The LysR-type transcriptional regulator CbbR controlling autotrophic CO2 fixation by Xanthobacter flavus is an NADPH sensor

Abstract

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.

FIG. 1

(A) Nucleotide sequences of the 277- and 56-bp DNA fragments used in the band shift assays. The five nucleotides of the 56-bp fragment derived from the vector are not shown. The positions of the putative binding sites of CbbR (IR₁ and IR₂) are indicated by arrows. The translations of cbbL and cbbR are shown below the nucleotide sequence. The translation of cbbL is from the reverse complement (lowercase letters). Putative ribosome-binding sites are underlined. The transcriptional start site of the cbb operon (19) is indicated by an arrow. The nucleotides protected by CbbR from DNase I digestion are boxed, and the position of the DNase I-hypersensitive nucleotide is indicated by the asterisk. (B) Alignment of IR₁ and IR₂, the putative binding sites of CbbR. Identical nucleotides are indicated by asterisks. The nucleotides making up the LysR motif (T-N₁₁-A) are boxed.

FIG. 2

Coomassie brilliant blue-stained denaturing polyacrylamide gel showing CbbR (10 μg) purified from IPTG-induced E. coli BL21(DE3)pLysE/pER500. The MW standards used are shown.

FIG. 3

DNase I footprint of a 464-bp DNA fragment containing the cbbR-cbbL intergenic region. The brackets indicate the positions of the strongly and weakly protected nucleotides from, respectively, −75 to −50 (I) and −44 to −29 (II). Lanes: 1, CbbR not added; 2, 0.34 μg of CbbR added; 3, 1.7 μg of CbbR added. The nucleotide sequence of the protected regions and the DNase I-hypersensitive site located between the two protected regions is shown in Fig. 1.

FIG. 4

Effects of pyridine dinucleotides (200 μM) on the DNA-binding characteristics of CbbR. A band shift assay was done with the 277-bp EcoRI-BamHI DNA fragment (10,000 cpm) of pTZ00 and purified CbbR (27 ng of CbbR). Lanes: 1, no CbbR added; 2, CbbR; 3, CbbR plus NADPH; 4, CbbR plus NADH; 5, CbbR plus NADP; 6, CbbR plus NAD. Arrows indicate the positions of the unbound DNA and the protein-DNA complexes of low (complex 1) and high (complex 2) electrophoretic mobility.

FIG. 5

Effect of NADPH concentration on the DNA-binding characteristics of purified CbbR. (A) Band shift assay using the 277-bp EcoRI-BamHI DNA fragment of pTZ00 and 17 ng of purified CbbR. Lanes: 1, no CbbR added; 2, 0 μM NADPH; 3, 10 μM NADPH; 4, 50 μM NADPH; 5, 100 μM NADPH; 6, 200 μM NADPH; 7,500 μM NADPH. Arrows indicate the positions of the unbound DNA and the protein-DNA complexes of low (complex 1) and high (complex 2) electrophoretic mobility. (B) Graphical representation of the band shift assay results in panel A. The percentages of total ³²P-labeled DNA present in protein-DNA complex 1 (•), protein-DNA complex 2 (▴), and the free-DNA fragment (▪) are plotted against the NADPH concentration. The amount of ³²P-labeled DNA was determined by quantifying the radioactivity in the bands with a PhosphorImager.

FIG. 6

DNA bending by CbbR in the absence (A) or presence (B) of 200 μM NADPH in the binding assay. Circular permutated DNA fragments of pLG168 containing both CbbR-binding sites were constructed by digestion with various restriction enzymes as described in Materials and Methods. The radiolabeled DNA fragments (10,000 cpm) were incubated with purified CbbR (1.7 μg) and analyzed on a nondenaturing acrylamide gel. Lanes: 1; no CbbR added, BglII; 2, BglII; 3, NheI; 4, XhoI; 5, EcoRV; 6, PvuII; 7, SmaI. (C) Graphical representation of CbbR bending of the cbbL promoter region in the absence (•) or presence (○) of 200 μM NADPH, showing electrophoretic mobility (in centimeters) plotted against the position (in nucleotides [nt]) of IR₁ and IR₂ with respect to the left end of the DNA fragment. The bending angle (α) was calculated as described in Materials and Methods from five experiments.