Role and Function of LitR, an Adenosyl B₁₂-Bound Light-Sensitive Regulator of Bacillus megaterium QM B1551, in Regulation of Carotenoid Production

Light-induced carotenoid production in Bacillus megaterium. UV-visible spectra are shown with regard to the crude carotenoid fraction extracted from cells of B. megaterium QM B1551 WT, ΔlitR, ΔlitR/litR, ΔBMQ_1998, and Δcrt strains and B. megaterium DSM319 grown at 37°C for 15 h under dark and light conditions.

Promoter regions of litR and crtI1. (A) Nucleotide sequences of the promoter regions preceding litR and crtI1. The C-terminal amino acid sequence of BMQ_4357 and the N-terminal amino acid sequences of LitR and CrtI1 are also shown. Possible −10 and −35 hexamer sequences of the litR and crtI1 promoters are indicated by lowercase letters. The transcriptional start sites (designated +1), determined by 5′-RACE, are indicated by bent arrows. (B) Alignment of LitR-binding sequences. The consensus sequence is also shown.

Transcriptional analysis of litR and crt promoters in B. megaterium QM B1551 (A) and in a heterologous host, B. subtilis 168 (B). (A) Quantification of transcripts using semiquantitative RT-PCR. The amounts of litR, crtI1, and sigA (control) transcripts produced in the wild-type strain (WT), the litR mutant (ΔlitR), and the genetically complemented litR mutant (ΔlitR/litR) were estimated. cDNA synthesis was performed in the presence (+) or absence (−) of reverse transcriptase (RT). ND, not detected due to elimination of the primer annealing site. (B) A chromosomal integration plasmid, pDG1661, was used to monitor the transcriptional activities of promoters preceding litR via measuring the β-galactosidase activity in B. subtilis 168. The promoter preceding rpoB (encoding the β-subunit of RNA polymerase) (PrpoB) was used as a control. The transformants were grown in liquid LB medium under dark conditions (circles) or light conditions (squares).

Absorption spectra of LitR recombinant proteins. Spectra are shown for free AdoB₁₂ (A), free OHB₁₂ (B), AdoB₁₂-treated LitR_H190A (C), and AdoB₁₂-LitR (D) under dark or illuminated conditions.

Light-inducible subunit dissociation of AdoB₁₂-LitR as analyzed by gel filtration (A) and analytical ultracentrifugation (B). (A) To estimate the relative molecular mass of the AdoB₁₂-LitR protein, the purified recombinant proteins were analyzed by gel filtration. The elution volumes of AdoB₁₂-LitR on a Superdex 200 HR 10/30 column are indicated. The panel shows the elution profiles for AdoB₁₂-LitR under dark and illuminated conditions. Each protein examined eluted at positions corresponding to the tetrameric (M_r, 13,900) and dimeric (M_r, 8,900) structures. (B) Sedimentation velocity experiments were performed with nonilluminated and illuminated AdoB₁₂-LitR. The sedimentation coefficients [c(s)], i.e., 4.35S and 6.45S for nonilluminated AdoB₁₂-LitR and 4.26S for illuminated AdoB₁₂-LitR, are shown. The molecular sizes of the proteins were estimated to be M_r of 60,700 and 109,000 and of 64,200 on the basis of the conversion of c(s) to c(M).

Gel shift assay of AdoB₁₂-LitR. (A) The amounts of AdoB₁₂-LitR and AdoB₁₂-LitR_H190A used in lanes 1, 2, 3, 4, and 5 were 0, 4, 8, 16, and 32 pmol, respectively. Purified AdoB₁₂-LitR or LitR_H190A was mixed with the probes for PlitR (135 bp), PcrtI1 (146 bp), and PsigA (230 bp) and applied to a nondenaturing polyacrylamide gel. Open and closed triangles indicate the probe and the protein-DNA complex, respectively. (B) AdoB₁₂-LitR (4 pmol) was incubated in the dark (9 min) or under white light conditions (0 to 9 min) prior to the addition of a ³²P-labeled probe for PlitR. Only the shifted band (the LitR-AdoB₁₂–DNA complex) is shown.

DNase I footprint analysis to determine the LitR-binding sites in the litR and crtI1 promoters. The assay was performed on the sense (+) and antisense (−) strands. The amounts of recombinant AdoB₁₂-LitR used were 0 pmol (lanes 1 and 7), 0.5 pmol (lanes 2), 1 pmol (lanes 3), 2 pmol (lanes 4), 4 pmol (lanes 5), and 8 pmol (lanes 6) for the litR promoter and 0 pmol (lanes 1 and 8), 0.25 pmol (lanes 2), 0.5 pmol (lanes 3), 1 pmol (lanes 4), 2 pmol (lanes 5), 4 pmol (lanes 6), and 8 pmol (lanes 7) for the crtI1 promoter. Positional numbering is based on the transcriptional start point of each promoter, numbered as +1. The DNase I digests were run with chemically cleaved probes (G+A lanes for both strands).

In vitro runoff transcription assay. (A) The indicated amounts of the E. coli RNA polymerase core enzyme containing B. megaterium σ^A (Eσ^A) and the AdoB₁₂-LitR recombinant protein were added to the reaction mixture with the promoter DNA fragments. Transcripts of the predicted lengths were detected: 87 bp for PlitR, M7, and M12; 91 bp for PcrtI1; and 87 bp for PpolA. (B) The AdoB₁₂-LitR_H190A recombinant protein was added to the reaction mixture. (C) The effects of AdoB₁₂, OHB₁₂ (hydroxocobalamin), CNB₁₂ (cyanocobalamin), and MeB₁₂ (methylcobalamin) were examined using AdoB₁₂-LitR and the DNA fragments containing PlitR. (D) The effects of different light wavelengths, i.e., white light, 365 nm, 450 nm, 530 nm, and 633 nm, were examined using the AdoB₁₂-LitR protein and PlitR. D and L denote dark and white light, respectively.