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

Abstract

A Brevibacterium ammoniagenes gene coding for glucose/mannose-specific enzyme II (EII(Glc)) 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 polypeptid of 661 amino acid residues and a TAA stop codon. The deduced amino acid sequence of the B. ammoniagenes EII(Glc) shows, at 46%, the highest degree of sequence similarity with the Corynebacterium glutamicum EII specific for both glucose and mannose. In addition, the EII(Glc) shares approximately 30% sequence similarities with sucrose-specific and β- glucoside-specific EIIs of the several bacteria belonging to the glucose-PTS class. The 161-amino-acid C-terminal sequence of EII(Glc) is also similar to that of E. coli enzyme IIA(Glc), specific for glucose (EIIA(Glc)). The B. ammoniagenes EII(Glc) consists of three domains; a hydrophobic region (EIIC) and two hydrophilic regions (EIIA, EIIB). The arrangement of structural domains, IIBCA, of the EII(Glc) is identical to those of EIIs specific for sucrose or β-glucoside. While the domain IIA was removed from the B. ammoniagenes EII(Glc), the remaining domains IIBC were found to restore the glucose and mannose-utilizing capacity of E. coli mutant lacking EII(Glc) activity with EIIA(Glc) of the E. coli mutant. EII(Glc) contains a histidine residue and a cysteine residue which are putative phosphorylation sites for the protein.