- Volume 155, Issue 12, 2009

Molecular biology has several distinct origins, but especially important are those contributed by fungal and yeast physiology, biochemistry and genetics. From the first gene action studies that became the basis of our understanding of the relationship between genes and proteins, through chromosome structure, mitochondrial genetics and membrane biogenesis, gene silencing and circadian clocks, studies with these organisms have yielded basic insight into these processes applicable to all eukaryotes. Examples are cited of pioneering studies with fungi that have stimulated new research in clinical medicine and agriculture; these studies include sexual interactions, cell stress responses, the cytoskeleton and pathogenesis. Studies with the yeasts and fungi have been effective in applying the techniques and insights gained from other types of experimental systems to research in fungal cell signalling, cell development and hyphal morphogenesis.

The study of fungal physiology is set to change dramatically in the next few years as highly scalable technologies are deployed allowing accurate measurement and identification of metabolites, proteins and transcripts within cells. The advent of next-generation DNA-sequencing technologies will also provide genome sequence information from large numbers of industrially relevant and pathogenic fungal species, and allow comparative genome analysis between strains and populations of fungi. When coupled with advances in gene functional analysis, protein–protein interaction studies, live cell imaging and mathematical modelling, this promises a step-change in our understanding of how fungal cells operate as integrated dynamic living systems.

Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, including organelles, are enclosed within autophagosomes and are degraded upon lysosome–vacuole fusion. In this study, we show that processing of GFP-tagged Atg8 can serve as a marker for autophagy in the fission yeast Schizosaccharomyces pombe. Using this marker, 13 Atg homologues were also found to be required for autophagy in fission yeast. In budding yeast, autophagy-deficient mutants are known to be sterile, whereas in fission yeast we found that up to 30 % of autophagy-defective cells with amino acid auxotrophy were able to recover sporulation when an excess of required amino acids was supplied. Furthermore, we found that approximately 15 % of the autophagy-defective cells were also able to sporulate when a prototrophic strain was subjected to nitrogen starvation, which suggested that fission yeast may store sufficient intracellular nitrogen to allow partial sporulation under nitrogen-limiting conditions, although the majority of the nitrogen source is supplied by autophagy. Monitoring of the sporulation process revealed that the process was blocked non-specifically at various stages in the atg1Δ and atg12Δ mutants, possibly due to a shortage of amino acids. Taking advantage of this partial sporulation ability of fission yeast, we sought evidence for the existence of a recycling system for nitrogen sources during starvation.

Glucose repression of the tricarboxylic acid (TCA) cycle in Saccharomyces cerevisiae was investigated under different environmental conditions using 13C-tracer experiments. Real-time quantification of the volatile metabolites ethanol and CO2 allowed accurate carbon balancing. In all experiments with the wild-type, a strong correlation between the rates of growth and glucose uptake was observed, indicating a constant yield of biomass. In contrast, glycerol and acetate production rates were less dependent on the rate of glucose uptake, but were affected by environmental conditions. The glycerol production rate was highest during growth in high-osmolarity medium (2.9 mmol g−1 h−1), while the highest acetate production rate of 2.1 mmol g−1 h−1 was observed in alkaline medium of pH 6.9. Under standard growth conditions (25 g glucose l−1 , pH 5.0, 30 °C) S. cerevisiae had low fluxes through the pentose phosphate pathway and the TCA cycle. A significant increase in TCA cycle activity from 0.03 mmol g−1 h−1 to about 1.7 mmol g−1 h−1 was observed when S. cerevisiae grew more slowly as a result of environmental perturbations, including unfavourable pH values and sodium chloride stress. Compared to experiments with high glucose uptake rates, the ratio of CO2 to ethanol increased more than 50 %, indicating an increase in flux through the TCA cycle. Although glycolysis and the ethanol production pathway still exhibited the highest fluxes, the net flux through the TCA cycle increased significantly with decreasing glucose uptake rates. Results from experiments with single gene deletion mutants partially impaired in glucose repression (hxk2, grr1) indicated that the rate of glucose uptake correlates with this increase in TCA cycle flux. These findings are discussed in the context of regulation of glucose repression.

In Saccharomyces cerevisiae, FLO11 encodes an adhesin that is associated with different phenotypes, such as adherence to solid surfaces, hydrophobicity, mat and air–liquid biofilm formation. In the present study, we analysed FLO11 allelic polymorphisms and FLO11-associated phenotypes of 20 flor strains. We identified 13 alleles of different lengths, varying from 3.0 to 6.1 kb, thus demonstrating that FLO11 is highly polymorphic. Two alleles of 3.1 and 5.0 kb were cloned into strain BY4742 to compare the FLO11-associated phenotypes in the same genetic background. We show that there is a significant correlation between biofilm-forming ability and FLO11 length both in different and in the same genetic backgrounds. Moreover, we propose a multiple regression model that allows prediction of air–liquid biofilm-forming ability on the basis of transcription levels and lengths of FLO11 alleles in a population of S. cerevisiae flor strains. Considering that transcriptional differences are only partially explained by the differences in the promoter sequences, our results are consistent with the hypothesis that FLO11 transcription levels are strongly influenced by genetic background and affect biofilm-forming ability.

The early endocytic patch protein Sla2 is important for morphogenesis and growth rates in Saccharomyces cerevisiae and Candida albicans, but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2 or ScSLA2 are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other components of early endocytic patches (Sla1 and Abp1) with those in strains lacking Sla2. Only sla2 strains had defects in actin cables, a known trigger of the morphogenesis checkpoint, yet all three strains exhibited Swe1-dependent phenotypes. Thus, Swe1 appears to monitor actin patch in addition to actin cable function. Furthermore, Swe1 contributed to virulence in a mouse model of disseminated candidiasis, implying a role for the morphogenesis checkpoint during the pathogenesis of C. albicans infections.

Recently, several pathogenic fungi were shown to produce extracellular vesicles that contain various components associated with virulence. In the human pathogenic fungus Cryptococcus neoformans, these components included laccase, an enzyme that catalyses melanin synthesis. Spherical melanin granules have been observed in the cell wall of C. neoformans. Given that melanin granules have dimensions that are comparable to those of extracellular vesicles, and that metazoan organisms produce melanin in vesicular structures known as melanosomes, we investigated the role of vesicles in cryptococcal melanization. Extracellular vesicles melanized when incubated with the melanin precursor l-3,4-dihydroxyphenylalanine (l-DOPA). The kinetics of substrate incorporation into cells and vesicles was analysed using radiolabelled l-DOPA. The results indicated that substrate incorporation was different for cells and isolated vesicles. Acid-generated melanin ghosts stained with lipophilic dyes, implying the presence of associated lipid. A model for C. neoformans melanization is proposed that accounts for these observations and provides a mechanism for the assembly of melanin into relatively uniform spherical particles stacked in an orderly arrangement in the cell wall.

The Aspergillus nidulans transcription factor AreA is a key regulator of nitrogen metabolic gene expression. AreA contains a C-terminal GATA zinc finger DNA-binding domain and activates expression of genes necessary for nitrogen acquisition. Previous studies identified AreB as a potential negative regulator of nitrogen catabolism showing similarity with Penicillium chrysogenum NreB and Neurospora crassa ASD4. The areB gene encodes multiple products containing an N-terminal GATA zinc finger and a leucine zipper motif. We deleted the areB gene and now show that AreB negatively regulates AreA-dependent nitrogen catabolic gene expression under nitrogen-limiting or nitrogen-starvation conditions. AreB also acts pleiotropically, with functions in growth, conidial germination and asexual development, though not in sexual development. AreB overexpression results in severe growth inhibition, aberrant cell morphology and reduced AreA-dependent gene expression. Deletion of either the DNA-binding domain or the leucine zipper domain results in loss of both nitrogen and developmental phenotypes.

The lysine biosynthetic pathway has to supply large amounts of α-aminoadipic acid for penicillin biosynthesis in Penicillium chrysogenum. In this study, we have characterized the P. chrysogenum L2 mutant, a lysine auxotroph that shows highly increased expression of several lysine biosynthesis genes (lys1, lys2, lys3, lys7). The L2 mutant was found to be deficient in homoaconitase activity since it was complemented by the Aspergillus nidulans lysF gene. We have cloned a gene (named lys3) that complements the L2 mutation by transformation with a P. chrysogenum genomic library, constructed in an autonomous replicating plasmid. The lys3-encoded protein showed high identity to homoaconitases. In addition, we cloned the mutant lys3 allele from the L2 strain that showed a G1534 to A1534 point mutation resulting in a Gly495 to Asp495 substitution. This mutation is located in a highly conserved region adjacent to two of the three cysteine residues that act as ligands to bind the iron–sulfur cluster required for homoaconitase activity. The L2 mutant accumulates homocitrate. Deletion of the lys1 gene (homocitrate synthase) in the L2 strain prevented homocitrate accumulation and reverted expression levels of the four lysine biosynthesis genes tested to those of the parental prototrophic strain. Homocitrate accumulation seems to act as a sensor of lysine-pathway distress, triggering overexpression of four of the lysine biosynthesis genes.

Maltose utilization and regulation in aspergilli is of great importance for cellular physiology and industrial fermentation processes. In Aspergillus oryzae, maltose utilization requires a functional MAL locus, composed of three genes: MALR encoding a regulatory protein, MALT encoding maltose permease and MALS encoding maltase. Through a comparative genome and transcriptome analysis we show that the MAL regulon system is active in A. oryzae while it is not present in Aspergillus niger. In order to utilize maltose, A. niger requires a different regulatory system that involves the AmyR regulator for glucoamylase (glaA) induction. Analysis of reporter metabolites and subnetworks illustrates the major route of maltose transport and metabolism in A. oryzae. This demonstrates that overall metabolic responses of A. oryzae occur in terms of genes, enzymes and metabolites when the carbon source is altered. Although the knowledge of maltose transport and metabolism is far from being complete in Aspergillus spp., our study not only helps to understand the sugar preference in industrial fermentation processes, but also indicates how maltose affects gene expression and overall metabolism.

Propionyl-CoA is an inhibitor of both primary and secondary metabolism in Aspergillus species and a functional methylcitrate cycle is essential for the efficient removal of this potentially toxic metabolite. Although the genomes of most sequenced fungal species appear to contain genes coding for enzymes of the methylcitrate cycle, experimental confirmation of pathway activity in filamentous fungi has only been provided for Aspergillus nidulans and Aspergillus fumigatus. In this study we demonstrate that pathogenic Fusarium species also possess a functional methylcitrate cycle. Fusarium solani appears highly adapted to saprophytic growth as it utilized propionate with high efficiency, whereas Fusarium verticillioides grew poorly on this carbon source. In order to elucidate the mechanisms of propionyl-CoA detoxification, we first identified the genes coding for methylcitrate synthase from both species. Despite sharing 96 % amino acid sequence identity, analysis of the two purified enzymes demonstrated that their biochemical properties differed in several respects. Both methylcitrate synthases exhibited low K m values for propionyl-CoA, but that of F. verticillioides displayed significantly higher citrate synthase activity and greater thermal stability. Activity determinations from cell-free extracts of F. solani revealed a strong methylcitrate synthase activity during growth on propionate and to a lesser extent on Casamino acids, whereas activity by F. verticillioides was highest on Casamino acids. Further phenotypic analysis confirmed that these biochemical differences were reflected in the different growth behaviour of the two species on propionyl-CoA-generating carbon sources.

Cryphonectria parasitica, the chestnut blight fungus, can be infected by virulence-attenuating mycoviruses of the family Hypoviridae. Previous studies have led to the hypothesis that the hypovirus-infected phenotype is partly due to metabolic changes induced by the viral infection. To investigate this, we measured the metabolic rate and respiration of C. parasitica colonies grown on solid medium. These experiments supported historical observations of other fungal species done in liquid cultures that the metabolic rate steadily declines with age and differentiation of the mycelium. Hypovirus infection increased metabolic rate in the youngest mycelium, but a subsequent decline was also observed as the mycelium aged. By measuring both CO2 production and O2 consumption, we also observed that changes occur in carbohydrate metabolism as a result of ageing in both infected and uninfected mycelium. Mycelium on the periphery of the colony exploited fermentation pathways extensively, before transitioning to aerobic carbohydrate metabolism and finally lipid metabolism in the interior regions, despite abundant remaining glucose. However, the hypovirus affected the extent of these changes, with infected mycelium apparently unable to utilize lipid-related metabolic pathways, leading to an increased depletion of glucose. Finally, we used metabolic profifiling to determine the changes in accumulation of primary metabolites in wild-type and hypovirus-infected mycelium and found that approximately one-third of the 164 detected metabolites were affected. These results are consistent with those expected from the physiological measurements, with significant alterations noted for compounds related to lipid and carbohydrate metabolism. Additionally, we observed an increase in the accumulation of the polyamine spermidine in the presence of hypovirus. Polyamines have been implicated in antiviral responses of mammalian systems; therefore this may suggest a novel antiviral response mechanism in fungi.

The putative Claviceps purpurea homologue of the Saccharomyces cerevisiae stretch-activated calcium ion channel Mid1 was investigated for its role in vegetative growth, differentiation and pathogenicity on rye (Secale cereale). Gene replacement mutants of Cl. purpurea mid1 were not affected in polar growth and branching in axenic culture but showed a significantly reduced growth rate. The growth defect could not be complemented by Ca2+ supplementation, in contrast to mid1 mutants in yeast, but the altered sensitivity of the mutants to changes in external and internal Ca2+ concentrations indicates some role of Mid1 in Ca2+ homeostasis. The major effect of mid1 deletion, however, was the complete loss of virulence: infected rye plants showed no disease symptoms at all. Detailed analyses of in vitro-infected rye ovaries demonstrated that the Δmid1 mutants had multiple apical branches and were unable to infect the host tissue, suggesting that Mid1 is essential for generating the necessary mechanical force for penetration. This is believed to be the first report of an essential role for a Mid1 homologue in the virulence of a plant-pathogenic fungus.

Asexual development in the filamentous fungus Aspergillus nidulans is governed by the timely expression and cellular localization of multiple transcription factors. Hence, factors mediating import and export across the nuclear pore complexes (karyopherins) are expected to play a key role in coordinating the developmental programme. Here we characterize KapI, a putative homologue of the Saccharomyces cerevisiae Kap121/Pse1p karyopherin. KapI is a non-essential importin-β-like protein located in the nucleus during vegetative growth and conidiophore development. The ΔkapI phenotype is aconidial with many aerial hyphae. This phenotype can be suppressed under abiotic stress. In this regard, it resembles that of the null allele of the bZIP transcription factor FlbB. However a ΔflbB; ΔkapI double mutant exhibited an additive phenotype with totally impaired conidiation, unresponsive to abiotic stress. In contrast to ΔflbB, the null kapI mutant is not a fluffy-low-bristle expression mutant. Taken together the findings indicate that KapI is required during asexual development, mediating the nuclear transport of factors acting in a different pathway(s) from those involving the upstream developmental activators.

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing system that downregulates target gene expression. Here, we provide several lines of evidence for RNA silencing in the industrial β-lactam antibiotic producer Penicillium chrysogenum using the DsRed reporter gene under the control of the constitutive trpC promoter or the inducible xylP promoter. The functional RNAi system was verified by detection of siRNAs that hybridized exclusively with gene-specific 32P-labelled RNA probes. Moreover, when RNAi was used to silence the endogenous PcbrlA morphogene that controls conidiophore development, a dramatic reduction in the formation of conidiospores was observed in 47 % of the corresponding transformants. Evidence that RNAi in P. chrysogenum is dependent on a Dicer peptide was provided with a strain lacking Pcdcl2. In the ΔPcdcl2 background, silencing of the PcbrlA gene was tested. None of the transformants analysed showed a developmental defect. The applicability of the RNAi system in P. chrysogenum was finally demonstrated by silencing the Pcku70 gene to increase homologous recombination frequency. This led to the generation of single and double knockout mutants.

Heterokaryon incompatibility (HI) is a nonself recognition phenomenon occurring in filamentous fungi that is important for limiting resource plundering and restricting viral transfer between strains. Nonself recognition and HI occurs during hyphal fusion between strains that differ at het loci. If two strains undergo hyphal fusion, but differ in allelic specificity at a het locus, the fusion cell is compartmentalized and undergoes a rapid programmed cell death (PCD). Incompatible heterokaryons show a macroscopic phenotype of slow growth and diminished conidiation, and a microscopic phenotype of hyphal compartmentation and cell death. To understand processes associated with HI and PCD, we used whole-genome microarrays for Neurospora crassa to assess transcriptional differences associated with induction of HI mediated by differences in het-c pin-c haplotype. Our data show that HI is a dynamic and transcriptionally active process. The production of reactive oxygen species is implicated in the execution of HI and PCD in N. crassa, as are several genes involved in phosphatidylinositol and calcium signalling pathways. However, genes encoding mammalian homologues of caspases or apoptosis-inducing factor (AIF) are not required for HI or programmed cell death. These data indicate that PCD during HI occurs via a novel and possibly fungal-specific mechanism, making this pathway an attractive drug target for control of fungal infections.

We have isolated serine protease inhibitors from the basidiomycete Clitocybe nebularis, CnSPIs, using trypsin affinity chromatography. Full-length gene and cDNA sequences were determined for one of them, named cnispin, and the recombinant protein was expressed in Escherichia coli at high yield. The primary structure and biochemical properties of cnispin are very similar to those of the Lentinus edodes serine protease inhibitor, until now the only member of the I66 family of protease inhibitors in the MEROPS classification. Cnispin is highly specific towards trypsin, with K i in the nanomolar range. It also exhibited weaker inhibition of chymotrypsin and very weak inhibition of subtilisin and kallikrein; other proteases were not inhibited. Inhibitory activity against endogenous proteases from C. nebularis revealed a possible regulatory role for CnSPIs in the endogenous proteolytic system. Another possible biological function in defence against predatory insects was indicated by the deleterious effect of CnSPIs on the development of larvae of Drosophila melanogaster. These findings, together with the biochemical and genetic characterization of cnispin, suggest a dual physiological role for this serine protease inhibitor of the I66 MEROPS family.

Mycobacteriophages have played an important role in the development of genetic tools and diagnostics for pathogenic mycobacteria, including Mycobacterium tuberculosis. However, despite the isolation of numerous phages that infect mycobacteria, the mechanisms of mycobacteriophage infection remain poorly understood, and knowledge about phage receptors is minimal. In an effort to identify the receptor for phage I3, we screened a library of Mycobacterium smegmatis transposon mutants for phage-resistant strains. All four phage I3-resistant mutants isolated were found to have transposon insertions in genes located in a cluster involved in the biosynthesis of the cell-wall-associated glycopeptidolipid (GPL), and consequently the mutants did not synthesize GPLs. The loss of GPLs correlated specifically with phage I3 resistance, as all mutants retained sensitivity to two other mycobacteriophages: D29 and Bxz1. In order to define the minimal receptor for phage I3, we then tested the phage sensitivity of previously described GPL-deficient mutants of M. smegmatis that accumulate biosynthesis intermediates of GPLs. The results indicated that, while the removal of most sugar residues from the fatty acyl tetrapeptide (FATP) core of GPL did not affect sensitivity to phage I3, a single methylated rhamnose, transferred by the rhamnosyltransferase Gtf2 to the FATP core, was critical for phage binding.