The Regulation of LexA on UV-Induced SOS Response in Myxococcus xanthus Based on Transcriptome Analysis.

SOS response is a conserved response to DNA damage in prokaryotes and is negatively regulated by LexA protein, which recognizes specifically an "SOS-box" motif present in the promoter region of SOS genes. Myxococcus xanthus DK1622 possesses a lexA gene, and while the deletion of lexA had no significant effect on either bacterial morphology, UV-C resistance, or sporulation, it did delay growth. UV-C radiation resulted in 651 upregulated genes in M. xanthus, including the typical SOS genes lexA, recA, uvrA, recN and so on, mostly enriched in the pathways of DNA replication and repair, secondary metabolism, and signal transduction. The UV-irradiated lexA mutant also showed the induced expression of SOS genes and these SOS genes enriched into a similar pathway profile to that of wild-type strain. Without irradiation treatment, the absence of LexA enhanced the expression of 122 genes that were not enriched in any pathway. Further analysis of the promoter sequence revealed that in the 122 genes, only the promoters of recA2, lexA and an operon composed of three genes (pafB, pafC and cyaA) had SOS box sequence to which the LexA protein is bound directly. These results update our current understanding of SOS response in M. xanthus and show that UV induces more genes involved in secondary metabolism and signal transduction in addition to DNA replication and repair; and while the canonical LexA-dependent regulation on SOS response has shrunk, only 5 SOS genes are directly repressed by LexA.

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.

High-mobility-group a-like CarD binds to a DNA site optimized for affinity and position and to RNA polymerase to regulate a light-inducible promoter in Myxococcus xanthus.

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 ß 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.

An anti-antisigma factor in the response of the bacterium Myxococcus xanthus to blue light.

Cells of the Gram-negative bacterium Myxococcus xanthus respond to blue light by producing carotenoids, pigments that play a protective role against the oxidative effects of light. Blue light triggers a network of regulatory actions that lead to the transcriptional activation of the structural genes for carotenoid synthesis. The product of carF, similar to a family of proteins of unknown function called Kua, is an early regulator of this process. Previous genetic data indicate that CarF participates in the light-dependent inactivation of the antisigma factor CarR. In the dark, CarR sequesters the ECF-sigma factor CarQ to the membrane, thereby preventing the activation of the structural genes for carotenoid synthesis. Using a bacterial two-hybrid system, we show here that both CarF and CarQ physically interact with CarR. These results, together with the finding that CarF is located at the membrane, support the hypothesis that CarF acts as an anti-antisigma factor. Comparison of CarF with other Kua proteins shows a remarkable conservation of a number of histidine residues. The effects on CarF function of several histidine to alanine substitutions and of the truncation of specific CarF domains are also reported here.

Light-induced carotenogenesis in Myxococcus xanthus: functional characterization of the ECF sigma factor CarQ and antisigma factor CarR.

Illumination of dark-grown Myxococcus xanthus with blue light leads to the induction of carotenoid synthesis. Central to this response is the activation of the light-inducible promoter, PcarQRS, and the transcription of three downstream genes, carQ, carR and carS. Sequence analysis predicted that CarQ is a member of the ECF (extracytoplasmic function) subfamily of RNA polymerase sigma factors, and that CarR is an inner membrane protein. Genetic analysis strongly implied that CarR is an antisigma factor that sequesters CarQ in a transcriptionally inactive complex. Using in vitro transcription run-off assays, we present biochemical evidence that CarQ functions as a bacterial sigma factor and is responsible for transcription initiation at PcarQRS. Similar experiments using the crtI promoter failed to implicate CarQ in direct transcription of the crtI gene. Experiments using the yeast two-hybrid system demonstrated a protein-protein interaction between CarQ and CarR, providing evidence of a CarQ-CarR complex. The yeast two-hybrid system data also indicated that CarR is capable of oligomerization. Fractionation of M. xanthus membranes with the detergent sarkosyl showed that CarR was associated with the inner membrane. Furthermore, CarR was found to be unstable in illuminated stationary phase cells, providing a possible mechanism by which the CarR-CarQ complex is disrupted.

ihfA gene of the bacterium Myxococcus xanthus and its role in activation of carotenoid genes by blue light.

Myxococcus xanthus responds to blue light by producing carotenoids. Several regulatory genes are known that participate in the light action mechanism, which leads to the transcriptional activation of the carotenoid genes. We had already reported the isolation of a carotenoid-less, Tn5-induced strain (MR508), whose mutant site was unlinked to the indicated regulatory genes. Here, we show that OmegaMR508::Tn5 affects all known light-inducible promoters in different ways. It blocks the activation of two of them by light but makes the activity of a third one light independent. The OmegaMR508 locus has been cloned and sequenced. The mutation had occurred at the promoter of a gene we propose is the M. xanthus ortholog of ihfA. This encodes the alpha subunit of the histone-like integration host factor protein. An in-frame deletion within ihfA causes the same effects as the OmegaMR508::Tn5 insertion. Like other IhfA proteins, the deduced amino acid sequence of M. xanthus IhfA shows much similarity to HU, another histone-like protein. Sequence comparison data, however, and the finding that the M. xanthus gene is preceded by gene pheT, as happens in other gram-negative bacteria, strongly argue for the proposed orthology relationship. The M. xanthus ihfA gene shows some unusual features, both from structural and physiological points of view. In particular, the protein is predicted to have a unique, long acidic extension at the carboxyl terminus, and it appears to be necessary for normal cell growth and even vital for a certain wild-type strain of M. xanthus.

A cluster of structural and regulatory genes for light-induced carotenogenesis in Myxococcus xanthus.

In the bacterium Myxococcus xanthus, several genes for carotenoid synthesis lie together at the carA-carB chromosomal locus and are co-ordinately activated by blue light. A 12-kb DNA stretch from wild-type M. xanthus has been sequenced that includes the entire carA-carB gene cluster. According to sequence analysis, the cluster contains 11 different genes. Intergenic distances are very short or nil (implying translational coupling), giving further support to previous evidence indicating that most (or all) of the genes in the cluster form a single operon. At the promoter region, a potential -35 site for the binding of sigma factors is found. However, the -10 region shows little similarity with analogous sites in other bacterial promoters. Five (possibly six) genes in the carA-carB operon code for enzymes acting on early or late steps of the pathway for carotenoid synthesis. Other genes in the operon show no overall similarity with previously known genes. However, peptide stretches in the predicted products of two genes exhibit strong similarity with the DNA binding domain of the MerR family of transcriptional regulators. At least one of the predicted DNA-binding domains is altered in a mutant strain affected in light-regulation of the car genes.

Two recA genes in Myxococcus xanthus.

Two recA genes, recA1 and recA2, in Myxococcus xanthus were cloned by using the recA gene of Escherichia coli, and their DNA sequences were determined. On the basis of deduced amino acid sequences, RecA1 and RecA2 have 67.0% identity to each other and 60.5 and 60.9% identities to E. coli RecA, respectively. Expression of recA2 was detected in both vegetative and developmental cells by Northern blot (RNA) analysis, and a threefold induction was observed when cells were treated with nalidixic acid. Repeated attempts to isolate a recA2 disruption mutant have failed, while a recA1 disruption mutant was readily isolated. Both the recA1 and recA2 genes expressed in E. coli complement the UV sensitivity of an E. coli recA strain.

A genetic link between light response and multicellular development in the bacterium Myxococcus xanthus.

The Gram-negative bacterium Myxococcus xanthus responds to blue light by producing carotenoid pigments (Car+ phenotype). Genes for carotenoid synthesis lie at two unlinked chromosomal sites, the carC and the carBA operon, but are integrated in a single "light regulon" by the action of common trans-acting regulatory elements. Three known regulatory genes are grouped together at the (light-inducible) carQRS operon. By screening the Car phenotype of a large collection of transposon-induced mutants, we have identified a new car locus that has been named carD (carD1 for the mutant allele). The carD gene product plays a critical role in the light regulon, as it is required for activation of the carQRS and carC promoters by blue light. The carD1 mutant is impaired in the (starvation-induced) developmental process that allows M. xanthus cells both to form multicellular fruiting bodies and to sporulate. Our results indicate that the carD gene product is also required for the expression of a particular set of development-specific genes that are normally activated through the action of intercellular signals.

Light-induced carotenogenesis in Myxococcus xanthus: DNA sequence analysis of the carR region.

The carR region encodes a light-inducible promoter, a negative regulator of the promoter and a trans-acting activator that controls the light-inducible Myxococcus xanthus carotenoid biosynthesis regulon. DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes, carQ, carR and carS. Sequencing of mutations demonstrated that carR encoded the negative regulator and was an integral membrane protein. Mutant construction and sequencing revealed that carS was the trans-acting activator and that carQ was a positive regulator of the promoter. Neither gene encodes proteins with known sequence-specific DNA-binding motifs. The sequence of the light-inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of the Escherichia coli stress response ('heat-shock') promoters.

Clustering and co-ordinated activation of carotenoid genes in Myxococcus xanthus by blue light.

Blue light activates carotenoid production in the non-photosynthetic, Gram-negative bacterium Myxococcus xanthus. Light is known to stimulate the expression of two unlinked genes for carotenoid synthesis, carB and carC, through a mechanism in which the regulatory genes carA, carQ and carR take part. Genes carQ and carR are linked together at a separate locus, whereas carA is linked to carB. We have introduced Tn5 at various sites between carA and carB. Chemical analyses of the mutant strains demonstrate the presence in this region of a cluster of genes for carotenoid synthesis. Gene expression analysis strongly argues for most (or all) of the genes in the cluster being transcribed from a single, light-inducible promoter under the control of genes carA, carQ and carR.

Light-induced carotenogenesis in Myxococcus xanthus: genetic analysis of the carR region.

Carotenogenesis is light-inducible in the non-photosynthetic, Gram-negative, bacterium Myxococcus xanthus. We report the characterization of the carR region which controls this phenomenon. Insertion of transposon Tn5 close to the carR region caused a dominant, carotenoid-constitutive mutation because of the presence of a constitutive, outward-reading promoter in the IS50L component of Tn5. In wild-type cells, a powerful, tightly-regulated, light-inducible promoter directs the transcription of two genetic functions. One of these functions is to activate transcription of the genetically unlinked carB gene, which is involved in carotenoid synthesis. The second function (carR) regulates the light-inducible promoter. We also report the mapping of two carotenoid constitutive mutations to the previously characterized carA locus.