Cloning, nucleotide sequencing, and characterization of the ptsG gene encoding glucose-specific enzyme II of the phosphotransferase system from Brevibacterium lactofermentum

Abstract

A Brevibacterium lactofermentum gene coding for a glucose-specific permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was cloned, by complementing an Escherichia coli mutation affecting a ptsG gene with the B. lactofermentum genomic library, and completely sequenced. The gene was identified as a ptsG, which enables an E. coli transformant to transport non-metabolizable glucose analogue 2-deoxyglucose (2DG). The ptsG gene of B. lactofermentum consists of an open reading frame, of 2,025 nucleotides encoding a polypeptide of 674 amino acid residues and a TAA stop codon. The 3' flanking region contains two stem-loop structures which may be involved in transcriptional termination. The deduced amino acid sequence of the B. lactofermentum enzyme II(Glc) specific to glucose (EII(Glc)) has a high homology with the Corynebacterium glutamicum enzyme II(Man) specific to glucose and mannose (EII(Man)), and the Brevibacterium ammoniagenes enzyme II(Glc) specific to glucose (EII(Glc)). The 171-amino-acid C-terminal sequence of the EII(Glc) is also similar to the Escherichia coli enzyme IIA(Glc) specific to glucose (IIA(Glc)). It is interesting that the arrangement of the structural domains, IIBCA, of the B. lactofermentum EII(Glc) protein is identical to that of EIIs specific to sucrose or β- glucoside. Several in vivo complementation studies indicated that the B. lactofermentum EII(Glc) protein could replace both EII(Glc) and EIIA(Glc) in an E. coli ptsG mutant or crr mutant, respectively.