A negative regulator of light-inducible carotenogenesis in Mucor circinelloides.

Mucor circinelloides responds to blue light by activating carotene biosynthesis. Wild-type strains grown in darkness contain minimal amounts of beta-carotene because of the low levels of transcription of the structural genes for carotenogenesis. When exposed to a light pulse, the level of transcription of these genes increases strongly, leading to the formation of high concentrations of beta-carotene. The crgA gene is involved in the regulation of light-induced carotenoid biosynthesis. This gene, originally identified as a 3'-truncated ORF which causes carotene over-accumulation in the dark, encodes a protein with a cysteine-rich, zinc-binding, RING-finger motif, as found in diverse groups of regulatory proteins. The expression of the crgA gene is activated by a light pulse, with a time course similar to that of the structural genes for carotenogenesis. To understand the regulatory role of the crgA gene in carotenogenesis, we have used a genetic approach based on the construction of crgA null mutants by gene replacement. Lack of the crgA function provokes the over-accumulation of carotenoids both in the dark and the light. Introduction of the wild-type crgA allele into these mutants restores the wild-type phenotype for carotenogenesis. The high levels of carotenoid accumulation shown by the null crgA mutants are correlated with an increase in the expression of carotenogenic structural genes. These results strongly indicate that crgA acts as a negative regulator of light-inducible carotenogenesis in M. circinelloides.

The structure of an ECF-sigma-dependent, light-inducible promoter from the bacterium Myxococcus xanthus.

Expression of the Myxococcus xanthus gene crtl is controlled by a light-inducible promoter. The activity of this promoter depends on CarQ, a sigma factor of the extracytoplasmic function (ECF) subfamily. Here, we show thatthe minimum DNA stretch reproducing normal expression of crtl extends from a few bases upstream of the -35 position to a site well downstream of the transcriptional start. The downstream DNA contains an enhancer-like element that remains active when displaced upstream of the promoter. Experimental evidence is provided for the activity of the crtl promoter being critically dependent on a pentanucleotide sequence centred at the -31 position. The similarity of this sequence with the consensus for ECF-sigma-dependent promoters from other bacteria is discussed. The activity of the crtl promoter also depends on certain basepairs at the -10 region. Hence, the operation of ECF-sigma-factors seems to require binding to two different DNA sites, although the -10 sequences of different ECF-sigma-dependent promoters are unrelated to one another, and the ECF-sigma-factors themselves lack the conserved domain known to mediate binding of other sigma-factors to the -10 DNA site.

High mobility group I(Y)-like DNA-binding domains on a bacterial transcription factor.

The bacterium Myxococcus xanthus responds to blue light by producing carotenoids. It also responds to starvation conditions by developing fruiting bodies, where the cells differentiate into myxospores. Each response entails the transcriptional activation of a separate set of genes. However, a single gene, carD, is required for the activation of both light- and starvation-inducible genes. Gene carD has now been sequenced. Its predicted amino acid sequence includes four repeats of a DNA-binding domain present in mammalian high mobility group I(Y) proteins and other nuclear proteins from animals and plants. Other peptide stretches on CarD also resemble functional domains typical of eukaryotic transcription factors, including a very acidic region and a leucine zipper. High mobility group yI(Y) proteins are known to bind the minor groove of A+T-rich DNA. CarD binds in vitro an A+T-rich element that is required for the proper operation of a carD-dependent promoter in vivo.

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 induction of gene expression in Myxococcus xanthus.

The synthesis of carotenoids by Myxococcus xanthus requires illumination with blue light. Mutations at two loci (carA and carR) remove the blue-light requirement and cause constitutive production of carotenoids. Mutations at a different locus (carB) prevent carotenogenesis in both wild-type and constitutive mutant strains. We describe here three independent car mutations induced by insertion of Tn5 lac, a transposon that carries a transcriptional probe for exogenous promoters. All three transposon insertions block carotenogenesis even in constitutive mutant strains. One insertion is in a previously unknown car gene and the other two are in the carB locus. One of the carB insertions expresses beta-galactosidase at very low levels in the dark but is strongly activated by light. When this Tn5 lac insertion is introduced in carA or carR mutants it expresses beta-galactosidase in dark- as well as light-grown cells. We conclude that carotenogenesis in M. xanthus is activated at the level of transcription by a light-induced mechanism in which the carA and the carR loci (or their gene products) take part. The potential usefulness of M. xanthus as a simple and sensitive tool for studies in photobiology is discussed.