The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

Genome sequences of Mx1, the first Myxococcus phage isolated, and Mx4, a generalized transducing myxophage.

Myxococcus xanthus is the best-studied member of the phylum Myxococcota, but the bacteriophages infecting it and their characterization remain limited. Here, we present complete genomes of Mx1, the first Myxococcus phage isolated, and of an Mx4 derivative widely used for generalized transduction, both unclassified Caudoviricetes with long, contractile tails.

Complete Genome Sequence Assembly and Annotation for Myxococcus xanthus Strains DK1050 and DK101.

Myxococcus xanthus is a social Gram-negative soil bacterium and the best studied member of the order Myxococcales in the class Deltaproteobacteria, which was recently reclassified as the phylum Myxococcota. Here, we report complete genomes, obtained using Illumina and PacBio sequencing, of M. xanthus strains DK1050 and DK101 (GenBank accession numbers CP104804 and CP104803, respectively).

Plasmalogens and Photooxidative Stress Signaling in Myxobacteria, and How it Unmasked CarF/TMEM189 as the Δ1'-Desaturase PEDS1 for Human Plasmalogen Biosynthesis.

Plasmalogens are glycerophospholipids with a hallmark sn-1 vinyl ether bond that endows them with unique physical-chemical properties. They have proposed biological roles in membrane organization, fluidity, signaling, and antioxidative functions, and abnormal plasmalogen levels correlate with various human pathologies, including cancer and Alzheimer's disease. The presence of plasmalogens in animals and in anaerobic bacteria, but not in plants and fungi, is well-documented. However, their occurrence in the obligately aerobic myxobacteria, exceptional among aerobic bacteria, is often overlooked. Tellingly, discovery of the key desaturase indispensable for vinyl ether bond formation, and therefore fundamental in plasmalogen biogenesis, emerged from delving into how the soil myxobacterium Myxococcus xanthus responds to light. A recent pioneering study unmasked myxobacterial CarF and its human ortholog TMEM189 as the long-sought plasmanylethanolamine desaturase (PEDS1), thus opening a crucial door to study plasmalogen biogenesis, functions, and roles in disease. The findings demonstrated the broad evolutionary sweep of the enzyme and also firmly established a specific signaling role for plasmalogens in a photooxidative stress response. Here, we will recount our take on this fascinating story and its implications, and review the current state of knowledge on plasmalogens, their biosynthesis and functions in the aerobic myxobacteria.

Vitamin B12 photoreceptors.

Photoreceptor proteins enable living organisms to sense light and transduce this signal into biochemical outputs to elicit appropriate cellular responses. Their light sensing is typically mediated by covalently or noncovalently bound molecules called chromophores, which absorb light of specific wavelengths and modulate protein structure and biological activity. Known photoreceptors have been classified into about ten families based on the chromophore and its associated photosensory domain in the protein. One widespread photoreceptor family uses coenzyme B12 or 5'-deoxyadenosylcobalamin, a biological form of vitamin B12, to sense ultraviolet, blue, or green light, and its discovery revealed both a new type of photoreceptor and a novel functional facet of this vitamin, best known as an enzyme cofactor. Large strides have been made in our understanding of how these B12-based photoreceptors function, high-resolution structural descriptions of their functional states are available, as are details of their unusual photochemistry. Additionally, they have inspired notable applications in optogenetics/optobiochemistry and synthetic biology. Here, we provide an overview of what is currently known about these B12-based photoreceptors, their discovery, distribution, molecular mechanism of action, and the structural and photochemical basis of how they orchestrate signal transduction and gene regulation, and how they have been used to engineer optogenetic control of protein activities in living cells.

Coenzyme B12 -dependent and independent photoregulation of carotenogenesis across Myxococcales.

Light-induced carotenogenesis in Myxococcus xanthus is controlled by the B12 -based CarH repressor and photoreceptor, and by a separate intricate pathway involving singlet oxygen, the B12 -independent CarH paralogue CarA and various other proteins, some eukaryotic-like. Whether other myxobacteria conserve these pathways and undergo photoregulated carotenogenesis is unknown. Here, comparative analyses across 27 Myxococcales genomes identified carotenogenic genes, albeit arranged differently, with carH often in their genomic vicinity, in all three Myxococcales suborders. However, CarA and its associated factors were found exclusively in suborder Cystobacterineae, with carA-carH invariably in tandem in a syntenic carotenogenic operon, except for Cystobacter/Melittangium, which lack CarA but retain all other factors. We experimentally show B12 -mediated photoregulated carotenogenesis in representative myxobacteria, and a remarkably plastic CarH operator design and DNA binding across Myxococcales. Unlike the two characterized CarH from other phyla, which are tetrameric, Cystobacter CarH (the first myxobacterial homologue amenable to analysis in vitro) is a dimer that combines direct CarH-like B12 -based photoregulation with CarA-like DNA binding and inhibition by an antirepressor. This study provides new molecular insights into B12 -dependent photoreceptors. It further establishes the B12 -dependent pathway for photoregulated carotenogenesis as broadly prevalent across myxobacteria and its evolution, exclusively in one suborder, into a parallel complex B12 -independent circuit.

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

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.

The Photoactive Excited State of the B12-Based Photoreceptor CarH.

We have used transient absorption spectroscopy in the UV-visible and X-ray regions to characterize the excited state of CarH, a protein photoreceptor that uses a form of B12, adenosylcobalamin (AdoCbl), to sense light. With visible excitation, a nanosecond-lifetime photoactive excited state is formed with unit quantum yield. The time-resolved X-ray absorption near edge structure difference spectrum of this state demonstrates that the excited state of AdoCbl in CarH undergoes only modest structural expansion around the central cobalt, a behavior similar to that observed for methylcobalamin rather than for AdoCbl free in solution. We propose a new mechanism for CarH photoreactivity involving formation of a triplet excited state. This allows the sensor to operate with high quantum efficiency and without formation of potentially dangerous side products. By stabilizing the excited electronic state, CarH controls reactivity of AdoCbl and enables slow reactions that yield nonreactive products and bypass bond homolysis and reactive radical species formation.

A bacterial light response reveals an orphan desaturase for human plasmalogen synthesis.

Plasmalogens are glycerophospholipids with a hallmark sn-1 vinyl ether bond. These lipids are found in animals and some bacteria and have proposed membrane organization, signaling, and antioxidant roles. We discovered the plasmanylethanolamine desaturase activity that is essential for vinyl ether bond formation in a bacterial enzyme, CarF, which is a homolog of the human enzyme TMEM189. CarF mediates light-induced carotenogenesis in Myxococcus xanthus, and plasmalogens participate in sensing photooxidative stress through singlet oxygen. TMEM189 and other animal homologs could functionally replace CarF in M. xanthus, and knockout of TMEM189 in a human cell line eliminated plasmalogens. Discovery of the human plasmanylethanolamine desaturase will spur further study of plasmalogen biogenesis, functions, and roles in disease.

B12-based photoreceptors: from structure and function to applications in optogenetics and synthetic biology.

Vitamin B12-based photoreceptor proteins sense ultraviolet (UV), blue or green light using 5'-deoxyadenosylcobalamin (AdoCbl). The prototype of this widespread bacterial photoreceptor family, CarH, controls light-dependent gene expression in photoprotective cellular responses. It represses transcription in the dark by binding to operator DNA as an AdoCbl-bound tetramer, whose disruption by light relieves operator binding to allow transcription. Structures of the 'dark' (free and DNA-bound) and 'light' CarH states and studies on the unusual AdoCbl photochemistry have provided fundamental insights into these photoreceptors. We highlight these, the plasticity within a conserved mode of action among CarH homologs, their distribution, and their promising applications in optogenetics and synthetic biology.

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

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 AdoCbl-bound tetramers, whose light-induced disassembly relieves repression. High-resolution structures of Thermus thermophilus CarHTt 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 CarHBm from Bacillus megaterium We found that apoCarHBm, unlike monomeric apoCarHTt, is an oligomeric molten globule that forms DNA-binding tetramers in the dark only upon AdoCbl binding, which requires a conserved W-X9-EH motif. Light relieved DNA binding by disrupting CarHBm tetramers to dimers, rather than to monomers as with CarHTt CarHBm operators resembled that of CarHTt, 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, CarHBm recognized the smaller CarHTt 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 B12-based CarH photoreceptors, important for their biological functions and development as optogenetic tools.

Multifactorial control of the expression of a CRISPR-Cas system by an extracytoplasmic function σ/anti-σ pair and a global regulatory complex.

Expression of CRISPR-Cas systems is a prerequisite for their defensive role against invading genetic elements. Yet, much remains unknown about how this crucial step is regulated. We describe a new mechanism controlling CRISPR-cas expression, which requires an extracytoplasmic function (ECF) σ factor (DdvS), its membrane-bound anti-σ (DdvA) and a global regulatory complex (CarD-CarG). Transcriptomic analyses revealed that the DdvS/CarD/CarG-dependent regulon comprises a type III-B CRISPR-Cas system in Myxococcus xanthus. We mapped four DdvS-driven CarD/CarG-dependent promoters, with one lying immediately upstream of the cas cluster. Consistent with direct action, DdvS and CarD-CarG localize at these promoters in vivo. The cas genes are transcribed as a polycistronic mRNA that reads through the leader into the CRISPR array, a putative σA-dependent promoter in the leader having negligible activity in vivo. Consequently, expression of the entire CRISPR-Cas system and mature CRISPR-RNA (crRNA) production is DdvS/CarD/CarG-dependent. DdvA likely uses its large C-terminal domain to sense and transduce the extracytoplasmic signal triggering CRISPR-cas expression, which we show is not starvation-induced multicellular development. An ECF-σ/anti-σ pair and a global regulatory complex provide an effective mechanism to coordinate signal-sensing with production of precursor crRNA, its processing Cas6 endoribonuclease and other Cas proteins for mature crRNA biogenesis and interference.

A New Facet of Vitamin B12: Gene Regulation by Cobalamin-Based Photoreceptors.

Living organisms sense and respond to light, a crucial environmental factor, using photoreceptors, which rely on bound chromophores such as retinal, flavins, or linear tetrapyrroles for light sensing. The discovery of photoreceptors that sense light using 5'-deoxyadenosylcobalamin, a form of vitamin B12 that is best known as an enzyme cofactor, has expanded the number of known photoreceptor families and unveiled a new biological role of this vitamin. The prototype of these B12-dependent photoreceptors, the transcriptional repressor CarH, is widespread in bacteria and mediates light-dependent gene regulation in a photoprotective cellular response. CarH activity as a transcription factor relies on the modulation of its oligomeric state by 5'-deoxyadenosylcobalamin and light. This review surveys current knowledge about these B12-dependent photoreceptors, their distribution and mode of action, and the structural and photochemical basis of how they orchestrate signal transduction and control gene expression.

Caulobacter crescentus CdnL is a non-essential RNA polymerase-binding protein whose depletion impairs normal growth and rRNA transcription.

CdnL is an essential RNA polymerase (RNAP)-binding activator of rRNA transcription in mycobacteria and myxobacteria but reportedly not in Bacillus. Whether its function and mode of action are conserved in other bacteria thus remains unclear. Because virtually all alphaproteobacteria have a CdnL homolog and none of these have been characterized, we studied the homolog (CdnLCc) of the model alphaproteobacterium Caulobacter crescentus. We show that CdnLCc is not essential for viability but that its absence or depletion causes slow growth and cell filamentation. CdnLCc is degraded in vivo in a manner dependent on its C-terminus, yet excess CdnLCc resulting from its stabilization did not adversely affect growth. We find that CdnLCc interacts with itself and with the RNAP ß subunit, and localizes to at least one rRNA promoter in vivo, whose activity diminishes upon depletion of CdnLCc. Interestingly, cells expressing CdnLCc mutants unable to interact with the RNAP were cold-sensitive, suggesting that CdnLCc interaction with RNAP is especially required at lower than standard growth temperatures in C. crescentus. Our study indicates that despite limited sequence similarities and regulatory differences compared to its myco/myxobacterial homologs, CdnLCc may share similar biological functions, since it affects rRNA synthesis, probably by stabilizing open promoter-RNAP complexes.

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.

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.

Structure-function dissection of Myxococcus xanthus CarD N-terminal domain, a defining member of the CarD_CdnL_TRCF family of RNA polymerase interacting proteins.

Two prototypes of the large CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-binding proteins, Myxococcus xanthus CarD and CdnL, have distinct functions whose molecular basis remain elusive. CarD, a global regulator linked to the action of several extracytoplasmic function (ECF) σ-factors, binds to the RNAP ß subunit (RNAP-ß) and to protein CarG via an N-terminal domain, CarDNt, and to DNA via an intrinsically unfolded C-terminal domain resembling eukaryotic high-mobility-group A (HMGA) proteins. CdnL, a CarDNt-like protein that is essential for cell viability, is implicated in σA-dependent rRNA promoter activation and interacts with RNAP-ß but not with CarG. While the HMGA-like domain of CarD by itself is inactive, we find that CarDNt has low but observable ability to activate ECF σ-dependent promoters in vivo, indicating that the C-terminal DNA-binding domain is required to maximize activity. Our structure-function dissection of CarDNt reveals an N-terminal, five-stranded ß -sheet Tudor-like domain, CarD1-72, whose structure and contacts with RNAP-ß mimic those of CdnL. Intriguingly, and in marked contrast to CdnL, CarD mutations that disrupt its interaction with RNAP-ß did not annul activity. Our data suggest that the CarDNt C-terminal segment, CarD61-179, may be structurally distinct from its CdnL counterpart, and that it houses at least two distinct and crucial function determinants: (a) CarG-binding, which is specific to CarD; and (b) a basic residue stretch, which is also conserved and functionally required in CdnL. This study highlights the evolution of shared and divergent interactions in similar protein modules that enable the distinct activities of two related members of a functionally important and widespread bacterial protein family.

Structural insights into RNA polymerase recognition and essential function of Myxococcus xanthus CdnL.

CdnL and CarD are two functionally distinct members of the CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-interacting proteins, which co-exist in Myxococcus xanthus. While CarD, found exclusively in myxobacteria, has been implicated in the activity of various extracytoplasmic function (ECF) σ-factors, the function and mode of action of the essential CdnL, whose homologs are widespread among bacteria, remain to be elucidated in M. xanthus. Here, we report the NMR solution structure of CdnL and present a structure-based mutational analysis of its function. An N-terminal five-stranded ß-sheet Tudor-like module in the two-domain CdnL mediates binding to RNAP-ß, and mutations that disrupt this interaction impair cell growth. The compact CdnL C-terminal domain consists of five α-helices folded as in some tetratricopeptide repeat-like protein-protein interaction domains, and contains a patch of solvent-exposed nonpolar and basic residues, among which a set of basic residues is shown to be crucial for CdnL function. We show that CdnL, but not its loss-of-function mutants, stabilizes formation of transcriptionally competent, open complexes by the primary σA-RNAP holoenzyme at an rRNA promoter in vitro. Consistent with this, CdnL is present at rRNA promoters in vivo. Implication of CdnL in RNAP-σA activity and of CarD in ECF-σ function in M. xanthus exemplifies how two related members within a widespread bacterial protein family have evolved to enable distinct σ-dependent promoter activity.

The CarD/CarG regulatory complex is required for the action of several members of the large set of Myxococcus xanthus extracytoplasmic function σ factors.

Extracytoplasmic function (ECF) σ factors are critical players in signal transduction networks involved in bacterial response to environmental changes. The Myxococcus xanthus genome reveals ∼45 putative ECF-σ factors, but for the overwhelming majority, the specific signals or mechanisms for selective activation and regulation remain unknown. One well-studied ECF-σ, CarQ, binds to its anti-σ, CarR, and is inactive in the dark but drives its own expression from promoter P(QRS) on illumination. This requires the CarD/CarG complex, the integration host factor (IHF) and a specific CarD-binding site upstream of P(QRS). Here, we show that DdvS, a previously uncharacterized ECF-σ, activates its own expression in a CarD/CarG-dependent manner but is inhibited when specifically bound to the N-terminal zinc-binding anti-σ domain of its cognate anti-σ, DdvA. Interestingly, we find that the autoregulatory action of 11 other ECF-σ factors studied here depends totally or partially on CarD/CarG but not IHF. In silico analysis revealed possible CarD-binding sites that may be involved in direct regulation by CarD/CarG of target promoter activity. CarD/CarG-linked ECF-σ regulation likely recurs in other myxobacteria with CarD/CarG orthologous pairs and could underlie, at least in part, the global regulatory effect of the complex on M. xanthus gene expression.