Maria Isabel Alvarez

Blue-light regulation of phytoene dehydrogenase (carB) gene expression in Mucor circinelloides.

Abstract. The carB gene, encoding the phytoene dehydrogenase of Mucor circinelloides, was isolated by heterologous hybridisation with a probe derived from the corresponding gene of Phycomyces blakesleeanus. The cDNA and genomic copies complemented phytoene dehydrogenase defects in Escherichia coli and in carB mutants of M. circinelloides, respectively. Fluence-response curves for transcript accumulation were constructed after different blue-light pulses. The level of carB mRNA accumulation reached values up to 150-fold higher than basal levels in darkness. Several elements in the promoter of this gene resemble a consensus sequence identified in Neurospora crassa (APE) which is essential for blue-light regulation. Comparison of the available phytoene dehydrogenase sequences from plants, fungi, algae and bacteria suggests that the two known types of phytoene dehydrogenase are more closely related to each other than previously thought.

Heterologous transformation of Mucor circinelloides with the Phycomyces blakesleeanus leu1 gene.

SummaryThe leu1 gene of Phycomyces blakesleeanus was isolated within a HindIII-HindIII genomic DNA fragment by heterologous hybridization screening of a cosmid library, making use of the Mucor circinelloides leuA gene as a probe. The complete nucleotide sequence of this fragment reveals a single 2070 bp ORF with no introns, which presents at least 68% homology with that of the leuA gene. The P. blakesleeanus leu1 gene has also been expressed in the M. circinelloides mutant R7B (leu-), which was used to isolate the leuA gene by complementation. The homology with other known sequences shows that the leu1 gene encodes the P. blakesleeanus α-IPM (isopropylmalate) isomerase.

Blue-Light Receptor Requirement for Gravitropism, Autochemotropism and Ethylene Response in Phycomyces*

Light, gravity and ethylene represent for plants and fungi important environmental cues for spatial orientation and growth regulation. Coordination of the frequently conflicting stimuli requires signal‐integration sites, which, however, remain largely unidentified. The genetic and physiological basis for signal integration was investigated with a set of phototropism mutants (genotype mad) of the UV‐ and blue‐light‐sensitive fungus Phycomyces blakes‐leeanus, which responds also to gravity, ethylene and nearby obstacles (autochemotropism or avoidance response). Both, class 1 and class 2 mutants display a reduced sensitivity to visible light. Class 1 mutants with defects in genes mad A, B, C, Z have preserved their sensitivity to gravity and ethylene, whereas class 2 mutants with defects in genes mad D, E, F, G, J have lost it. We found that the phototropic sensitivity of class 1 mutants is affected roughly to the same extent in far UV and blue light. In contrast, the sensitivity loss of class 2 mutants is restricted mainly to the near‐UV and the blue‐light region, whereas the sensitivity to far UV is only mildly affected. This behavior of the class 2 mutants indicates that different photoreceptors mediate phototropism in far‐UV and in near‐UVhlue light. The photogravitropic action spectra for two class 2 mutants with defects in genes mad F and mad J display distortions between 342 and 530 nm and a bathochromic shift relative to the action spectrum of the wild type. These features indicate that the mad F and mad J mutants are affected at the level of the blue‐light photoreceptor system. As an implication we infer that an intact near‐UVhlue‐light photoreceptor system is required even in darkness for negative gravitropism, the ethylene response and autochemotropism. In Phycomyces, signal integration occurs, at least in part, at the level of the near‐U Vhlue‐light photoreceptor system.

Expression of the carG gene, encoding geranylgeranyl pyrophosphate synthase, is up-regulated by blue light in Mucor circinelloides

Abstract A new structural gene, carG, involved in the biosynthesis of carotenoids in the fungus Mucor circinelloides was isolated by heterologous hybridisation, using a probe derived from the Gibberella fujikuroi ggs1 gene. Functional analyses in Escherichia coli showed that the encoded protein has geranylgeranyl pyrophosphate (GGPP) synthase activity. A comparison of the deduced protein with other GGPP synthases suggested that the carG gene might have evolved from other larger genes present in some fungi. The analysis of carG mRNA accumulation after blue light irradiation showed that the expression of this gene is up-regulated by blue light, as happens with the other structural genes involved in carotenogenesis in M. circinelloides. Analysis of the promoter region revealed the presence of several APE-like sequences, which participate in the blue-light regulation of the expression of different fungal genes. These sequences are also present in the above-mentioned Mucor genes and strongly support the idea that this gene plays an important role in the regulation of carotenoid synthesis, despite belonging to a more general metabolic pathway.

A genetic map of Phycomyces blakesleeanus

SummaryComplementation tests among Phycomyces auxotrophic strains revealed the existence of four genes with mutants requiring riboflavin, three genes with purine auxotrophs, two with nicotinic acid auxotrophs, and two with lysine auxotrophs. A total of 134 sexual crosses between strains carrying mutations affecting phototropism (madA-madE), carotenoid biosynthesis (carA), auxotrophy (ribA-ribD, purA-purC, lysA and lysB, nicA and nicB, and leuA) and resistance to 5-fluorouracil (furA and furB) were studied; mating type (sex) was also included as a marker. The results from random spore analysis, tetrad analysis, and gene-centromere distances shows that these markers are distributed into 11 linkage groups.

Carotenoid biosynthesis in wild type and mutant strains of Mucor circinelloides.

Carotenoid biosynthesis in wild type Mucor circinelloides has been investigated and the biochemical characterisation of the MS1 and MS9 mutant strains, impaired in carotenoid formation, carried out. In liquid cultures, all strains produced carotenoids (mainly beta-carotene, but also xi-carotene, lycopene and gamma-carotene) at the onset of stationary phase of growth. Carotenogenesis was light dependent. In liquid cultures carotenoid formation in wild type was affected by diphenylamine, which prevented desaturation, nicotine, resulting in reduced carotenoid levels, but CPTA caused an increase in the total carotenoid content but a reduced beta-carotene level, with the accumulation of lycopene and gamma-carotene. The mutant strains MS1 and MS9 contained only 5.0 and 11.5% of wild type carotenoid levels, respectively. Cell extracts of light-grown mycelia, incubated with 3(R)-[2-14C] mevalonic acid, produced beta-carotene, but incorporations into carotenoids were substantially reduced in the cell extracts of MS1 and MS9. Analysis of prenyl diphosphate intermediates indicated that, compared to wild type, geranylgeranyl diphosphate accumulated in MS1. MS9 extracts produced a larger amount of prenyl phosphates and a more even distribution of radioactivity from mevalonic acid into farnesyl and geranylgeranyl diphosphates. Squalene and long chain prenyl phosphates were formed by the cell extracts of all strains. It is proposed that the MS1 strain possesses a mutation in a gene responsible for phytoene formation, whilst a regulatory mutation, affecting prenyl transferase activities has occurred in MS9.

Isolation and characterization of phototropism mutants of Phycomyces insensitive to ultraviolet light

Phototropism mutants of the zygomycete fungus Phycomyces blakesleeanus were isolated on the basis of their loss of responsivity to UV light. Four of these mutants had retained a partial sensitivity to near-UV and to blue light. Gravitropism and the avoidance response were unaffected in these mutants. One mutant, A909, had lost most of its sensitivity to near-UV and blue light while the sensitivity to far-UV light was only slightly affected. Additionally, the gravitropic and the avoidance responses were significantly reduced in A909. A complementation analysis of the five strains of Phycomyces with known phototropism mutants indicated that strains A896, A897, and A898 were defective in the madA gene, and that A905 was affected in the madC gene. In strain A909 the input, as well as the output, of the transduction chain is affected.

A new gene (madI) involved in the phototropic response of Phycomyces

SummaryOnly eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.

Mutants of Phycomyces with abnormal phototropism induced by ICR-170

SummaryTwenty two mutants with abnormal phototrophism (genotype mad) were obtained from the wild type by mutagenesis with ICR-170. These mutants were tested for their sensory responses and grouped into two classes, based on the phenotypic classification scheme of Bergman et al. (1973). There were twenty class 1 mutants and two class 2 mutants. Complementation tests revealed that all class 2 mutants carry a mutation in the madD gene and all class 1 mutants tested carry a mutation in the madC gene. Recombination analysis between several madC alleles showed that all the madC alleles tested were in the same locus. No new gene affecting the light sensory pathway was found and none of these mutants were totally blind.

Mating type heterokaryons inPhycomyces blakesleeanus

A simple method is described for construction of mating type heterokaryons inPhycomyces blakesleeanus. The specific morphology of these heterokaryons is used as a phenotypic marker which reveals the presence of nuclei of different mating types in the same mycelium. These heterokaryons can be successfully used in complementation tests involving both nutritional and phototropic markers. Complementation between mutants with abnormal phototropism belonging to seven previously established complementation groups showed that in 54 different mating type heterokaryons the pattern of complementation was the same as that earlier described in heterokaryons involving nuclei of the same mating type. Complementation tests involving riboflavin auxotrophs showed the presence of two complementation groups.