Molecular analysis of two ScrR repressors and of a ScrR-FruR hybrid repressor for sucrose and D-fructose specific regulons from enteric bacteria.
The scr regulon of pUR400 and the chromosomally encoded scr regulon of Klebsiella pneumoniae KAY2026 are both negatively controlled by a specific repressor (ScrR). As deduced from the nucleotide sequences, both scrR genes encode polypeptides of 334 residues (85.5% identical base pairs, 91.3% identical amino acids), containing an N-terminal helix-turn-helix motif. Comparison with other regulatory proteins revealed 30.6% identical amino acids to FruR, 27.0% to Lacl and 28.1% to GalR. Six scrRs super-repressor mutations define the inducer-binding domain. The scr operator sequences were identified by in vivo titration tests of the sucrose repressor and by in vitro electrophoretic mobility shift assays. D-fructose, an intracellular product of sucrose transport and hydrolysis, and D-fructose 1-phosphate were shown to be molecular inducers of both scr regulons. An active ScrR-FruR hybrid repressor protein was constructed with the N-terminal part of the sucrose repressor of K. pneumoniae and the C-terminal part of the fructose repressor of Salmonella typhimurium LT2. Gel retardation assays showed that the hybrid protein bound to scr-specific operators, and that D-fructose 1-phosphate, the inducer for FruR, was the only inducer. In vivo, neither the operators of the fru operon nor of the pps operon, the natural targets for FruR, were recognized, but the scr operators were. These data and the data obtained from the super-repressor alleles confirm previous models on the binding of repressors of the Lacl family to their operators.
Jahreis
K
K
Arbeitsgruppe Genetik, Universität Osnabrück, Germany.
Lengeler
J W
JW
D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002241:Carbohydrates / Q000494:pharmacology; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005631:Fructokinases / Q000235:genetics*; D005632:Fructose / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D006026:Glycoside Hydrolases / Q000235:genetics*; D007711:Klebsiella pneumoniae / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D018272:Porins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D012486:Salmonella typhimurium / Q000235:genetics*; D016415:Sequence Alignment; D017385:Sequence Homology; D013395:Sucrose / Q000378:metabolism*; D013403:Sugar Phosphates / Q000494:pharmacology; D043324:beta-Fructofuranosidase; D056968:Lac Repressors
D001426:Bacterial Proteins; D002241:Carbohydrates; D029968:Escherichia coli Proteins; D005631:Fructokinases; D005632:Fructose; D006026:Glycoside Hydrolases; D018272:Porins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D013395:Sucrose; D013403:Sugar Phosphates; D043324:beta-Fructofuranosidase; C489416:FruR protein, E coli; C489789:ScrY protein, E coli; C079242:ScrY protein, bacteria; C083535:scrR protein, bacteria; C068837:FruR protein, Bacteria; D056968:Lac Repressors; C046173:fructokinase