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PubMed1993

Heterologous expression of mating-type genes in filamentous fungi.

Podospora anserina and Neurospora crassa, two filamentous heterothallic ascomycetes, have a single mating-type locus with two alternate forms called mat+ and mat- and A and a, respectively. Mating type controls entry into the sexual cycle, events subsequent to fertilization, and, in N. crassa, prevents the formation of mixed mating-type heterokaryons. The mating types of these two organisms display similarity in their DNA structure and in the encoded polypeptides involved in fertilization. Here we show that this molecular similarity reflects a functional homology with respect to mating identity. Transformation experiments show that the N. crassa mating-type genes can provide the fertilization functions in P. anserina strains devoid of mating specificity as well as in mat+ and mat- strains. Reciprocally, the introduction of P. anserina mating-type genes confers mating activity in N. crassa. Functional identity between the mating types is not observed for vegetative incompatibility or for post-fertilization events such as meiosis and ascosporogenesis.

DNA-Binding Proteins↗
PubMed1994

Pulsed growth of fungal hyphal tips.

Somatic fungal hyphae are generally assumed to elongate at steady linear rates when grown under constant environmental conditions with ample nutrients. However, patterns of pulsed hyphal elongation were detected during apparent steady growth of hyphal tips in fungi from several major taxonomic groups (Oomycetes, Pythium aphanidermatum and Saprolegnia ferax; Zygomycetes, Gilbertella persicaria; Deuteromycetes, Trichoderma viride; Ascomycetes, Neurospora crassa and Fusarium culmorum; Basidiomycetes, Rhizoctonia solani). Growing hyphal tips were recorded with video-enhanced phase-contrast microscopy at high magnification, and digital images were measured at very short time intervals (1-5 s). In all fungi tested, the hyphal elongation rate was never perfectly steady but fluctuated continuously with alternating periods of fast and slow growth at more or less regular intervals. Pulsed growth was observed in fungi differing in cell diameter, overall growth rate, taxonomic position, and presence and pattern of Spitzenkörper organization, suggesting that this is a general phenomenon. Frequency and amplitude of the pulses varied among the test organisms. T. viride and N. crassa showed the most frequent pulses (average of 13-14 per min), and F. culmorum the least frequent (2.7 per min). Average pulse amplitude varied from 0.012 microns/s for F. culmorum to 0.068 microns/s for G. persicaria. In F. culmorum and T. viride, the fast phase of the growth pulses was correlated with the merger of satellite Spitzenkörper with the main Spitzenkörper. These findings are consistent with a causal relationship between fluctuations in the overall rate of secretory vesicle delivery/discharge at the hyphal apex and the fluctuations in hyphal elongation rate.

Activity Cycles↗
PubMed1994

Tagged mutations at the Tox1 locus of Cochliobolus heterostrophus by restriction enzyme-mediated integration.

We have used the restriction enzyme-mediated integration insertional mutagenesis procedure to tag the Tox1 locus in the filamentous Ascomycete Cochliobolus heterostrophus. Mutations at other, unselected, loci were also identified and a high proportion (30-50%) of them were tagged. This procedure may be of general utility for simultaneously mutating and tagging genes in fungi and in other eukaryotes. The Tox1 locus of C. heterostrophus has been defined by Mendelian analysis as a single genetic element that controls production of T toxin, a linear polyketide involved in virulence of the fungus to its host plant, corn. To tag Tox1, protoplasts of a Tox1+ (T-toxin producing) strain were transformed with a linearized, nonhomologous plasmid along with an excess of the restriction enzyme used to linearize the plasmid. Of 1310 transformants recovered, two produced no detectable T toxin in culture or on corn plants. In each of these transformants, the Tox- mutation mapped at Tox1, was tagged with the selectable marker (hygB) on the transforming plasmid, and was tightly linked to the other tagged Tox- mutation. The two mutations, however, represent two different points of plasmid insertion at the Tox1 locus.

Ascomycota↗
PubMed1997

Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis.

Simple cladogenetic theory suggests that gene genealogies can be used to detect mixis in a population and delineate reproductively isolated groups within sexual taxa. We have taken this approach in a study of Coccidioides immitis, an ascomycete fungus responsible for a recent epidemic of coccidioidomycosis (Valley fever) in California. To test whether this fungus represents a single sexual species throughout its entire geographic range, we have compared genealogies from fragments of five nuclear genes. The five genealogies show multiple incompatibilities indicative of sex, but also share a branch that partitions the isolates into two reproductively isolated taxa, one centered in California and the other outside California. We conclude that coccidioidomycosis can be caused by two distinct noninterbreeding taxa. This result should aid the future study of the disease and illustrates the utility of the genealogical approach in population genetics.

Amino Acid Sequence↗
PubMed1998

Cryptic speciation and recombination in the aflatoxin-producing fungus Aspergillus flavus.

Aspergillus flavus, like approximately one-third of ascomycete fungi, is thought to be cosmopolitan and clonal because it has uniform asexual morphology. A. flavus produces aflatoxin on nuts, grains, and cotton, and assumptions about its life history are being used to develop strategies for its biological control. We tested the assumptions of clonality and conspecificity in a sample of 31 Australian isolates by assaying restriction site polymorphisms from 11 protein encoding genes and DNA sequences from five of those genes. A. flavus isolates fell into two reproductively isolated clades (groups I and II). The lack of concordance among gene genealogies among isolates in one of the clades (group I) was consistent with a history of recombination. Our analysis included five strains of the closely related industrial fungus A. oryzae, all of which proved to be clonally related to group I.

Aflatoxins↗
PubMed1999

Evolution of the fungal self-fertile reproductive life style from self-sterile ancestors.

In most fungal ascomycetes, mating is controlled by a single locus (MAT). Fungi requiring a partner to mate are heterothallic (self-sterile); those not requiring a partner are homothallic (self-fertile). Structural analyses of MAT sequences from homothallic and heterothallic Cochliobolus species support the hypothesis that heterothallism is ancestral. Homothallic species carry both MAT genes in a single nucleus, usually closely linked or fused, in contrast to heterothallic species, which have alternate MAT genes in different nuclei. The structural organization of MAT from all heterothallic species examined is highly conserved; in contrast, the organization of MAT in each homothallic species is unique. The mechanism of conversion from heterothallism to homothallism is a recombination event between islands of identity in otherwise dissimilar MAT sequences. Expression of a fused MAT gene from a homothallic species confers self-fertility on a MAT-null strain of a heterothallic species, suggesting that MAT alone is sufficient to change reproductive life style.

DNA-Binding Proteins↗
PubMed1995

Gibberellin biosynthesis in gib mutants of Gibberella fujikuroi.

The Ascomycete Gibberella fujikuroi synthesizes gibberellins, fujenal, carotenoids, and other terpenoids. Twelve gib mutants, isolated through the modified gibberellin fluorescence of their culture media, were subjected to chemical and biochemical analyses. Two mutants were specifically defective in the hydroxylation of carbon 13; their total gibberellin production was normal, but their main gibberellin was GA7 instead of GA3. Four mutants were blocked in the early reactions between geranylgeranyl pyrophosphate and 7-hydroxy-kaurenoic acid; two of them could not synthesize kaurene and another one was blocked in several oxidative steps. Six mutants had partial defects in early reactions, leading to the production of one-fifth to one-third of the wild type amounts of gibberellins and fujenal. Two of these produced considerable amounts of kaurenolides due to a defect in the conversion of kaurenoic acid to 7-hydroxykaurenoic acid. Another one produced no carotenoids, but attempts to isolate mutants of reactions shared by the carotenoid and gibberellin pathways failed. The gib mutations did not modify the ability of the fungus to live as a saprophyte.

Gibberella↗
PubMed1995

Secretion and maturation study of endothiapepsin in Saccharomyces cerevisiae. A first step toward improving its substrate specificity.

The gene encoding endothiapepsin (EAP), an extracellular aspartic proteinase from the filamentous ascomycete Cryphonectria parasitica, was expressed into Saccharomyces cerevisiae. Efficient secretion of an active and correctly processed enzyme was achieved when expressing the entire cDNA encoding prepro-EAP under the control of the galactose-inducible GRAP1 yeast promoter. Since three independent, site-directed mutations of EAP, including the substitution of an aspartyl catalytic residue, resulted in the intracellular accumulation of zymogen forms, we assumed that the EAP propeptide was autocatalytically processed. As a prerequisite to further improve the specificity of EAP, we therefore attempted to bypass this self-processing step in three different ways: 1) introduction of a Kex2-like recognition site between the pro and the mature part, 2) deletion of the prosequence (pre-EAP), and 3) co-expression in trans of the pre-EAP with its preprosequence. No improvement in the secretion of mutant enzymes was obtained in any of these experiments. As an alternative, we finally replaced the EAP processing site by the chymosin cleavage sequence of kappa-casein. Such a modification remained efficient in directing the secretion of active EAP only when a putative alpha-helix structural motif was conserved at the C terminus of the pro region.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of a gene encoding a homogentisate dioxygenase from Aspergillus nidulans and identification of its human and plant homologues.

We report here the first characterization of a gene encoding a homogentisate dioxygenase, the Aspergillus nidulans hmgA gene. The HmgA protein catalyzes an essential step in phenylalanine catabolism, and disruption of the gene results in accumulation of homogentisate in broths containing phenylalanine. hmgA putatively encodes a 448-residue polypeptide (Mr = 50,168) containing 21 histidine and 23 tyrosine residues. This polypeptide has been expressed in Escherichia coli as a fusion to glutathione S-transferase, and the affinity-purified protein has homogentisate dioxygenase activity. A. nidulans, an ascomycete amenable to classical and reverse genetic analysis, is a good metabolic model to study inborn errors in human Phe catabolism. One such disease, alkaptonuria, was the first human inborn error recognized (Garrod, A. E. (1902) Lancet 2, 1616-1620) and results from loss of homogentisate dioxygenase. Here we take advantage of the high degree of conservation between the amino acid sequences of the fungal and higher eukaryote enzymes of this pathway to identify expressed sequence tags encoding human and plant homologues of HmgA. This is a significant advance in characterizing the genetic defect(s) of alkaptonuria and illustrates the usefulness of our fungal model.

Amino Acid Sequence↗
PubMed1997

Overexpression of nreB, a new GATA factor-encoding gene of Penicillium chrysogenum, leads to repression of the nitrate assimilatory gene cluster.

To investigate the mechanism of nitrogen metabolite repression in the biotechnologically important fungus Penicillium chrysogenum a polymerase chain reaction approach was employed to identify transcription factors involved in this regulatory circuit, leading to the isolation of a new gene (nreB) encoding a 298 amino acid protein. Despite a low overall amino acid sequence identity of approximately 30%, it shares several features with Dal80p/Uga43p and Gzf3p/Nil2p, both repressors in nitrogen metabolism in Saccharomyces cerevisiae. All three proteins contain an N-terminal GATA-type zinc finger motif, displaying 86% amino acid sequence identity, and a putative leucine zipper motif in the C terminus. Northern blot analysis revealed the presence of two nreB transcripts, 1.8 and 1.5 kilobases in length, that differ in polyadenylation sites. The steady state level of both transcripts is subject to nitrogen metabolite repression. The putative DNA binding domain of NREB, expressed as a fusion protein in Escherichia coli, binds in vitro to GATA sites of its own 5'-upstream region as well as in the promoter of the nitrate assimilation gene cluster. Consistent with a role in the regulation of nitrogen metabolism, overexpression of nreB leads to repression of nitrate assimilatory genes. Hence, the simple view of nitrogen regulation by four GATA factors in yeast, but only one key regulator in filamentous ascomycetes seems no longer valid.

Amino Acid Sequence↗
PubMed2001

Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans.

In the Ascomycete fungus Aspergillus nidulans, the ratio of conidia (asexual spores) to ascospores (sexual spores) is affected by linoleic acid moieties including endogenous sporogenic factors called psi factors. Deletion of odeA (Delta odeA), encoding a Delta-12 desaturase that converts oleic acid to linoleic acid, resulted in a strain depleted of polyunsaturated fatty acids (18:2 and 18:3) but increased in oleic acid (18:1) and total percent fatty acid content. Linoleic acid-derived psi factors were absent in this strain but oleic acid-derived psi factors were increased relative to wild type. The Delta odeA strain was reduced in conidial production and mycelial growth; these effects were most noticeable when cultures were grown at 26 degrees C in the dark. Under these environmental conditions, the Delta odeA strain was delayed in ascospore production but produced more ascospores than wild type over time. This suggests a role for oleic acid-derived psi factors in affecting the asexual to sexual spore ratio in A. nidulans. Fatty acid composition and spore development were also affected by veA, a gene previously shown to control light driven conidial and ascospore development. Taken together our results indicate an interaction between veA and odeA alleles for fatty acid metabolism and spore development in A. nidulans.

Aspergillus↗
PubMed2001

A new yeast metabolon involving at least the two first enzymes of arginine biosynthesis: acetylglutamate synthase activity requires complex formation with acetylglutamate kinase.

Open reading frame YJL071W of Saccharomyces cerevisiae was shown to be ARG2 and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis. The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents. The measured synthase activity did not increase with the number of ARG2 gene copies unless the number of ARG5,6 gene copies was increased similarly. ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway. The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active. Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed. This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R. W., Jacobson, J. A., Weiss, R. L., and Davis, R. H. (1986) J. Biol. Chem. 261, 5848-5852). In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase. Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase. Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway. Hypotheses regarding the biological significance of this interaction are discussed.

Acetyltransferases↗
PubMed2002

Phosphorylation positively regulates DNA binding of the carbon catabolite repressor Cre1 of Hypocrea jecorina (Trichoderma reesei).

Cre1 of the ascomycete Hypocrea jecorina is a Cys(2)His(2) zinc finger DNA-binding protein functioning as regulator for carbon catabolite repression. It represents the functional equivalent of yeast Mig1, known to be negatively regulated by the Snf1-kinase at the nuclear import level. We demonstrate that Cre1 is also a phosphoprotein, and identify Ser(241) within an acidic protein region as phosphorylation target. In contrast to Mig1 phosphorylation is required for DNA binding of Cre1. A S241E mutation mimics phosphorylation, whereas a S241A mutant protein shows phosphorylation-independent DNA binding activity, suggesting that phosphorylation is required to release Cre1 from an inactive conformation involving unphosphorylated Ser(241). Retransformation of a H. jecorina cre1-non functional mutant with Cre1-S241A leads to permanent carbon catabolite repression in cellobiohydrolase I expression. Contrary to Mig1, the amino acid sequence surrounding Ser(241) (HSNDEDD) suggests that phosphorylation may occur by a casein kinase II-like protein. This is supported by a mutation of E244V leading to loss of phosphorylation, loss of DNA binding, and gain of carbon catabolite derepression. Our results imply that the regulation of carbon catabolite repression at the level of DNA binding strongly differs between Saccharomyces cerevisiae and H. jecorina.

Amino Acid Sequence↗
PubMed2006

Kinetic and mechanistic basis of the nonprocessive Kinesin-3 motor NcKin3.

Kinesin-3 motors have been shown to transport cellular cargo along microtubules and to function according to mechanisms that differ from the conventional hand-over-hand mechanism. To find out whether the mechanisms described for Kif1A and CeUnc104 cover the full spectrum of Kinesin-3 motors, we characterize here NcKin3, a novel member of the Kinesin-3 family that localizes to mitochondria of ascomycetes. We show that NcKin3 does not move in a K-loop-dependent way as Kif1A or in a cluster-dependent way as CeUnc104. Its in vitro gliding velocity ranges between 0.30 and 0.64 mum/s and correlates positively with motor density. The processivity index (k(bi,ratio)) of approximately 3 reveals that not more than three ATP molecules are hydrolyzed per productive microtubule encounter. The NcKin3 duty ratio of 0.03 indicates that the motor spends only a minute fraction of the ATPase cycle attached to the filament. Unlike other Kinesin-3 family members, NcKin3 forms stable dimers, but only one subunit releases ADP in a microtubule-dependent fashion. Together, these data exclude a processive hand-over-hand mechanism of movement and suggest a power-stroke mechanism where nucleotide-dependent structural changes in a single motor domain lead to displacement of the motor along the filament. Thus, NcKin3 is the first plus end-directed kinesin motor that is dimeric but moves in a nonprocessive fashion to its destination.

Adenosine Triphosphate↗
PubMed2002

Yeasts associated with fresh and frozen pulps of Brazilian tropical fruits.

The occurrence of yeasts on ripe fruits and frozen pulps of pitanga (Eugenia uniflora L), mangaba (Hancornia speciosa Gom.), umbu (Spondias tuberosa Avr. Cam.), and acerola (Malpighia glaba L) was verified. The incidence of proteolytic, pectinolytic, and mycocinogenic yeasts on these communities was also determined. A total of 480 colonies was isolated and grouped in 405 different strains. These corresponded to 42 ascomycetous and 28 basidiomycetous species. Candida sorbosivorans, Pseudozyma antarctica, C. spandovensis-like, C. spandovensis, Kloeckera apis, C. parapsilosis, Rhodotorula graminis, Kluyveromyces marxianus, Cryptococcus laurentii, Metchnikowia sp (isolated only from pitanga ripe fruits), Issatchenkia occidentalis and C. krusei (isolated only from mangaba frozen pulps), were the most frequent species. The yeast communities from pitanga ripe fruits exhibited the highest frequency of species, followed by communities from acerola ripe fruits and mangaba frozen pulps. Yeast communities from frozen pulp and ripe fruits of umbu had the lowest number of species. Except the yeasts from pitanga, yeast communities from frozen pulp exhibited higher number of yeasts than ripe fruit communities. Mycocinogenic yeasts were found in all of the substrates studied except in communities from umbu ripe fruits and pitanga frozen pulps. Most of the yeasts found to produce mycocins were basidiomycetes and included P. antarctica, Cryptococcus albidus, C. bhutanensis-like, R. graminis and R. mucilaginosa-like from pitanga ripe fruits as well as black yeasts from pitanga and acerola ripe fruits. The umbu frozen pulps community had the highest frequency of proteolytic species. Yeasts able to hydrolyse casein at pH 5.0 represented 38.5% of the species isolated. Thirty-seven percent of yeast isolates were able to hydrolyse casein at pH 7.0. Pectinolytic yeasts were found in all of the communities studied, excepted for those of umbu frozen pulps. The highest frequency of pectinolytic activity was found in mangaba frozen pulp communities. Around 30% of all isolates produced pectinases. The ability to split arbutin was observed in all communities ranging from 8% in yeasts from pitanga frozen pulps to 40.6% in acerola ripe fruit communities. Among 432 species tested, 125 were active for beta-glucosidase production, and Kloeckera apis, P. antarctica, C. sorbosivorans, and C. spandovensis-like were the most active species.

Arbutin↗
PubMed2002

Molecular cell biology and molecular genetics of Histoplasma capsulatum.

Histoplasma capsulatum is a dimorphic ascomycete which is capable of producing a broad spectrum of disease ranging from mild asymptomatic, pulmonary illness to severe, life-threatening systemic mycosis. Regulatory mechanisms that use temperature and other environmental cues are paramount to the successful adaptation of the organism as an effective intracellular pathogenic yeast. Although the biochemistry and phenomenology of reversible morphogenesis have been well examined in Histoplasma, the identification and functional characterization of genes and their products that are required for early establishment or maintenance of the parasitic yeast phase in intracellular host compartments have only recently been fruitful. Advances in the molecular biology of Histoplasma, including approaches to introduce telomeric plasmids, reporter fusion constructs, and gene disruption cassettes into the fungus are poised to solidify the pre-eminence of this fungus as a model system which can be applied to other dimorphic fungal pathogens that exhibit similar cellular and immunological complexities. This review centers on recent developments in the molecular cell biology and molecular genetics of Histoplasma capsulatum that provide important new avenues for examining the mold-to-yeast phase transition beyond the historical, binary view of dimorphism and the implications that these successful approaches may have on seminal issues in fungal pathogenesis.

Gene Expression Regulation, Fungal↗
PubMed2002

Pneumocystis.

Pneumocystis organisms can cause pneumonia in mammals that lack a strong immune defense. The genus Pneumocystis contains many different organisms that can be distinguished by DNA sequence analysis. These different organisms are different species of yeast-like fungi that are most closely related to the ascomycete, Schizosaccharomyces pombe. Each species of Pneumocystis appears to be specific for the mammal in which it is found. The species that infects humans is Pneumocystis jiroveci. P. jiroveci has not been found in any other mammal and the species of Pneumocystis found in other mammals have not been seen in humans. Genetic variation among P. jiroveci samples is common, suggesting that there are many strains. Strain analysis shows that adults can be infected by more than one strain, and suggests that pneumonia can be the result of infection occurring proximal to the time of disease, rather than to reactivation of dormant organisms acquired in early childhood. Nevertheless, long-term colonisation may be occurring. A large fraction of normal children and animals show evidence of infection. A Pneumocystis species that grows in rats has been shown to possess a complex genetic system for surface antigen variation, a strategy employed by other microbes that dwell in immunocompetent hosts. These findings, together with strong host specificity, suggest that Pneumocystis species may be obligate parasites. The source of infection is not clear. Pneumocystis DNA is detectable in the air, but is scarce except in environments occupied by individuals with Pneumocystis pneumonia. In a few cases, there is direct evidence of person to person transmission. In general, however, patients and their contacts have been found to have different strains of P. jiroveci.

Animals↗
PubMedDate not supplied

Isolation and characterization of a calmodulin-encoding cDNA from the pathogenic fungus Histoplasma capsulatum.

We describe in this paper the isolation and complete sequence of a calmodulin (CaM) encoding cDNA from the dimorphic pathogenic fungus Histoplasma capsulatum (GenBank accession U12505). The deduced amino acid sequence was identical to the CaM of Aspergillus nidulans and had only one amino acid difference from the CaM of Neurospora crassa. Saccharomyces cerevisiae CaM, however, has only about 60% amino acid identity compared with H. capsulatum. These data further support the close relationship of Histoplasma to the filamentous ascomycetes. Histoplasma CaM was expressed as a single major transcript of approximately 1200 nt. in both the yeast and mould growth forms. CaM mRNA levels were approximately two-fold greater in the yeast than in the mould form of the organism. Southern blot analysis indicated that the H. capsulatum CaM gene is present in a single copy.

Amino Acid Sequence↗