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 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 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 IR1 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. IR3 represents the main low-affinity binding site of CbbR. Binding to IR3 occurs in a cooperative manner, since mutations preventing the binding of CbbR to IR1 also prevent binding to the low-affinity site. Although mutations in IR3 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 IR3 partially overlaps with the -35 region of the cbb operon promoter. Mutations in IR2 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 IR2 binding site is therefore critical for transcriptional activation by CbbR.

FIG. 1.

Alignment of DNA sequences of the cbbR-cbbL intergenic regions of X. flavus (XF), T. ferrooxidans (TF), and R. eutropha (RE). The CbbR binding motif is given below the alignment. ∗, identical nucleotides. Brackets, positions of LysR motifs. Inverted and direct repeats are in boldface. Positions of the −35 and −10 regions of the cbb promoter are indicated. +1, cbbL transcription start site. The start codon of the cbbR gene of X. flavus is underlined.

FIG. 2.

(A) Gel retardation assays of wild-type (wt) and mutant IR₁, IR₂, and IR₃ cbb promoters performed with identical increasing amounts (as indicated by the triangles) of purified CbbR (34, 68, or 136 ng of CbbR per assay) with or without the CbbR inducer NADPH (final concentration, 200 μM). F, free (unbound) DNA; I, complex I (DNA bound by two CbbR dimers); II, complex II (DNA bound by one CbbR dimer). (B) Percentages of bound DNA in complex I and complex II for wild-type and mutated cbb promoters.

FIG. 3.

Site-directed mutagenesis of the cbb promoter of X. flavus. Mutation positions are given relative to the transcriptional start site of the cbb promoter (+1). The half-sites of the three inverted repeats are in boldface. The start codon of cbbR is underlined. The DNA fragments used to determine the binding of CbbR to IR₁ (SRΔ16) or IR₂ and IR₃ (SR8) are indicated by solid bars above the nucleotide sequence. Every tenth nucleotide upstream from the transcriptional start site is indicated by a dot above the nucleotide sequence.

FIG. 4.

In vivo activation of wild-type (wt) and cbb promoter mutants. (A and B) Normalized levels of β-galactosidase activities expressed by cbbL-lacZ fusions in X. flavus H4-14 grown autotrophically (A) or heterotrophically (B). β-Galactosidase activities driven by the wild-type cbb promoter were, respectively, 7,285 and 250 nmol per min per mg of protein and were set at 100%. Empty, promoter probe vector pBC3 without an insert. (C) Normalized levels of β-galactosidase activities expressed by cbbL-lacZ fusions in X. flavus R22 grown heterotrophically. The β-galactosidase activity driven by the wild-type cbb promoter was 59 nmol per min per mg of protein and was set at 100%.

FIG. 5.

(A) Gel retardation assays of mutant cbb promoters with either 5 (+5) or 10 (+10) nucleotides inserted between CbbR binding sites IR₁ and IR₂. F, I, and II and the symbols for the concentrations of CbbR and NADPH are as defined in the legend for Fig. 2. (B) Percentages of bound DNA in complex I and complex II for wild-type (wt) and mutated cbb promoters.

FIG. 6.

Activities of β-galactosidase in extracts of X. flavus H4-14 (wild-type; •) and R22 (cbbR; ○) containing a cbbR-lacZ fusion and growing on 5 mM gluconate. The results before and following the induction of the Calvin cycle by the addition of 20 mM formate and automatic titration with formic acid (25% [vol/vol] at 0 h) are shown. Enzyme amounts are expressed as nanomoles per minute per milligram of protein.

FIG. 7.

Proposed model of CbbR-mediated positive and negative regulation of the cbb operon and the cbbR gene and the response to the inducer NADPH. (1) No CbbR bound to the cbbR-cbbL intergenic region. cbbR is expressed highly, and the cbb operon is silent. (2) One CbbR dimer bound to IR₁. (3) Two CbbR dimers bound to IR₁ and IR₃, resulting in the bending of DNA at an angle of 64°. cbbR expression is downregulated, and the cbb operon remains silent. (3) Two CbbR dimers bound to sites IR₁ and IR₂, relaxing the DNA bend by 9° to 55° in the presence of the CbbR inducer NADPH. cbbR expression is further downregulated, and the cbb operon is expressed highly. The sizes of wave patterns above cbbR or below the cbb operon are an indication of the expression of the respective genes.