Montserrat Elías-Arnanz

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.

Bacteriophage φ29 DNA replication arrest caused by codirectional collisions with the transcription machinery

The consequences on replication of collisions between φ29 DNA polymerase, a monomeric replicase endowed with strand displacement capacity, and the transcription machinery have been studied in vitro. Codirectional collisions with stalled transcription ternary complexes at four different promoters in the φ29 genome were found to block replication fork progression. Upon collision, the DNA polymerase remained on the template and was able to resume elongation once the RNA polymerase was allowed to move. Collisions with RNA polymerase molecules moving in the same direction also interfered with replication, causing a decrease in the replication rate. These results lead to the proposal that in bacteriophage φ29 a transcription complex physically blocks the progression of a replication fork. We suggest that temporal regulation of transcription and the low probability that the replication and transcription processes colocalize in vivo contribute to achieving minimal interference between the two events.

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.

Resolution of head‐on collisions between the transcription machinery and bacteriophage Φ29 DNA polymerase is dependent on RNA polymerase translocation

The outcome of collisions between Bacillus subtilis phage Φ29 DNA polymerase and oppositely oriented transcription complexes has been studied in vitro. We found that the replication fork was unable to go past a transcription ternary complex stalled head‐on. However, head‐on collisions did not lead to a deadlock. Both DNA and RNA polymerase remained bound to the template and, when the halted transcription complex was allowed to move, the replication machinery resumed normal elongation. These results suggested that a replication fork that encounters an RNA polymerase head‐on whose movement is not impeded would bypass the transcription machinery. Our results for head‐on collisions between concurrently moving replication and transcription complexes are indeed consistent with the existence of a resolving mechanism. The ability of Φ29 DNA polymerase to resolve head‐on collisions with itself during symmetrical replication of Φ29 DNA in vivo is likely to be related to its ability to pass a head‐on oriented RNA polymerase.

Bacteriophage ø29 DNA replication arrest caused by codirectional and head-on collisions with the transcription machinery

The consequences on replication of collisions between φ29 DNA polymerase, a monomeric replicase endowed with strand displacement capacity, and the transcription machinery have been studied in vitro. Codirectional collisions with stalled transcription ternary complexes at four different promoters in the φ29 genome were found to block replication fork progression. Upon collision, the DNA polymerase remained on the template and was able to resume elongation once the RNA polymerase was allowed to move. Collisions with RNA polymerase molecules moving in the same direction also interfered with replication, causing a decrease in the replication rate. These results lead to the proposal that in bacteriophage φ29 a transcription complex physically blocks the progression of a replication fork. We suggest that temporal regulation of transcription and the low probability that the replication and transcription processes colocalize in vivo contribute to achieving minimal interference between the two events.

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.