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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., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

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 β subunit (RNAP-β) 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-β 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 β -sheet Tudor-like domain, CarD1-72, whose structure and contacts with RNAP-β mimic those of CdnL. Intriguingly, and in marked contrast to CdnL, CarD mutations that disrupt its interaction with RNAP-β 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.

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 β-sheet Tudor-like module in the two-domain CdnL mediates binding to RNAP-β, 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.

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., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

CdnL, an essential protein in Myxococcus xanthus and several other bacteria, is a member of the large CarD_TRCF family of bacterial proteins that interact with RNA polymerase. Structural analyses of the 164-residue M. xanthus CdnL by NMR is complicated because of broadening, and hence overlap, of the signals due to the self-association and the monomer-dimer equilibrium that occurs in solution. Here, we report (1)H, (13)C and (15)N assignments for CdnL achieved by analyzing its NMR spectra on the basis of the complete assignment obtained in this study for the 68-residue N-terminal fragment of CdnL (CdnLNt) together with those we described previously for the stable, protease-resistant, 110-residue C-terminal domain (CdnLCt). This approach relied on our observation that many of the CdnLNt and CdnLCt chemical shifts matched closely with those of the equivalent residues in the full-length protein. Our assignments provide the crucial first step in the structural analysis of CdnL and this functionally important family of bacterial proteins.

The CarD-CarG complex controls various cellular processes in the bacterium Myxococcus xanthus including fruiting body development and light-induced carotenogenesis. The CarD N-terminal domain, which defines the large CarD_CdnL_TRCF protein family, binds to CarG, a zinc-associated protein that does not bind DNA. The CarD C-terminal domain resembles eukaryotic high-mobility-group A (HMGA) proteins, and its DNA binding AT hooks specifically recognize the minor groove of appropriately spaced AT-rich tracts. Here, we investigate the determinants of the only known CarD binding site, the one crucial in CarD-CarG regulation of the promoter of the carQRS operon (P(QRS)), a light-inducible promoter dependent on the extracytoplasmic function (ECF) σ factor CarQ. In vitro, mutating either of the 3-bp AT tracts of this CarD recognition site (TTTCCAGAGCTTT) impaired DNA binding, shifting the AT tracts relative to P(QRS) had no effect or marginally lowered DNA binding, and replacing the native site by the HMGA1a binding one at the human beta interferon promoter (with longer AT tracts) markedly enhanced DNA binding. In vivo, however, all of these changes deterred P(QRS) activation in wild-type M. xanthus, as well as in a strain with the CarD-CarG pair replaced by the Anaeromyxobacter dehalogenans CarD-CarG (CarD(Ad)-CarG(Ad)). CarD(Ad)-CarG(Ad) is functionally equivalent to CarD-CarG despite the lower DNA binding affinity in vitro of CarD(Ad), whose C-terminal domain resembles histone H1 rather than HMGA. We show that CarD physically associates with RNA polymerase (RNAP) specifically via interactions with the RNAP β subunit. Our findings suggest that CarD regulates a light-inducible, ECF σ-dependent promoter by coupling RNAP recruitment and binding to a specific DNA site optimized for affinity and position.

Blue light triggers carotenogenesis in the nonphototrophic bacterium Myxococcus xanthus by inducing inactivation of an anti-σ factor, CarR, and the consequent liberation of the cognate extracytoplasmic function (ECF) σ factor, CarQ. CarF, the protein implicated earliest in the response to light, does not resemble any known photoreceptor. It interacts physically with CarR and is required for its light-driven inactivation, but the mechanism is unknown. Blue-light sensing in M. xanthus has been attributed to the heme precursor protoporphyrin IX (PPIX), which can generate the highly reactive singlet oxygen species ((1)O(2)) by energy transfer to oxygen. However, (1)O(2) involvement in M. xanthus light-induced carotenogenesis remains to be established. Here, we present genetic evidence of the involvement of PPIX as well as (1)O(2) in light-induced carotenogenesis in M. xanthus and of how these are linked to CarF in the signal transduction pathway. Response to light was examined in carF-bearing and carF-deficient M. xanthus strains lacking endogenous PPIX due to deletion of hemB or accumulating PPIX due to deletion of hemH (hemB and hemH are early- and late-acting heme biosynthesis genes, respectively). This demonstrated that light induction of the CarQ-dependent promoter, P(QRS), correlated directly with cellular PPIX levels. Furthermore, we show that P(QRS) activation is triggered by (1)O(2) and is inhibited by exogenously supplied hemin and that CarF is essential for the action of (1)O(2). Thus, our findings indicate that blue light interaction with PPIX generates (1)O(2), which must be transmitted via CarF to trigger the transcriptional response underlying light-induced carotenogenesis in M. xanthus.

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.

A global regulatory complex made up of two unconventional transcriptional factors, CarD and CarG, is implicated in the control of various processes in Myxococcus xanthus, a Gram-negative bacterium that serves as a prokaryotic model system for multicellular development and the response to blue light. CarD has a unique two-domain architecture composed of: (1) a C-terminal DNA-binding domain that resembles eukaryotic high mobility group A (HMGA) proteins, which are relatively abundant, nonhistone components of chromatin that remodel DNA and prime it for the assembly of multiprotein-DNA complexes essential for various DNA transactions, and (2) an N-terminal domain involved in interactions with CarG and RNA polymerase, which is also the founding member of the large CarD_TRCF family of bacterial proteins. CarG, which does not bind DNA directly, has a zinc-binding motif of the type found in the archaemetzincin class of metalloproteases that, in CarG, appears to play a purely structural role. This review aims to provide an overview of the known molecular details and insights emerging from the study of the singular CarD-CarG prokaryotic regulatory complex and its parallels with enhanceosomes, the higher order, nucleoprotein transcription complexes in eukaryotes.

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.

Histone H1 and high-mobility group A (HMGA) proteins compete dynamically to modulate chromatin structure and regulate DNA transactions in eukaryotes. In prokaryotes, HMGA-like domains are known only in Myxococcus xanthus CarD and its Stigmatella aurantiaca ortholog. These have an N-terminal module absent in HMGA that interacts with CarG (a zinc-associated factor that does not bind DNA) to form a stable complex essential in regulating multicellular development, light-induced carotenogenesis, and other cellular processes. An analogous pair, CarD(Ad) and CarG(Ad), exists in another myxobacterium, Anaeromyxobacter dehalogenans. Intriguingly, the CarD(Ad) C terminus lacks the hallmark HMGA DNA-binding AT-hooks and instead resembles the C-terminal region (CTR) of histone H1. We find that CarD(Ad) alone could not replace CarD in M. xanthus. By contrast, when introduced with CarG(Ad), CarD(Ad) functionally replaced CarD in regulating not just 1 but 3 distinct processes in M. xanthus, despite the lower DNA-binding affinity of CarD(Ad) versus CarD in vitro. The ability of the cognate CarD(Ad)-CarG(Ad) pair to interact, but not the noncognate CarD(Ad)-CarG, rationalizes these data. Thus, in chimeras that conserve CarD-CarG interactions, the H1-like CTR of CarD(Ad) could replace the CarD HMGA AT-hooks with no loss of function in vivo. More tellingly, even chimeras with the CarD AT-hook region substituted by human histone H1 CTR or full-length H1 functioned in M. xanthus. Our domain-swap analyses showing functional equivalence of HMGA AT-hooks and H1 CTR in prokaryotic transcriptional regulation provide molecular insights into possible modes of action underlying their biological roles.

CdnL, a 164-residue protein essential for Myxococcus xanthus viability, is a member of a large family of bacterial proteins of unknown structure and function. Here, we report the (1)H, (13)C and (15)N backbone and side chain assignments for the stable C-terminal domain of CdnL identified by limited proteolysis.

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 B12 and CarH, a MerR-type transcriptional repressor, together downregulate a photoinducible promoter. Using this system, we confirmed that M. xanthus rpoN, encoding sigma(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. B12-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 sigma(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.

A light-inducible promoter, P(B), drives expression of the carB operon in Myxococcus xanthus. Repressed by CarA in the dark, P(B) 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 P(B). Intriguingly, P(B) 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 P(B) 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.

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 alpha2 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.

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.

Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dis-mantle CarA-DNA complexes. Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.

The carB operon encodes all except one of the enzymes involved in light-induced carotenogenesis in Myxococcus xanthus. Expression of its promoter (P(B)) is repressed in the dark by sequence-specific DNA binding of CarA to a palindrome (pI) located between positions -47 and -64 relative to the transcription start site. This promotes subsequent binding of CarA to additional sites that remain to be defined. CarS, produced in the light, interacts physically with CarA, abrogates CarA-DNA binding, and thereby derepresses P(B). In this study, we delineate the operator design that exists for CarA by precisely mapping out the second operator element. For this, we examined how stepwise deletions and site-directed mutagenesis in the region between the palindrome and the transcription start site affect CarA binding around P(B) in vitro and expression of P(B) in vivo. These revealed the second operator element to be an imperfect interrupted palindrome (pII) spanning positions -26 to -40. In vitro assays using purified M. xanthus RNA polymerase showed that CarA abolishes P(B)-RNA polymerase binding and runoff transcription and that both were restored by CarS, thus rationalizing the observations in vivo. CarA binding to pII (after association with pI) effectively occludes RNA polymerase from P(B) and so provides the operative mechanism for the repression of the carB operon by CarA. The bipartite operator design, whereby transcription is blocked by the low affinity CarA-pII binding and is readily restored by CarS, may have evolved to match the needs for a rapid and an effective response to light.

Myxococcus xanthus, a member of the Proteobacteria delta-class, has two independent recA genes, recA1 and recA2, but only recA2 is DNA damage-inducible. The lexA gene has been isolated from M. xanthus by PCR amplification with oligonucleotides designed after sequence identification by tblastn analysis of its genome at the Cereon Microbial Sequence Database. The M. xanthus purified LexA protein is shown to bind specifically to the consensus sequence CTRHAMRYBYGTTCAGS present upstream of lexA and recA2. A degenerate copy of this motif but with important differences can be identified in the region upstream of the recA1 gene. A knock-out lexA(Def) mutant that has been generated does not differ significantly from wild type in morphology, growth rate, light-induced carotenogenesis or development. Using transcriptional lacZ fusions and quantitative RT-PCR analysis, it has been demonstrated that expression of both lexA and recA2 genes is constitutive in the lexA(Def) mutant, whereas the transcription of the DNA damage non-inducible recA1 gene is not affected in this strain. recN and ssb, whose expression in Escherichia coli are LexA-regulated, are induced by DNA damage in the M. xanthus lexA(Def) mutant. These data reveal the existence of different regulatory mechanisms for DNA damage-inducible genes in bacteria belonging to different phyla.

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 (P(B)) in the dark, and CarS counteracts this on illumination. The CarA sequence revealed a helix-turn-helix DNA-binding motif of the type found in bacterial MerR transcriptional factors, whereas CarS contains no known DNA-binding motif. Here, we examine the molecular interplay between CarA and CarS. We demonstrate the following. (i) Whereas CarS exhibits no DNA binding in vitro, CarA binds specifically to a region encompassing P(B) to form at least two distinct complexes. (ii) A palindrome located between positions -46 and -63 relative to the transcription start point is essential but not sufficient for the formation of the two CarA-DNA complexes observed. (iii) CarS abrogates the specific DNA binding of CarA. CarA is therefore a repressor and CarS an antirepressor. (iv) CarS physically interacts with CarA; thus, the functional interaction between them is mediated by protein-protein interactions.

Summary: 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. [ABSTRACT FROM AUTHOR]

Extracytoplasmic function ( ECF) σ factors are critical players in signal transduction networks involved in bacterial response to environmental changes. The M yxococcus 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 PQRS on illumination. This requires the CarD/ CarG complex, the integration host factor ( IHF) and a specific CarD-binding site upstream of PQRS. 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. [ABSTRACT FROM AUTHOR]

CdnL, an essential protein in Myxococcus xanthus and several other bacteria, is a member of the large CarD_TRCF family of bacterial proteins that interact with RNA polymerase. Structural analyses of the 164-residue M. xanthus CdnL by NMR is complicated because of broadening, and hence overlap, of the signals due to the self-association and the monomer-dimer equilibrium that occurs in solution. Here, we report H, C and N assignments for CdnL achieved by analyzing its NMR spectra on the basis of the complete assignment obtained in this study for the 68-residue N-terminal fragment of CdnL (CdnLNt) together with those we described previously for the stable, protease-resistant, 110-residue C-terminal domain (CdnLCt). This approach relied on our observation that many of the CdnLNt and CdnLCt chemical shifts matched closely with those of the equivalent residues in the full-length protein. Our assignments provide the crucial first step in the structural analysis of CdnL and this functionally important family of bacterial proteins. [ABSTRACT FROM AUTHOR]

The CarS antirepressor activates a photo-inducible promoter in Myxococcus xanthus by physically interacting with the CarA repressor and eliminating the latter’s binding to operator DNA. Interestingly, interactions with both CarS and operator are crucially dependent on the DNA recognition helix of the CarA winged-helix DNA-binding domain. The CarA–CarS and the CarA-operator interfaces therefore overlap, and CarS may have structural features that mimic operator DNA. CarS has no known sequence homologues and its Gly and Pro contents are unusually high. Here, we report 1H, 13C and 15N backbone and side chain assignments of CarS1, an 86-residue truncated yet fully functional variant of CarS. Secondary structural elements inferred from these data differ from those predicted from sequence. [ABSTRACT FROM AUTHOR]

CdnL, a 164-residue protein essential for Myxococcus xanthus viability, is a member of a large family of bacterial proteins of unknown structure and function. Here, we report the 1H, 13C and 15N backbone and side chain assignments for the stable C-terminal domain of CdnL identified by limited proteolysis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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[sub Sa], that is very similar to Card and that occurs in the bacterium Stigmatella aurantiaca. CarD[sub 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[sub 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[sub 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. [ABSTRACT FROM AUTHOR]