Plasticity in oligomerization, operator architecture, and DNA binding in the mode of action of a bacterial B12-based photoreceptor

Abstract

Edited by Ruma Banerjee 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 AdoCblbound tetramers, whose light-induced disassembly relieves repression. High-resolution structures of Thermus thermophilus CarH Tt 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 CarH Bm from Bacillus megaterium. We found that apo-CarH Bm , unlike monomeric apoCarH Tt , is an oligomeric molten globule that forms DNA-binding tetramers in the dark only upon AdoCbl binding, which requires a conserved W-X 9-EH motif. Light relieved DNA binding by disrupting CarH Bm tetramers to dimers, rather than to monomers as with CarH Tt. CarH Bm operators resembled that of CarH Tt , 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, CarH Bm recognized the smaller CarH Tt 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 B 12based CarH photoreceptors, important for their biological functions and development as optogenetic tools.

Light-inducible carotenoid production controlled by a MarR-type regulator in Corynebacterium glutamicum

“…In non-phototrophic bacteria, the control of carotenoid production is classified into three types: constitutive, light-inducible, and cryptic manner 7 . We and other groups have been studying the phenomena and molecular mechanism of light-inducible production of carotenoids of phylogenetically different bacteria including S. coelicolor A3(2) 8 , Thermus thermophilus HB27 9–14 , Bacillus megaterium QM B1551 15,16 , Mycobacterium marinum 17 , and Arthrobacter arilaitensis 18 . Our study has revealed that LitR/CarH family proteins, a MerR family transcriptional regulator retained by S .…”

Interplay between chromophore binding and domain assembly by the B12-dependent photoreceptor protein, CarH

“…Interestingly, apo-CarH from Bacillus megaterium has also been shown to self-interact independently of AdoCbl, but to a signicantly greater extent than for TtCarH. 9 apo-BmCarH elutes in SEC as a molten globule (i.e., loosely packed) oligomeric species with a molecular weight consistent with a trimer, although these oligomeric states do not bind DNA. AdoCbl-binding to BmCarH induces tetramer formation, which is much more rigidly structured (and binds DNA in vivo) and dissociates into dimers instead of monomers upon illumination.…”

“…7 AdoCbl photochemistry in CarH results in transcriptional activation in bacteria, which leads to the biosynthesis of carotenoids in response to photo-oxidative stress. Mechanistic details about its function are now beginning to emerge 4,[6][7][8][9][10][11] and it is already showing great promise and versatility as the basis of photoactivated, biomolecular tools. [12][13][14][15][16] The size and structural complexity of AdoCbl reect a long and expensive biosynthetic pathway 17 and mean that its uptake 18 and subsequent binding to its dependent enzymes 19 are oen tightly regulated.…”

Light-Triggered Carotenogenesis in Myxococcus xanthus: New Paradigms in Photosensory Signaling, Transduction and Gene Regulation

“…Both homologs turned out to be B 12 -dependent like CarH. Studies of the homolog in the Gram-negative Thermus thermophilus , CarH Tt , yielded valuable biochemical [ 20 ], structural [ 19 ] and photochemical insights [ 61 , 62 , 63 ] that were further extended with CarH Bm , the homolog in the Gram-positive Bacillus megaterium [ 64 , 65 ]. These findings, reviewed elsewhere [ 21 , 22 ], are briefly highlighted here.…”

“…Studies of CarH Tt and CarH Bm revealed that light-dependent regulation of transcription relies on modulation of their oligomeric state by AdoCbl and light ( Figure 2 c) [ 19 , 20 , 21 , 22 , 64 , 65 , 66 ]. AdoCbl-free apoCarH Tt is a monomer and apoCarH Bm is a loosely folded molten globule tetramer, and both bind poorly to operator DNA.…”