Abstract
Newly discovered bacterial photoreceptors called CarH sense light by using 5'-deoxyadenosylcobalamin (AdoCbl). They repress their own expression and that of genes for carotenoid synthesis by binding in the dark to operator DNA as AdoCbl-bound tetramers, whose light-induced disassembly relieves repression. High-resolution structures of Thermus thermophilus CarHTt have provided snapshots of the dark and light states and have revealed a unique DNA-binding mode whereby only three of four DNA-binding domains contact an operator comprising three tandem direct repeats. To gain further insights into CarH photoreceptors and employing biochemical, spectroscopic, mutational, and computational analyses, here we investigated CarHBm from Bacillus megaterium We found that apoCarHBm, unlike monomeric apoCarHTt, is an oligomeric molten globule that forms DNA-binding tetramers in the dark only upon AdoCbl binding, which requires a conserved W-X9-EH motif. Light relieved DNA binding by disrupting CarHBm tetramers to dimers, rather than to monomers as with CarHTt CarHBm operators resembled that of CarHTt, but were larger by one repeat and overlapped with the -35 or -10 promoter elements. This design persisted in a six-repeat, multipartite operator we discovered upstream of a gene encoding an Spx global redox-response regulator whose photoregulated expression links photooxidative and general redox responses in B. megaterium Interestingly, CarHBm recognized the smaller CarHTt operator, revealing an adaptability possibly related to the linker bridging the DNA- and AdoCbl-binding domains. Our findings highlight a remarkable plasticity in the mode of action of B12-based CarH photoreceptors, important for their biological functions and development as optogenetic tools.
Highlights
Discovered bacterial photoreceptors called CarH sense light by using 5-deoxyadenosylcobalamin (AdoCbl)
Sequence alignment (Fig. S1D) indicates that CarHBm retains: (a) almost every residue of the conserved R-X-WE-XRY-X6-R-X5-R-X-Y motif (X: any amino acid) in the DNA-binding domain (DBD) involved in contacts with operator DNA in the structure of the CarHTt-DNA complex, hinting at a shared mode of DNA recognition; (b) the W-X9-EH-X32–39-E-X-H-XX-G-X41-S-X-T/ V-X(22–30)-GG signature found in the C-terminal domain of putative AdoCbl-binding light-sensing modules (12)
CarHBm conserves only Gly-192 as Gly-205 (Fig. S1D), suggesting that despite conserved secondary structures, DNA- and AdoCbl-binding motifs, CarHBm and CarHTt may differ in dimer and tetramer assembly
Summary
Discovered bacterial photoreceptors called CarH sense light by using 5-deoxyadenosylcobalamin (AdoCbl). A newly discovered and widespread family of bacterial photoreceptors uses as its chromophore one of the two biologically relevant forms of vitamin B12, 5Ј-deoxyadenosylcobalamin (AdoCbl).[4] In AdoCbl a 5Ј-deoxyadenosyl (5ЈdAdo) group is covalently bound through its 5Ј-carbon to the cobalt atom in the B12 corrin ring, as the upper axial ligand The prototype of this family, CarH, is a transcription factor that regulates light-dependent expression, typically of its own gene and of those for the synthesis of carotenoids (8 –13), which quench singlet oxygen and other reactive oxygen species to mitigate photooxidative damage (2, 6). Our findings indicate a plasticity, within otherwise conserved modes of AdoCbl binding, oligomerization, operator design, and DNA binding, which underlies the action of a B12-based CarH photoreceptor in light-dependent gene expression
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