Cooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals.
Carbon catabolite repression (CCR) of several operons in Bacillus subtilis and Bacillus megaterium is mediated by the cis-acting cre sequence and trans-acting catabolite control protein (CcpA). We describe purification of CcpA from B. megaterium and its interaction with regulatory sequences from the xyl operon. Specific interaction of CcpA with cre as scored by DNase I footprints at concentrations similar to the in vivo situation requires the presence of effectors. We have found two molecular effectors for CcpA activity, which lead to different recognition modes of DNA. The heat-stable phosphotransfer protein HPr from the PTS sugar uptake system triggers non-cooperative binding of CcpA to cre when phosphorylated at Ser46 (HPr-Ser46-P). Glucose 6-phosphate (Glc-6-P) triggers cooperative binding of CcpA to cre and two auxiliary cre* sites, one of which overlaps the -35 box of the xyl promoter. Binding to cre* depends on the presence of the functional cre sequence. A mutation in cre abolishes carbon catabolite repression in vivo and binding of CcpA to cre and cre* in vitro, indicating looping of the intervening DNA. The two triggers are not simultaneously active. The acidity of the buffer determines which of them activates CcpA when both are present in vitro. Glc-6-P is preferred at pH values below 5.4, and HPr-Ser46-P is preferred at neutral pH. The Ccpa dimers present at neutral pH form tetramers and higher oligomers at pH 4.6, explaining cooperativity of binding to DNA. CcpA is the first member of the LacI/GalR family of regulators, for which oligomerization without the leucine zipper at the C terminus is demonstrated.
Gösseringer
R
R
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universität Erlangen-Nürnberg, FRG.
Küster
E
E
Galinier
A
A
Deutscher
J
J
Hillen
W
W
D001410:Bacillus megaterium / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism*; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D005947:Glucose / Q000494:pharmacology; D019298:Glucose-6-Phosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009875:Operator Regions, Genetic; D009876:Operon / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; D019298:Glucose-6-Phosphate; D008156:Luciferases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D005947:Glucose