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 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.

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.

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.

Growth phase dependence of the activation of a bacterial gene for carotenoid synthesis by blue light.

Myxococcus xanthus responds to blue light by producing carotenoid pigments. A mutation at a gene named carC is known to block the metabolism of phytoene, a carotenoid precursor, and this gene has now been cloned and sequenced. We show here that gene carC, which is homologous to phytoene dehydrogenase genes from other organisms, is tightly regulated by light through a mechanism that operates only when the cells have reached the stationary phase or are starved of a carbon source. A genetic element that mediates the effect of the growth phase has been identified. Gene carC is integrated with another unlinked carotenogenic gene in a single 'light regulon' controlled by common trans-acting genetic elements. A potential -35 site for the binding of sigma factors has been found upstream of the carC transcriptional start. However, the -10 region shows no similarity with analogous sites at promoters of other Gram-negative bacteria.

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.

Abnormal motility and fruiting behavior of Myxococcus xanthus bacteriophage-resistant strains induced by a clear-plaque mutant of bacteriophage Mx8.

Myxococcus xanthus mutants resistant to a clear-plaque derivative of phage Mx8 were isolated. A significant fraction of the mutants, easily recognizable by their colony morphology, were induced by the presence of the phage and may correspond to low-frequency lysogens. They were all defective in cell motility and showed the same nonfruiting phenotype under starvation conditions.

Construction of chimeric plasmids containing histone H5 cDNA from hen erythrocyte. DNA sequence of a fragment derived from the 5' region of H5 mRNA.

We report that construction and characterization of chicken erythrocyte histone H5 cDNA recombinant plasmids. cDNA was synthesized from poly(A)+ polysomal RNA enriched in H5 mRNA and inserted into the PstI site of pBR322. Several clones containing H5 cDNA sequences were obtained and one of them (p541), expressing H5 antigenic determinants, was sequenced. The DNA insert of p541 contains 118 nucleotides from the 5' non-translated region of H5 mRNA and sequences coding for up to residue 46 of the N-terminus of the arginine (position 15) H5 variant. There is a strikingly high number of repeated sequences both in the leader and coding region; among these, the octanucleotide 5' GCG GCG GC 3' is found five times along the sequence. Although the H5 mRNA 5' leader is GC-rich (66%), there is an AT-rich region, about 16 nucleotides long, which shares strong homology with the leaders of sea urchin histone H1 mRNAs.

An Escherichia coli mutant refractory to nitrosoguanidine mutagenesis.

A newly-isolated Escherichia coli mutant suffers only about 10% as many mutations as normal strains on exposure to nitrosoguanidine. The responsible mutation, inm-1, maps at approximately minute 79 in the current E. coli genetic map. The mutant is normal for overall growth, nitrosoguanidine lethality, spontaneous mutagenesis, ultraviolet light lethality and mutagenesis, ethyl methanesulfonate lethality and mutagenesis, and the adaptive repair induced by alkylating agents. The existence of this mutation proves that nitrosoguanidine mutagenesis is not merely the result of reactions between the chemical and DNA, but requires specific cellular function(s), and underscores the peculiarity of nitrosoguanidine as a mutagen.

A mutagen assay detecting forward mutations in an arabinose-sensitive strain of Salmonella typhimurium.

Strain SV3 of Salmonella typhimurium is sensitive to arabinose, that is, unable to grow in a medium containing arabinose plus glycerol as carbon source. Arabinose resistance is the consequence of the mutational inactivation of one of at least three different genes. The selection of arabinose-resistant mutants provides a simple and sensitive assay for the detection of weak mutagens and for refined quantitative studies of strong ones. The assay is not influenced by experimental artifacts derived from physiological or lethal effects or from differences in plating density. Such artifacts are common with other bacterial mutagen assays, including those using strains analogous to SV3. As practical examples, the assay was used with N-methyl-N'-nitro-N-nitrosoguanidine and the fungicide captafol.