Analysis of DNA Binding and Transcriptional Activation by the LysR-Type Transcriptional Regulator CbbR of Xanthobacter flavus

Abstract

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 CO 2 fixation. The cbb operon promoter of this chemoautotrophic bacterium contains three potential CbbR binding sites, two of which partially overlap. Site-directed mutagenesis and subsequent analysis of DNA binding by CbbR and cbb promoter activity were used to show that the potential CbbR binding sequences are functional. Inverted repeat IR 1 is a high-affinity CbbR binding site. The main function of this repeat is to recruit CbbR to the cbb operon promoter. In addition, it is required for negative autoregulation of cbbR expression. IR 3 represents the main low-affinity binding site of CbbR. Binding to IR 3 occurs in a cooperative manner, since mutations preventing the binding of CbbR to IR 1 also prevent binding to the low-affinity site. Although mutations in IR 3 have a negative effect on the binding of CbbR to this site, they result in an increased promoter activity. This is most likely due to steric hindrance of RNA polymerase by CbbR since IR 3 partially overlaps with the ؊35 region of the cbb operon promoter. Mutations in IR 2 do not affect the DNA binding of CbbR in vitro but have a severe negative effect on the activity of the cbb operon promoter. This IR 2 binding site is therefore critical for transcriptional activation by CbbR.Xanthobacter flavus is a chemoautotrophic bacterium which uses the Calvin cycle to assimilate carbon dioxide (9, 12). The energy to drive carbon dioxide fixation is provided by the oxidation of compounds such as methanol, formate, and H 2 . The majority of the genes encoding the Calvin cycle enzymes constitute three transcriptional units: the cbb and gap-pgk operons and the tpi gene. The cbb operon encodes the key enzymes of the Calvin cycle, ribulose bisphosphate carboxylase/ oxygenase and phosphoribulokinase, and in addition a number of enzymes required for the regeneration of ribulose bisphosphate. Glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase are encoded by the gap-pgk operon and play a role in both the Calvin cycle and glycolysis. The tpi gene encodes triosephosphate isomerase (10,11,13,14,24). During heterotrophic growth on, for instance, succinate, the cbb operon is not expressed and the gap-pgk operon is transcribed at a low constitutive level. A transition from heterotrophic to autotrophic growth is accompanied by a rapid induction of the cbb operon and a superinduction of the gap-pgk operon (11,14). The first two genes of the cbb operon encode the CO 2 -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO).The induction and superinduction of, respectively, the cbb and gap-pgk operons are completely dependent on the presence of the transcriptional regulator CbbR, which is encoded upstream and whose gene is transcribed divergently from the cbb operon (14,25). This transcriptional regulator is encountered in many photoautot...

The role of two CbbRs in the transcriptional regulation of three ribulose-1,5-bisphosphate carboxylase/oxygenase genes in Hydrogenovibrio marinus strain MH-110

“…The gel shift assay showed that CbbR1 bound to the overlapping promoter region between cbbLS-1 and cbbR1, suggesting that the expression of CbbR1 is subjected to autoregulation. Negative autoregulation has also been demonstrated for the LTTR including CbbR (Schell, 1993;Vichivanives et al, 2000;van Keulen et al, 2003). The amount of the cbbRm transcripts did not vary significantly under any of the CO 2 conditions.…”

“…Another possible explanation for the cross-regulation is that an accumulation of certain intermediates or a change in certain cellular states caused by the loss of CbbLS-1 and/or CbbM is the trigger for the induction of another RubisCO. It has been reported that the DNA binding of CbbR is altered in the presence of certain metabolites in other bacteria (van Keulen et al, 1998(van Keulen et al, , 2003Grzeszik et al, 2000;Terazono et al, 2001;Dubbs et al, 2004). In R. capsulatus, the binding of both CbbRI and CbbRII to their cognate promoter was enhanced in the presence of certain metabolites formed by the CBB cycle, as well as other intermediates, including ribulose-1,5-bisphosphate, 3-phosphoglycerate and PEP (Dubbs et al, 2004).…”

“…It has been reported that NADPH enhances the DNA-binding ability of the CbbR of Xanthobacter flavus (van Keulen et al, 1998Keulen et al, , 2003. NADPH also changes the binding ability of the CbbR of Hydrogenophilus thermoluteolus (Terazono et al, 2001).…”

“…CbbRm also gave two shifted bands for the cbbM promoter at higher protein concentrations. It has been shown that CbbR binds to the target promoter with two subsites and forms a dimer of dimers on the promoter in some bacteria (Kusano & Sugawara, 1993;Kusian & Bowien, 1995;van Keulen et al, 1998van Keulen et al, , 2003. Thus, CbbR1 and CbbRm of H. marinus probably bind to each target promoter as a dimer of dimers.…”

Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins

“…Co-inducers are recognized as being important for the function of LTTRs and often appear to contribute to a feedback loop in which a product or intermediate of a given metabolic/synthesis pathway (usually activated by an LTTR) acts as the co-inducer necessary for transcriptional activation or repression ( Fig. 1) (Celis, 1999;van Keulen et al, 2003;Picossi et al, 2007).…”