The LysR-type transcriptional regulator CbbR controlling autotrophic CO2 fixation by Xanthobacter flavus is an NADPH sensor.
نویسندگان
چکیده
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 microM NADPH, but not NADH, resulted in a threefold increase in DNA binding. The apparent K(dNADPH) of CbbR was determined to be 75 microM. By using circular permutated DNA fragments, it was shown that CbbR introduces a 64 degree bend in the DNA. The presence of NADPH in the DNA-bending assay resulted in a relaxation of the DNA bend by 9 degree. 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.
منابع مشابه
CbbR, a LysR-type transcriptional activator, is required for expression of the autotrophic CO2 fixation enzymes of Xanthobacter flavus.
Xanthobacter flavus is able to grow autotrophically with the enzymes of the Calvin cycle for the fixation of CO2, which are specified by the cbbLSXFP gene cluster. Previously, the 5' end of an open reading frame (cbbR), displaying a high sequence similarity to the LysR family of regulatory proteins and transcribed divergently from cbbLSXFP, was identified (W. G. Meijer, A. C. Arnberg, H. G. Ene...
متن کاملAnalysis of DNA binding and transcriptional activation by the LysR-type transcriptional regulator CbbR of Xanthobacter flavus.
The LysR-type transcriptional regulator CbbR controls the expression of the cbb and gap-pgk operons in Xanthobacter flavus, which encode the majority of the enzymes of the Calvin cycle required for autotrophic CO2 fixation. The cbb operon promoter of this chemoautotrophic bacterium contains three potential CbbR binding sites, two of which partially overlap. Site-directed mutagenesis and subsequ...
متن کاملUniversity of Groningen Analysis of DNA binding and transcriptional activation by the LysR-type transcriptional regulator CbbR of Xanthobacter flavus
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Expression of the triosephosphate isomerase and the tkt encoded transketolase of Xanthobacter flavus is independent of the autotrophic regulator CbbR
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Xanthobacter flavus employs a single triosephosphate isomerase for heterotrophic and autotrophic metabolism.
The expression of the cbb and gap-pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR. In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion between the promoter of the cbb operon and the reporter gene lacZ. This mutant was unable to grow autot...
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ورودعنوان ژورنال:
- Journal of bacteriology
دوره 180 6 شماره
صفحات -
تاریخ انتشار 1998