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J Bacteriol, March 1998, p. 1411-1417, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The LysR-Type Transcriptional Regulator CbbR Controlling Autotrophic CO2 Fixation by Xanthobacter flavus Is an NADPH Sensor

G. van Keulen, L. Girbal, E. R. E. van den Bergh, L. Dijkhuizen, and W. G. Meijer*

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands

Received 2 June 1997/Accepted 13 January 1998

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. Maximal induction of the cbb and gap-pgk operons encoding enzymes of the Calvin cycle occurs in the absence of multicarbon substrates and the presence of methanol, formate, hydrogen, or thiosulfate. The LysR-type transcriptional regulator CbbR regulates the expression of the cbb and gap-pgk operons, but it is unknown to what cellular signal CbbR responds. In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein was expressed in Escherichia coli and subsequently purified to homogeneity. CbbR of X. flavus is a dimer of 36-kDa subunits. DNA-binding assays suggested that two CbbR molecules bind to a 51-bp DNA fragment on which two inverted repeats containing the LysR motif are located. The addition of 200 µM NADPH, but not NADH, resulted in a threefold increase in DNA binding. The apparent KdNADPH of CbbR was determined to be 75 µM. By using circular permutated DNA fragments, it was shown that CbbR introduces a 64° bend in the DNA. The presence of NADPH in the DNA-bending assay resulted in a relaxation of the DNA bend by 9°. From the results of these in vitro experiments, we conclude that CbbR responds to NADPH. The in vivo regulation of the cbb and gap-pgk operons may therefore be regulated by the intracellular concentration of NADPH.

* Corresponding author. Present address: Department of Industrial Microbiology, University College Dublin, Belfield Campus, Dublin 4, Ireland. Phone: 353-1-7061512. Fax: 353-1-7061183. E-mail: wim.meijer{at}ucd.ie.




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