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 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(Glc)1 and EIICBA(Glc)2 show 69% identity at the protein level, and despite the common primary substrate glucose, they have different specificities towards glucosides as substrate. EIICBA(Glc)1 phosphorylates glucose in a PEP-dependent reaction with a Km of 12 microM; the reaction can be inhibited by 2-deoxyglucose and methyl beta-D-glucoside. EIICBA(Glc)2 phosphorylates glucose with a Km of 19 microM and this reaction is inhibited by methyl alpha-D-glucoside, methyl beta-D-glucoside, p-nitrophenyl alpha-D-glucoside, o-nitrophenyl beta-D-glucoside and salicin, but unlike other glucose permeases, including EIICBA(Glc)1, 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(Glc)1 nor EIICBA(Glc)2, 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.
Christiansen
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Department of Microbiology, Ruhr-Universität Bochum, Germany.
Hengstenberg
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D003847:Deoxyglucose / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008757:Methylglucosides / Q000494:pharmacology; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering; D013210:Staphylococcus / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005960:Glucosides; D008757:Methylglucosides; D009097:Multienzyme Complexes; D010455:Peptides; C025193:4-nitrophenyl beta-D-glucoside; C019502:4-nitrophenyl alpha-glucoside; D003847:Deoxyglucose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase; D005947:Glucose