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

CarD, a global transcriptional regulator in Myxococcus xanthus, interacts with CarG via CarDNter, its N-terminal domain, and with DNA via a eukaryotic HMGA-type C-terminal domain. Genomic analysis reveals a large number of standalone proteins resembling CarDNter. These constitute, together with the RNA polymerase (RNAP) interacting domain, RID, of transcription–repair coupling factors, the CarD_TRCF protein family. We show that one such CarDNter-like protein, M. xanthus CdnL, cannot functionally substitute CarDNter (or vice versa) nor interact with CarG. Unlike CarD, CdnL is vital for growth, and lethality due to its absence is not rescued by homologs from various other bacteria. In mycobacteria, with no endogenous DksA, the function of the CdnL homolog mirrors that of Escherichia coli DksA. Our finding that CdnL, like DksA, is indispensable in M. xanthus implies that they are not functionally redundant. Cells are normal on CdnL overexpression, but divide aberrantly on CdnL depletion. CdnL localizes to the nucleoid, suggesting piggyback recruitment by factors such as RNAP, which we show interacts with CdnL, CarDNter and RID. Our study highlights a complex network of interactions involving these factors and RNAP, and points to a vital role for M. xanthus CdnL in an essential DNA transaction that affects cell division.

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 transcriptional factor CarD participates in carotenogenesis and fruiting body formation. It is the only reported prokaryotic protein having adjacent "AT-hook" DNA-binding and acidic regions characteristic of eukaryotic high mobility group A (HMGA) proteins. The latter are small, unstructured, nonhistone nuclear proteins that function as architectural factors to remodel DNA and chromatin structure and modulate various DNA binding activities. We find CarD to be predominantly dimeric with two stable domains: (a) an N-terminal domain of defined secondary and tertiary structure which is absent in eukaryotic HMGA proteins; (b) a C-terminal domain formed by the acidic and AT-hook segments and lacking defined structure. CarD, like HMGA proteins, binds specifically to the minor-groove of AT-rich DNA present in two appropriately spaced tracts. As in HMGA proteins, casein kinase II can phosphorylate the CarD acidic region, and this dramatically decreases the DNA binding affinity of CarD. The acidic region, in addition to modulating DNA binding, confers structural stability to CarD. We discuss how the structural and functional plasticity arising from domain organization in CarD could be linked to its role as a general transcriptional factor in M. xanthus.

Myxococcus xanthus responds to blue light by producing carotenoid pigments. A mutation at a gene named carC is known to block the metabolism of phytoene, a carotenoid precursor, and this gene has now been cloned and sequenced. We show here that gene carC, which is homologous to phytoene dehydrogenase genes from other organisms, is tightly regulated by light through a mechanism that operates only when the cells have reached the stationary phase or are starved of a carbon source. A genetic element that mediates the effect of the growth phase has been identified. Gene carC is integrated with another unlinked carotenogenic gene in a single ‘light regulon’ controlled by common trans‐acting genetic elements. A potential −35 site for the binding of sigma factors has been found upstream of the carC transcriptional start. However, the −10 region shows no similarity with analogous sites at promoters of other Gram‐negative bacteria.

Enhanceosome assembly in eukaryotes often requires high mobility group A (HMGA) proteins. In prokaryotes, the only known transcriptional regulator with HMGA-like physical, structural and DNA-binding properties is Myxococcus xanthus CarD. Here, we report that every CarD-regulated process analysed also requires the product of gene carG, located immediately downstream of and transcriptionally coupled to carD. CarG has the zinc-binding H/C-rich metallopeptidase motif found in archaemetzincins, but with Q replacing a catalytically essential E. CarG, a monomer, binds two zinc atoms, shows no apparent metallopeptidase activity, and its stability in vivo absolutely requires the cysteines. This indicates a strictly structural role for zinc-binding. In vivo CarG localizes to the nucleoid but only if CarD is also present. In vitro CarG shows no DNA-binding but physically interacts with CarD via its N-terminal and not HMGA domain. CarD and CarG thus work as a single, physically linked, transcriptional regulatory unit, and if one exists in a bacterium so does the other. Like zinc-associated eukaryotic transcriptional adaptors in enhanceosome assembly, CarG regulates by interacting not with DNA but with another transcriptional factor.

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The light-inducible carB operon encodes all but one of the structural genes for carotenogenesis in Myxococcus xanthus. It is transcriptionally controlled by two proteins expressed from two unlinked genetic loci: CarS from the light-inducible carQRS operon, and CarA from the light-independent carA operon. CarA represses transcription from the carB promoter (