S. Padmanabhan

Light-dependent gene regulation by a coenzyme B12-based photoreceptor

Cobalamin (B12) typically functions as an enzyme cofactor but can also regulate gene expression via RNA-based riboswitches. B12-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 B12 for activity even though both have a canonical B12-binding motif. Unanswered were what underlies this striking difference, what is the specific cobalamin used, and how it acts. Here, we show that coenzyme B12 (5′-deoxyadenosylcobalamin, AdoB12), specifically dictates CarH function in the dark and on exposure to light. In the dark, AdoB12-binding to the autonomous domain containing the B12-binding motif foments repressor oligomerization, enhances operator binding, and blocks transcription. Light, at various wavelengths at which AdoB12 absorbs, dismantles active repressor oligomers by photolysing the bound AdoB12 and weakens repressor–operator binding to allow transcription. By contrast, AdoB12 alters neither CarA oligomerization nor operator binding, thus accounting for its B12-independent activity. Our findings unveil a functional facet of AdoB12 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 B12-based molecular mechanism for photoregulation may be widespread in bacteria.

Structural basis for gene regulation by a B12-dependent photoreceptor.

Photoreceptor 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 visualizations 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.

Vitamin B12 partners the CarH repressor to downregulate a photoinducible promoter in Myxococcus xanthus

A 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 cobalamin‐binding 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.

The Stigmatella aurantiaca Homolog of Myxococcus xanthus High-Mobility-Group A-Type Transcription Factor CarD: Insights into the Functional Modules of CarD and Their Distribution in Bacteria

Transcriptional factor CarD is the only reported prokaryotic analog of eukaryotic high-mobility-group A (HMGA) proteins, in that it has contiguous acidic and AT hook DNA-binding segments and multifunctional roles in Myxococcus xanthus carotenogenesis and fruiting body formation. HMGA proteins are small, randomly structured, nonhistone, nuclear architectural factors that remodel DNA and chromatin structure. Here we report on a second AT hook protein, CarD(Sa), that is very similar to CarD and that occurs in the bacterium Stigmatella aurantiaca. CarD(Sa) has a C-terminal HMGA-like domain with three AT hooks and a highly acidic adjacent region with one predicted casein kinase II (CKII) phosphorylation site, compared to the four AT hooks and five CKII sites in CarD. Both proteins have a nearly identical 180-residue N-terminal segment that is absent in HMGA proteins. In vitro, CarD(Sa) exhibits the specific minor-groove binding to appropriately spaced AT-rich DNA that is characteristic of CarD or HMGA proteins, and it is also phosphorylated by CKII. In vivo, CarD(Sa) or a variant without the single CKII phosphorylation site can replace CarD in M. xanthus carotenogenesis and fruiting body formation. These two cellular processes absolutely require that the highly conserved N-terminal domain be present. Thus, three AT hooks are sufficient, the N-terminal domain is essential, and phosphorylation in the acidic region by a CKII-type kinase can be dispensed with for CarD function in M. xanthus carotenogenesis and fruiting body development. Whereas a number of hypothetical proteins homologous to the N-terminal region occur in a diverse array of bacterial species, eukaryotic HMGA-type domains appear to be confined primarily to myxobacteria.

Light-dependent gene regulation in nonphototrophic bacteria

Bacteria sense and respond to light, a fundamental environmental factor, by employing highly evolved machineries and mechanisms. Cellular systems exist to harness light energy usefully as in phototrophic bacteria, to combat photo-oxidative damage stemming from the highly reactive species generated on absorption of light energy, and to link the light stimulus to DNA repair, taxis, development, and virulence. Recent findings on the genetic response to light in nonphototrophic bacteria highlight the ingenious transcriptional regulatory mechanisms and the panoply of factors that have evolved to perceive and transmit the signal, and to bring about finely tuned gene expression.

CdnL, a member of the large CarD-like family of bacterial proteins, is vital for Myxococcus xanthus and differs functionally from the global transcriptional regulator CarD

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.

Recruitment of a novel zinc-bound transcriptional factor by a bacterial HMGA-type protein is required for regulating multiple processes in Myxococcus xanthus.

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.

A bacterial antirepressor with SH3 domain topology mimics operator DNA in sequestering the repressor DNA recognition helix

Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor–antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel β-sheet fold resembling SH3 domains, protein–protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein–protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.

The photochemical mechanism of a B12-dependent photoreceptor protein.

The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

Structural basis for operator and antirepressor recognition by Myxococcus xanthus CarA repressor.

Blue light induces carotenogenesis in Myxococcus xanthus. The carB operon encodes all but one of the structural genes involved, and its expression is regulated by the CarA‐CarS repressor‐antirepressor pair. In the dark, CarA‐operator binding represses carB. CarS, produced on illumination, interacts physically with CarA to dismantle the CarA‐operator complex and activate carB. Both operator and CarS bind to the autonomously folded N‐terminal domain of CarA, CarA(Nter), which in excess represses carB. Here, we report the NMR structure of CarA(Nter), and map residues that interact with operator and CarS by NMR chemical shift perturbations, and in vivo and in vitro analyses of site‐directed mutants. We show CarA(Nter) adopts the winged‐helix topology of MerR‐family DNA‐binding domains, and conserves the majority of the helix‐turn‐helix and wing contacts with DNA. Tellingly, helix α2 in CarA, a key element in operator DNA recognition, is also critical for interaction with CarS, implying that the CarA‐CarS protein‐protein and the CarA‐operator protein–DNA interfaces overlap. Thus, binding of CarA to operator and to antirepressor are mutually exclusive, and CarA may discern structural features in the acidic CarS protein that resemble operator DNA. Repressor inactivation by occluding the DNA‐binding region may be a recurrent mechanism of action for acidic antirepressors.