Previous sequence analysis of the glucose-specific PTS gene locus from revealed the unexpected finding of two adjacent, highly similar ORFs, and ,each encoding a glucose-specific membrane permease EIICBA. and show 73% identity at the nucleotide level and is located 131 bp downstream from . Each of the genes is flanked by putative regulatory elements such as a termination stem–loop, promoter and ribosome-binding site, suggesting independent regulation. The finding of putative -active operator sequences, CRE (catabolite-responsive elements) suggests additional regulation by carbon catabolite repression. As described previously by the authors, both genes can be expressed in under control of their own promoters. Two putative promoters are located upstream of , and both were found to initiate transcription in . Although the two permeases EIICBA1 and EIICBA2 show 69% identity at the protein level, and despite the common primary substrate glucose, they have different specificities towards glucosides as substrate. EIICBA1 phosphorylates glucose in a PEP-dependent reaction with a of 12 μM; the reaction can be inhibited by 2-deoxyglucose and methyl β-D-glucoside. EIICBA2 phosphorylates glucose with a of 19 μM and this reaction is inhibited by methyl α-D-glucoside, methyl β-D-glucoside, -nitrophenyl α-D-glucoside, -nitrophenyl β-D-glucoside and salicin, but unlike other glucose permeases, including EIICBA1, not by 2-deoxyglucose. Natural mono- or disaccharides, such as mannose or -acetylglucosamine, that are transported by other glucose transporters are not phosphorylated by either EIICBA1 nor EIICBA2, indicating a high specificity for glucose. Together, these findings support the suggestion of evolutionary development of different members of a protein family, by gene duplication and subsequent differentiation. C-terminal fusion of a histidine hexapeptide to both gene products did not affect the activity of the enzymes and allowed their purification by Ni-NTA affinity chromatography after expression in a (EIICB) deletion mutant of . Upstream of , the 3’ end of a further ORF encoding 138 amino acid residues of a putative antiterminator of the BglG family was found, as well as a putative target DNA sequence (RAT), which indicates a further regulation by glucose specific antitermination.


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