Light regulates a Phycomyces blakesleeanus gene family similar to the carotenogenic repressor gene of Mucor circinelloides.

The transcription of about 5-10 % of the genes in Phycomyces blakesleeanus is regulated by light. Among the most up-regulated, we have identified four genes, crgA-D, with similarity to crgA of Mucor circinelloides, a gene encoding a repressor of light-inducible carotenogenesis. The four proteins have the same structure with two RING RING Finger domains and a LON domain, suggesting that they could act as ubiquitin ligases, as their M. circinelloides homolog. The expression of these genes is induced by light with different thresholds as in other Mucoromycotina fungi like Blakeslea trispora and M. circinelloides. Only the P. blakesleeanus crgD gene could restore the wild type phenotype in a M. circinelloides null crgA mutant suggesting that P. blakesleeanus crgD is the functional homolog of crgA in M. circinelloides. Despite their sequence similarity it is possible that the P. blakesleeanus Crg proteins do not participate in the regulation of beta-carotene biosynthesis since none of the carotene-overproducing mutants of P. blakesleeanus had mutations in any of the crg genes. Our results provide further support of the differences in the regulation of the biosynthesis of beta-carotene in these two Mucoromycotina fungi.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

Pulsed light inactivation of naturally occurring moulds on wheat grain.

BACKGROUND: Pulsed light (PL) is emerging as a non-thermal technology with excellent prospects for the decontamination of foods and food contact surfaces. Its application for mould inactivation on cereal grains would allow a reduction of storage losses as well as the prevention of mycotoxin contamination at a post-harvest level. The potential of PL for the decontamination of naturally occurring moulds on wheat grain was investigated in this study. RESULTS: Treatments of up to 40 flashes of a fluence of 0.4 J cm⁻² per pulse were applied to both sides of the grain, with an overall energy release ranging from 6.4 to 51.2 J g⁻¹. The most powerful treatment applied to wheat in this study (51.2 J g⁻¹) resulted in a mould reduction of approximately 4 log cycles on samples displaying an initial mould contamination level of 2.2 × 105 CFU g⁻¹. At the same time, the seed germination percentage was only slightly affected. For PL treatments causing an inactivation of 3-4 log cycles, only 14-15% of the germination power of the wheat seeds was lost. CONCLUSION: The PL treatments attained greater microbial reductions for higher treatment times and lower initial mould loads. The absence of the UV portion of the radiation spectrum was found to significantly reduce the treatment effectiveness.

[Blue-light induced expression of S-adenosy-L-homocysteine hydrolase-like gene in Mucor amphibiorum RCS1].

OBJECTIVE: To determine blue-light induced expression of S-adenosyl-L-homocysteine hydrolase-like (sahhl) gene in fungus Mucor amphibiorum RCS1. METHODS: In the random process of PCR, a sequence of 555 bp was obtained from M. amphibiorum RCS1. The 555 bp sequence was labeled with digoxin to prepare the probe for northern hybridization. By northern hybridization, the transcription of sahhl gene was analyzed in M. amphibiorum RCS1 mycelia culture process from darkness to blue light to darkness. Simultaneously real-time PCR method was used to the sahhl gene expression analysis. RESULTS: Compared with the sequence of sahh gene from Homo sapiens, Mus musculus and some fungi species, a high homology of the 555 bp sequence was confirmed. Therefore, the preliminary confirmation has supported that the 555 bp sequence should be sahhl gene from M. amphibiorum RCS1. Under the dark pre-culture in 24 h, a large amounts of transcript of sahhl gene in the mycelia can be detected by northern hybridization and real-time PCR in the condition of 24 h blue light. But a large amounts of transcript of sahhl gene were not found in other detection for the dark pre-culture of 48 h, even though M. amphibiorum RCS1 mycelia were induced by blue light. CONCLUSION: Blue light can induce the expression of sahhl gene in the vigorous growth of M. amphibiorum RCS1 mycelia.

[The effect of hydrogen peroxide on the growth of microscopic mycelial fungi isolated from habitats with different levels of radioactive contamination].

The effect of hydrogen peroxide ( 10(-9)-10(-1) M) on the mycelial growth of the fungi Alternaria alternata, Cladosporium cladosporioides, Mucor hiemalis, and Paecilomyces lilacinus has been studied. The growth of fungi isolated from habitats with a background level of radioactive contamination was stopped by H2O2 concentrations equal to 10(-3) and 10(-2) M, whereas the growth of fungi that were isolated from habitats with high levels of radioactive contamination was only arrested by 10(-1) M H2O2. The response of the different fungi to hydrogen peroxide was of three types: (1) a constant growth rate of fungal hyphae at H2O2 concentrations between 10(-9) and 10(-4) M and a decrease in this rate at 10(-3) M H2O2, (2) a gradual decrease in the growth rate as the H2O2 concentration was increased, and (3) an increase in the growth rate as the H2O2 concentration was increased from 10(-7) to 10(2)-5 M. The melanin-containing species A. alternata and C. cladosporioides exhibited all three types of growth response to hydrogen peroxide, whereas the light-pigmented species M. hiemalis and P. lilacinus showed only the first type of growth response. A concentration of hydrogen peroxide equal to 10(-1) M was found to be lethal to all of the fungi studied. The most resistant to hydrogen peroxide was found to be the strain A. alternata 56, isolated from the exclusion zone of the Chernobyl Nuclear Power Plant.

cigA, a light-inducible gene involved in vegetative growth in Mucor circinelloides is regulated by the carotenogenic repressor crgA.

A strategy based on the differential-display technique has been applied to identify and to isolate potential target genes controlled by crgA, a negative regulator of the light-inducible carotenogenesis in the fungus Mucor circinelloides. This approach resulted in the identification and cloning of the gene cigA, whose expression is repressed by the crgA gene. After a light pulse, there is an immediate though transient accumulation of transcripts of cigA gene. A similar result was obtained when mycelia were exposed to continuous illumination, suggesting the existence of a photoadaptation mechanism in M. circinelloides. The protein sequence deduced from the nucleotide sequence of cigA shows homology to the mouse and human GDP-fucose protein O-fucosyltransferases. Null cigA mutants were generated by gene replacement. Lack of cigA function does not affect carotenogenesis but provokes a slight reduction in vegetative growth that is independent of light. Introduction of wild-type cigA alleles into a cigA- strain restored the wild-type phenotype for vegetative growth. These results suggest that cigA is involved in vegetative growth and, moreover, that crgA controls cellular processes other than carotenogenesis.

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.

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

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.

Overexpression of the crgA gene abolishes light requirement for carotenoid biosynthesis in Mucor circinelloides.

This work describes the isolation and characterization of crgA, a Mucor circinelloides gene, which has a dominant-positive effect on light-regulated carotenogenesis. The crgA gene was originally identified in a transformation experiment as a 3'-truncated open reading frame which caused carotenoid overaccumulation in the dark. The complete cloning and sequencing of crgA revealed that its putative product presented several recognizable structural domains: a RING-finger zinc binding domain near the N-terminus, a putative nuclear localization signal, two stretches of acidic amino acids, glutamine-rich regions and a putative isoprenylation motif. The expression of exogenous copies of the complete crgA gene or two different 3'-truncated versions, produced a similar dominant-positive effect on the light-inducible carotenogenesis of M. circinelloides. The presence of these exogenous sequences also caused a missregulation of the endogenous crgA gene, resulting in its overexpression. Collectively, these observations suggest that crgA is involved in the regulation of carotenoid biosynthesis by light.

Cobalt 60 radiation and growth of eleven species of micro-fungi from Evolution Canyon, Lower Nahal Oren, Israel.

Eleven micro-fungal species isolated from both the north facing slope (NFS) and the south facing slope (SFS) of Evolution Canyon, Lower Nahal Oren, Israel, were examined for growth rates before and after exposure to 60Co irradiation. Species of Alternaria, Aspergillus, Humicola, Oidiodendron, and Staphylotrichum from SFS grew faster than the NFS isolates while Fusarium, Sordaria, and Stachybotrys grew at greater rates from the NFS than from the SFS. Mucor and Ulocladium isolates grew at the same rate from both SFS and NFS. The eleven isolates from each slope were next subjected to 60Co irradiation. At 40,000 rads exposure, Alternaria, Fusarium, and Stachybotrys grew more rapidly when isolated from the NFS, while Humicola and Staphylotrichum grew at a faster rate when isolated from the SFS. Aspergillus, Mucor, Sordaria, and Ulocladium from both the NFS and the SFS had relatively the same growth rate at 40,000 rads exposure. At 400,000 rads exposure, growth rates remained much the same for both the N and S exposed isolates as they were at 40,000 rads. Above 10(6) rads, growth ceased but recovery occurred at various times for individual isolates of the same species from opposing canyon slopes.