COOPERATIVE AND NONCOOPERATIVE DNA-BINDING MODES OF CATABOLITE CONTROL PROTEIN CCPA FROM BACILLUS-MEGATERIUM RESULT FROM SENSING 2 DIFFERENT SIGNALS

Carbon catabolite repression (CCR) of several operons in Bacillus subt ilis and Bacillus megaterium is mediated by the cis-acting cre sequenc e and trans-acting catabolite control protein (CcpA). We describe puri fication of CcpA from B. megaterium and its interaction with regulator y sequences from the xyl operon. Specific interaction of CcpA with cre as scored by DNase I footprints at concentrations similar to the in v ivo situation requires the presence of effecters. We have found two mo lecular effecters for CcpA activity, which lead to different recogniti on modes of DNA. The heat-stable phosphotransfer protein HPr from the PTS sugar uptake system triggers non-cooperative binding of CcpA to cr e when phosphorylated at Ser46 (HPr-Ser46-P). Glucose 6-phosphate (Glc -6-P) triggers cooperative binding of CcpA to cre and two auxiliary cr e sites, one of which overlaps the -35 box of the xyl promoter. Bindi ng to cre depends on the presence of the functional cre sequence. A m utation in cre abolishes carbon catabolite repression in vivo and bind ing of CcpA to eve and cre in vitro, indicating looping of the interv ening DNA. The two triggers are not simultaneously active. The acidity of the buffer determines which of them activates CcpA when both are p resent 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 cooperat ivity of binding to DNA. CcpA is the first member of the LacI/GalR fam ily of regulators, for which oligomerization without the leucine zippe r at the C terminus is demonstrated. (C) 1997 Academic Press Limited.