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

Department of Microbiology, Ruhr-Universität Bochum, NDEF-06, D-44780 Bochum, Germany¹

Author for correspondence: Wolfgang Hengstenberg. Tel: +49 234 7004247. Fax: +49 234 709 4620. e-mail: wolfgang.hengstenberg{at}ruhr-uni-bochum.de

Previous sequence analysis of the glucose-specific PTS gene locus from Staphylococcus carnosus revealed the unexpected finding of two adjacent, highly similar ORFs, glcA and glcB, each encoding a glucose-specific membrane permease 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 flanked by putative regulatory elements such as a termination stem–loop, promoter and ribosome-binding site, suggesting independent regulation. The finding of putative cis-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 Escherichia coli under control of their own promoters. Two putative promoters are located upstream of glcA, and both were found to initiate transcription in E. coli. Although the two permeases EIICBA^Glc1 and EIICBA^Glc2 show 69% identity at the protein level, and despite the common primary substrate glucose, they have different specificities towards glucosides as substrate. EIICBA^Glc1 phosphorylates glucose in a PEP-dependent reaction with a K_m of 12 µM; the reaction can be inhibited by 2-deoxyglucose and methyl ß-D-glucoside. EIICBA^Glc2 phosphorylates glucose with a K_m of 19 µM and this reaction is inhibited by methyl -D-glucoside, methyl ß-D-glucoside, p-nitrophenyl -D-glucoside, o-nitrophenyl ß-D-glucoside and salicin, but unlike other glucose permeases, including EIICBA^Glc1, not by 2-deoxyglucose. Natural mono- or disaccharides, such as mannose or N-acetylglucosamine, that are transported by other glucose transporters are not phosphorylated by either EIICBA^Glc1 nor EIICBA^Glc2, 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 Ni²⁺-NTA affinity chromatography after expression in a ptsG (EIICB^Glc) 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 BglG family was found, as well as a putative target DNA sequence (RAT), which indicates a further regulation by glucose specific antitermination.

Keywords: glucose-specific phosphotransferase system, Staphylococcus carnosus, membrane protein, regulation, kinetics

Abbreviations: CRE, catabolite-responsive element; NTA, nitrilotriacetic acid; PEP, phosphoenolpyruvate; PTS, phosphotransferase system

^a Present address: Department of Microbiology, Biozentrum, University of Basel, Basel, Switzerland.

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