SummaryPhotoreceptor proteins enable organisms to sense and respond to light. The newly discovered CarH-type photoreceptors use a vitamin B12 derivative, adenosylcobalamin, as the light-sensing chromophore to mediate light-dependent gene regulation. Here, we present crystal structures of Thermus thermophilus CarH in all three relevant states: in the dark, both free and bound to operator DNA, and after light exposure. These structures provide a visualization of how adenosylcobalamin mediates CarH tetramer formation in the dark, how this tetramer binds to the promoter −35 element to repress transcription, and how light exposure leads to a large-scale conformational change that activates transcription. In addition to the remarkable functional repurposing of adenosylcobalamin from an enzyme cofactor to a light sensor, we find that nature also repurposed two independent protein modules in assembling CarH. These results expand the biological role of vitamin B12 and provide fundamental insight into a new mode of light-dependent gene regulation.

Cobalamin (B 12 ) typically functions as an enzyme cofactor but can also regulate gene expression via RNA-based riboswitches. B 12 -directed gene regulatory mechanisms via protein factors have, however, remained elusive. Recently, we reported down-regulation of a light-inducible promoter in the bacterium Myxococcus xanthus by two paralogous transcriptional repressors, of which one, CarH, but not the other, CarA, absolutely requires B 12 for activity even though both have a canonical B 12 -binding motif. Unanswered were what underlies this striking difference, what is the specific cobalamin used, and how it acts. Here, we show that coenzyme B 12 (5′-deoxyadenosylcobalamin, AdoB 12 ), specifically dictates CarH function in the dark and on exposure to light. In the dark, AdoB 12 -binding to the autonomous domain containing the B 12 -binding motif foments repressor oligomerization, enhances operator binding, and blocks transcription. Light, at various wavelengths at which AdoB 12 absorbs, dismantles active repressor oligomers by photolysing the bound AdoB 12 and weakens repressor-operator binding to allow transcription. By contrast, AdoB 12 alters neither CarA oligomerization nor operator binding, thus accounting for its B 12 -independent activity. Our findings unveil a functional facet of AdoB 12 whereby it serves as the chromophore of a unique photoreceptor protein class acting in light-dependent gene regulation. The prevalence of similar proteins of unknown function in microbial genomes suggests that this distinct B 12 -based molecular mechanism for photoregulation may be widespread in bacteria.carotenogenesis | Thermus thermophilus | antirepressor | MerR | TtCarH

SummaryA light-inducible promoter, PB, drives expression of the carB operon in Myxococcus xanthus. Repressed by CarA in the dark, PB is activated when CarS, produced in the light, sequesters CarA to prevent operator-CarA binding. The MerR-type, N-terminal domain of CarA, which mediates interactions with both operator and CarS, is linked to a C-terminal oligomerization module with a predicted cobalaminbinding motif. Here, we show that although CarA does bind vitamin B12, mutating the motif involved has no effect on its ability to repress PB. Intriguingly, PB could be repressed in the dark even with no CarA, so long as B12 and an intact CarA operator were present. We have discovered that this effect of B12 depends on the gene immediately downstream of carA. Its product, CarH, also consists of a MerR-type, N-terminal domain that specifically recognizes the CarA operator and CarS, linked to a predicted B12-binding C-terminal oligomerization module. The B12-mediated repression of PB in the dark is relieved by deleting carH, by mutating the DNA-or B12-binding residues of CarH, or by illumination. Our findings unveil parallel regulatory circuits that control a light-inducible promoter using a transcriptional factor repertoire that includes a paralogous gene pair and vitamin B12.

Myxococcus xanthus is a prokaryotic model system for the study of multicellular development and the response to blue light. The previous analyses of these processes and the characterization of new genes would benefit from a robust system for controlled gene expression, which has been elusive so far for this bacterium. Here, we describe a system for conditional expression of genes in M. xanthus based on our recent finding that vitamin B 12 and CarH, a MerR-type transcriptional repressor, together downregulate a photoinducible promoter. Using this system, we confirmed that M. xanthus rpoN, encoding 54 , is an essential gene, as reported earlier. We then tested it with ftsZ and dksA. In most bacteria, ftsZ is vital due to its role in cell division, whereas null mutants of dksA, whose product regulates the stringent response via transcriptional control of rRNA and amino acid biosynthesis promoters, are viable but cause pleiotropic effects. As with rpoN, it was impossible to delete endogenous ftsZ or dksA in M. xanthus except in a merodiploid background carrying another functional copy, which indicates that these are essential genes. B 12 -based conditional expression of ftsZ was insufficient to provide the high intracellular FtsZ levels required. With dksA, as with rpoN, cells were viable under permissive but not restrictive conditions, and depletion of DksA or 54 produced filamentous, aberrantly dividing cells. dksA thus joins rpoN in a growing list of genes dispensable in many bacteria but essential in M. xanthus.

The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these caUi. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg 1-~ of BA and 0.186 mg I J NAA + 2.25 mg 1-l BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.

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.

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Myxobacteria are Gram-negative δ-proteobacteria found predominantly in terrestrial habitats and often brightly colored due to the biosynthesis of carotenoids. Carotenoids are lipophilic isoprenoid pigments that protect cells from damage and death by quenching highly reactive and toxic oxidative species, like singlet oxygen, generated upon growth under light. The model myxobacterium Myxococcus xanthus turns from yellow in the dark to red upon exposure to light because of the photoinduction of carotenoid biosynthesis. How light is sensed and transduced to bring about regulated carotenogenesis in order to combat photooxidative stress has been extensively investigated in M. xanthus using genetic, biochemical and high-resolution structural methods. These studies have unearthed new paradigms in bacterial light sensing, signal transduction and gene regulation, and have led to the discovery of prototypical members of widely distributed protein families with novel functions. Major advances have been made over the last decade in elucidating the molecular mechanisms underlying the light-dependent signaling and regulation of the transcriptional response leading to carotenogenesis in M. xanthus. This review aims to provide an up-to-date overview of these findings and their significance.