THE LYSR-TYPE TRANSCRIPTIONAL REGULATOR CBBR CONTROLLING AUTOTROPHIC CO2 FIXATION BY XANTHOBACTER-FLAVUS IS AN NADPH SENSOR

Autotrophic growth of Xanthobacter flavus is dependent on the fixation of carbon dioxide via the Calvin cycle and on the oxidation of simple organic and inorganic compounds to provide the cell with energy. Maxi mal induction of the cbb and gap-pgk operons encoding enzymes of the C alvin cycle occurs in the absence of multicarbon substrates and the pr esence of methanol, formate, hydrogen, or thiosulfate. The LysR-type t ranscriptional regulator CbbR regulates the expression of the cbb and gap-pgk operons, but it is unknown to what cellular signal CbbR respon ds. In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein aas expressed in Escherichia coil and subsequently purified to homogeneity. CbbR of X. flavus is a dimer of 36-kDa subunits. DNA-binding assays suggested th at two CbbR molecules bind to a 51-bp DNA fragment on which two invert ed repeats containing the LysR motif are located. The addition of 200 mu M NADPH, but not NADH, resulted in a threefold increase in DNA bind ing. The apparent K-dNADPH of CbbR was determined to be 75 mu M. By us ing circular permutated DNA fragments, it was shown that CbbR introduc es a 64 degrees bend in the DNA. The presence of NADPH in the DNA-bend ing assay resulted in a relaxation of the DNA bend by 9 degrees. From the results of these in vitro experiments, we conclude that CbbR respo nds to NADPH. The in vivo regulation of the cbb and gap-pgk operons ma y therefore be regulated by the intracellular concentration of NADPB.