Biomedical subjects

F J Murillo

Publications and source records attributed to F J Murillo.

At least 19 recordsLinked to original sources

PubMed2001

Domain architecture of a high mobility group A-type bacterial transcriptional factor.

Myxococcus xanthus transcriptional factor CarD participates in carotenogenesis and fruiting body formation. It is the only reported prokaryotic protein having adjacent "AT-hook" DNA-binding and acidic regions characteristic of eukaryotic high mobility group A (HMGA) proteins. The latter are small, unstructured, nonhistone nuclear proteins that function as architectural factors to remodel DNA and chromatin structure and modulate various DNA binding activities. We find CarD to be predominantly dimeric with two stable domains: (a) an N-terminal domain of defined secondary and tertiary structure which is absent in eukaryotic HMGA proteins; (b) a C-terminal domain formed by the acidic and AT-hook segments and lacking defined structure. CarD, like HMGA proteins, binds specifically to the minor-groove of AT-rich DNA present in two appropriately spaced tracts. As in HMGA proteins, casein kinase II can phosphorylate the CarD acidic region, and this dramatically decreases the DNA binding affinity of CarD. The acidic region, in addition to modulating DNA binding, confers structural stability to CarD. We discuss how the structural and functional plasticity arising from domain organization in CarD could be linked to its role as a general transcriptional factor in M. xanthus.

Amino Acid Sequence↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed1998

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.

Binding Sites↗

PubMed1996

Prt1, an unusual retrotransposon-like sequence in the fungus Phycomyces blakesleeanus.

This work reports the isolation and structural characterization of Prt1, a 4.7 kb retrotransposon-like sequence from the filamentous fungus Phycomyces blakesleeanus. Two open reading frames are found within Prt1. The first shows no similarity with known genes. The second encodes peptide stretches similar to the reverse transcriptase and RNaseH domains of the Ty3/gypsy family of LTR-retrotransposons. Prt1 lacks long terminal repeats, having instead short (54 bp) terminal inverted repeats. No target site duplication has been found. A single copy of Prt1 was detected in the genome of P. blakesleeanus. Adjacent to this sole copy of Prt1, a cluster of various short sequence repeats, both direct and inverted, is found. These sequences, which are reminiscent of defective, non-retroviral transposable elements, are also represented in other regions of the P. blakesleeanus genome.

Amino Acid Sequence↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1995

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.

Alkyl and Aryl Transferases↗

PubMed1995

PkpA, a novel Phycomyces blakesleeanus serine/threonine protein kinase.

This work reports the cloning and sequencing of pkpA, a gene of the filamentous fungus Phycomyces blakesleeanus, whose expression seems to be coupled to vegetative growth. This gene encodes a putative serine/threonine-specific protein kinase, whose sequence is related to that of the yeast protein STE20, involved in pheromone-response pathways, and to a number of MAPK kinase proteins. However, detailed analysis of the kinase sequence suggests that PkpA is a novel serine/threonine protein kinase that probably participates as an intermediate in an intracellular system controlling nuclear proliferation in P. blakesleeanus.

Amino Acid Sequence↗

PubMed1994

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.

Carotenoids↗

PubMed1994

Isolation of the facA (acetyl-CoA synthetase) gene of Phycomyces blakesleeanus.

A 5.6 kb DNA fragment from the fungus Phycomyces blakesleeanus has been cloned and sequenced. The fragment contains a gene that probably codes for the enzyme acetyl-coenzyme A synthetase (facA). The amino acid sequence deduced for the P. blakesleeanus protein is highly homologous to those of acetyl-coA-synthetases from other organisms. When placed under the control of a constitutive promoter from Aspergillus nidulans, the cloned gene complemented a facA- mutation of this organism. In P. blakesleeanus, the expression of facA is induced by acetate.

Acetate-CoA Ligase↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

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.

Carbon↗

PubMed1990

Accumulation of carotenoids in structural and regulatory mutants of the bacterium Myxococcus xanthus.

Accumulation of carotenoids in Myxococcus xanthus is absolutely dependent on illumination with blue light. We report the analysis of the carotenoids of dark- and light-grown cultures of the wild type and several previously characterized mutants. A carR mutant produces the same carotenoids in the dark as the wild type grown in the light. This agrees with previous evidence indicating that the carR gene codes for a general negative regulator of the system. A cis-dominant mutation in the gene carA causes constitutive expression of the light-inducible gene carB, which is linked to carA. In the dark, the carA mutant produces high levels of phytoene, the first C40 colourless carotenoid precursor; in the light, it produces the same carotenoids as the wild type. Since a mutation in carB blocks accumulation of phytoene, we propose that carB, and probably other linked genes also controlled by carA, code for enzymes involved in the synthesis of phytoene. This is virtually the only carotene accumulated by strains mutated in the gene carC, which is unlinked to the others. Thus carC codes for phytoene dehydrogenase, the enzyme that converts phytoene into coloured carotenoids. The results presented here also provide evidence for control of carotenogenesis by an endproduct that is independent of the blue light effect.

Carotenoids↗

PubMed1990

A cytoplasmically inherited mutation in the fungus Phycomyces blakesleeanus.

Fourteen mutants of the fungus Phycomyces blakesleeanus, showing high levels of resistance to copper, were isolated. In all the mutants, copper resistance behaved as a very variable and unstable trait. In the mutant strain MU102, the mutation was demonstrated to be cytoplasmically inherited. In addition, this mutant strain differed from the wild-type in growth, respiration rate, and shape and viability of spores.

Copper↗

PubMed1988

Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanus.

A plasmid, carrying the Tn5 gene for kanamycin resistance lacking its own promoter, has successfully been used in the selection of DNA sequences of the fungus Phycomyces blakesleeanus having promoter activity in Escherichia coli. Many of these sequences were also effective in promoting resistance to kanamycin when the corresponding chimeric plasmids were introduced in the fungus via spheroplast transformation. The selected phenotype was easily propagated through vegetative spores and behaved as a stable character since it was not appreciably lost in the absence of selection.

DNA Transposable Elements↗

PubMed1987

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.

Journal Article↗

PubMed1986

A method for the selection of mutants of Phycomyces blakesleeanus defective in germination.

A simple method for the physical separation of dormant and germinated cells of Phycomyces blakesleeanus has been designed. Separation was attained by isopycnic centrifugation in a preformed discontinuous density gradient of Urografin. In this gradient, dormant spores and active cells at two different stages of germination were separated in three homogeneous and clearly distinct bands. The method has been used to follow the kinetics of the variation of cellular density during germination of wild type spores and to select for mutants temperature-sensitive for heat-shock induced germination. Sixty-nine mutants have been isolated by various procedures which include a separation step in the described Urografin gradient.

Centrifugation, Density Gradient↗

PubMed1986

Characterization of Phycomyces blakesleeanus mutants temperature-sensitive for heat-shock induced germination.

Sixty-nine mutants of the fungus Phycomyces blakesleeanus, temperature-sensitive for heat-shock induced germination, have been characterized. All of them show a low viability at 26 degrees C and normal viability at 16 degrees C. Eleven mutants recover the wild type phenotype if yeast extract is added to the minimal medium; the mutant phenotype of eight of these mutants is also suppressed by the addition of putrescine or other polyamines. The majority of the mutants are affected very early in germination. Spontaneous, heat-shock and acetate induced germination are not equally impaired by some of the mutations, so specific and independent steps seem to be involved in part of the activation mechanism of germination.

Genes, Fungal↗

PubMed1984

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.

Bacteriophages↗