Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system - two highly similar glucose permeases in Staphylococcus carnosus with different glucoside specificity: protein engineering in vivo

Previous sequence analysis of the glucose-specific PTS gene locus from Stap hylococcus carnosus revealed the unexpected finding of two adjacent, highly similar ORFs, glcA and glcB, each encoding a glucose-specific membrane per mease EIICBA(Glc). glcA and glcB show 73 % identity at the nucleotide level and glcB is located 131 bp downstream from glcA. Each of the genes is flan ked by putative regulatory elements such as a termination stem-loop, promot er and ribosome-binding site, suggesting independent regulation. The findin g of putative cia-active operator sequences, CRE (catabolite-responsive ele ments) suggests additional regulation by carbon catabolite repression. As d escribed previously by the authors, both genes can be expressed in Escheric hia coil under control of their own promoters. Two putative promoters are l ocated upstream of glcA, and both were found to initiate transcription in E . coli. Although the two permeases EIICBA(Glc)1 and EIICBA(Glc)2 show 69 % identity at the protein level, and despite the common primary substrate glu cose, they have different specificities towards glucosides as substrate. EI ICBA(Glc)1 phosphorylates glucose in a PEP-dependent reaction with a K-m of 12 mu M; the reaction can be inhibited by 2-deoxyglucose and methyl beta-D -glucoside. EIICBA(Glc)2 phosphorylates glucose with a K-m of 19 mu M and t his reaction is inhibited by methyl alpha-D-glucoside, methyl beta-D-glucos ide, p-nitrophenyl alpha-D-glucoside, o-nitrophenyl beta-D-glucoside and sa licin, but unlike other glucose permeases, including EIICBA(Glc)1, not by 2 -deoxyglucose. Natural mono- or disaccharides, such as mannose or N-acetylg lucosamine, that are transported by other glucose transporters are not phos phorylated by either EIICBA(Glc)1 nor EIICBA(Glc)2, indicating a high speci ficity for glucose. Together, these findings support the suggestion of evol utionary development of different members of a protein family, by gene dupl ication and subsequent differentiation. C-terminal fusion of a histidine he xapeptide to both gene products did not affect the activity of the enzymes and allowed their purification by Ni2+-NTA affinity chromatography after ex pression in a ptsG (EIICBGlc) deletion mutant of E. coli. Upstream of glcA, the 3' end of a further ORF encoding 138 amino acid residues of a putative antiterminator of the BgIG family was found, as well as a putative target DNA sequence (RAT), which indicates a further regulation by glucose specifi c antitermination.