glucoside-specific EIIs of the several bacteria belonging to the glucose-PTS class. The 161-amino-acid C-terminal sequence of EIIGlc is also similar to that of E. coli enzyme IIA(Glc), specific for glucose (EIIA(Glc)). The B. ammoniagenes EIIGlc consists of three domains; a hydrophobic region (EIIC) and two hydrophilic regions (EIIA, EIIB). The arrangement of structural domains, IIBCA, of the EIIGlc is identical to those of EIIs specific for sucrose or beta-glucoside. While the domain IIA was removed from the B. ammoniagenes EIIGlc, the remaining domains IIBC were found to restore the glucose and mannose-utilizing capacity of E. coli mutant lacking EIIGlc activity with EIIA(Glc) of the E. coli mutant. EIIGlc contains a histidine residue and a cysteine residue which are putative phosphorylation sites for the protein.