Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic transition in which non-pathogenic, yeast-like cells mate to form a pathogenic, filamentous dikaryon. Northern analysis indicated that two chitin synthase genes, chs1 and chs2, from U. maydis are expressed at similar levels in yeast-like cells and in cells undergoing the mating reaction leading to the filamentous cell type. A mutation was constructed in each of the chitin synthase genes by targeted gene disruption. Each mutant showed a reduction in the level of trypsin-activated enzyme activity, compared with a wild-type strain, but retained the wild-type morphology, the ability to mate and the ability to form the filamentous pathogenic cell type.     

Effects of different growth forms of Mucor indicus on cultivation on dilute-acid lignocellulosic hydrolyzate, inhibitor tolerance, and cell wall composition

The dimorphic fungus Mucor indicus was grown in different forms classified as purely filamentous, mostly filamentous, mostly yeast-like and purely yeast-like, and the relationship between morphology and metabolite production, inhibitor tolerance and the cell wall composition was investigated. Low concentrations of spores in the inoculum with subsequent aeration promoted filamentous growth, whereas higher spore concentrations and anaerobic conditions promoted yeast-like growth. Ethanol was the main metabolite with glycerol next under all conditions tested. The yields of ethanol from glucose were between 0.39 and 0.42 g g⁻¹ with productivities of 3.2–5.0 g l⁻¹ h⁻¹. The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0 g l⁻¹ h⁻¹ by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6 g l⁻¹ furfural and 10 g l⁻¹ acetic acid and assimilate the sugars, although with different consumption rates. The cell wall content of the fungus measured as alkali insoluble materials (AIM) of the purely yeast-like cells was 26% of the biomass, compared to 8% of the pure filaments. However, the chitosan concentration of the filaments was 29% of the AIM, compared to 6% of the yeast-like cells.     

Yeasts and fungi in excrements of the sand hill snail,Theba pisana

Eight microbial species were isolated and identified from excrements of the sand hill snail,Theba pisana, viz. 3 filamentous fungi, 3 basidiomycetous yeasts and 2 yeast-like ascomycetous fungi.     

Effect of antioxidants, pro-oxidants, respiration inhibitors and substrates on the formation of autonomic secondary colonies of Fusarium bulbigenum var. blasticola

Autonomous secondary colonies are formed on the primary colonies of Fusarium bulbigenum var. blasticola grown in a synthetic Rieder medium. The secondary colonies are similar to genuine neoplasia in the following characteristics: dedifferentiation of the mycelium filamentous structure to yield single yeast-like cells, selective growth, faulty differentiation. Just as antioxidants and respiration substrates inhibit tumour formation and growth in higher organisms, they either prevent or inhibit the formation of secondary colonies in microorganisms. In contrast, their formation is stimulated when prooxidants and respiration inhibitors are added to the growth medium.     

Effect of chloramphenicol,antimycin A and hydroxamate on the morphogenetic development of the dimorphic ascomyceteEndomycopsis capsularis

Mitochondrial protein synthesis, primary (antimycin-sensitive) respiration and secondary (antimycin-insensitive, salicyl-hydroxamate-sensitive) respiration, have been characterized in the dimorphic yeastEndomycopsis capsularis. The inhibition by chloramphenicol (CAP) of the morphogenetic development from the yeast-like form to the mycelial structure in this yeast could represent the intervention in the morphogenetic process of mitochondrial protein synthesis, since chloramphenicol blocks in vivo and in vitro mitochondrial protein synthesis. In fact, other functions such as primary and secondary respiration, do not seem to play a role in the morphogenetic development since their inhibition by antimycin A (AA) or by salicyl-hydroxamic acid (SHAM) does not affect the process. In addition, mitochondrial protein synthesis has been shown to be uninhibited by the two respiratory inhibitors.     

Mycological profile of crew during 56-day simulated orbital flight

Mycological analyses were made on specimens obtained from the skin, oral rinse, urine, and feces of three subjects participating in a 56 day Skylab simulation test. Samples were taken over a 175 day period divided into three phases consisting of a prechamber, intrachamber, and postchamber period. A total of 69 species of filamentous fungi and 22 species of yeast and yeast-like fungi were recovered and identified. There was a marked decrease in the isolation incidence of both the filamentous and yeast and yeast-like fungi during the intrachamber period. This, taken with the fact that 71% of the filamentous species and 55% of the yeast and yeast-like species were isolated no more than twice, reflects the transitory nature of the relationship between the human body and most fungi. However, there was a relative increase in the potential pathogens Candida albicans and Candida parapsilosis during the intrachamber period. Over 50% of all fungi recovered were isolated from the nasal and oral cavities. There was no evidence of intercrew transfer of a particular species during the intrachamber period nor was there any evidence of microbial shock during the postchamber period.     

Control of mating and development inUstilago maydis

InUstilago maydis thea andb mating type loci control pathogenicity as well as sexual development. We review the function of these loci in controlling the cell fusion step, the switch from yeast-like to filamentous growth and subsequent pathogenic development. Our special emphasis will be the role of pheromones and pheromone signaling in these processes.     

Comparison of morphogenetic networks of filamentous fungi and yeast.

Fungi generally display either of two growth modes, yeast-like or filamentous, whereas dimorphic fungi, upon environmental stimuli, are able to switch between the yeast-like and the filamentous growth mode. Signal transduction pathways have been elucidated in the budding yeast Saccharomyces cerevisiae, establishing a morphogenetic network that links cell-cycle events with cellular morphogenesis. Recent molecular genetic studies in several filamentous fungal model systems revealed key components required for distinct steps from fungal spore germination to the maintenance of polar hyphal growth, mycelium formation, and nuclear division. This allows a mechanistic comparison of yeast-like and hyphal growth and the establishment of a core model morphogenetic network for filamentous growth including signaling via the cAMP pathway, Rho modules, and cell cycle kinases. Appreciating similarities between morphogenetic networks of the unicellular yeasts and the multicellular filamentous fungi will open new research directions, help in isolating the central network components, and ultimately pave the way to elucidate the central differences (of many) that distinguish, e.g., the growth mode of filamentous fungi from that of their yeast-like relatives, the role of cAMP signaling, and nuclear division.     

Characterization and Molecular Genetic Complementation of Mutants Affecting Dimorphism in the FungusUstilago maydis

Ustilago maydis,the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a unicellular, nonpathogenic yeast-like form and a dikaryotic, pathogenic filamentous form. Previously, a constitutively filamentous haploid mutant was obtained. Complementation of this mutant led to the isolation of the gene encoding adenylate cyclase,uac1.Secondary mutagenesis of auac1disruption strain allowed the isolation of a large number of suppressor mutants, termedubc,forUstilagobypass of cyclase, lacking the filamentous phenotype. Analysis of one of these suppressor mutants previously led to the identification of theubc1gene, encoding the regulatory subunit of cAMP-dependent protein kinase. In this report we describe the isolation of cosmids containing three newubcgenes, termedubc2, ubc3,andubc4.We also describe the morphology of theubc2, ubc3,andubc4mutants in auac1⁻background as well as in a background with a functionaluac1gene. In addition, we describe several mutant strains not complemented with any of the genes currently in hand and that are thus presumed to possess mutations in additionalubcgenes.     

Cyanide-resistant alternative respiration is strictly correlated to intracellular peroxide levels in Acremonium Chrysogenum

A strict correlation between the intensity of the cyanide-resistant alternative respiratory pathway and the intracellular peroxide levels in the cephalosporin C producer filamentous fungus Acremonium chrysogenum was demonstrated. Intracellular peroxide levels increased in a dose-dependent manner after addition of H₂O₂ to the culture media. A similar phenomenon was observed due to the specific inhibition of catalase by salicylic acid. In both cases, cyanide-resistant respiration was markedly stimulated. On the other hand, both cyanide-resistant respiration and intracellular peroxide levels were effectively suppressed by the lipid peroxyl radical scavenger DL-α-tocopherol, which breaks lipid peroxidation chains effectively. Our findings firmly supported the assumption that there is a connection between the intracellular peroxide levels and the intensity of the alternative respiratory pathway in fungi.     

Culturable Fungi of Stored ‘Golden Delicious’ Apple Fruits: A One-Season Comparison Study of Organic and Integrated Production Systems in Switzerland

The effects of organic and integrated production systems on the culturable fungal microflora of stored apple fruits from five matched pairs of certified organic and integrated ‘Golden Delicious’ farms were studied at five representative production sites in Switzerland. Isolated fungi were identified morphologically. Colonization frequency (percentage of apples colonized), abundance (colony numbers), and diversity (taxon richness) were assessed for each orchard. The standard quality of the stored fruits was comparable for both organic and integrated apples and complied with national food hygiene standards. Yeasts (six taxa) and the yeast-like fungus Aureobasidium pullulans were the dominant epiphytes, filamentous fungi (21 taxa) the dominant endophytes. The most common fungi occurred at all sites and belonged to the “white” and “pink” yeasts, yeast-like A. pullulans, filamentous fungi Cladosporium spp., Alternaria spp., and sterile filamentous fungi. Canonical correspondence analysis of the total fungal community revealed a clear differentiation among production systems and sites. Compared to integrated apples, organic apples had significantly higher frequencies of filamentous fungi, abundance of total fungi, and taxon diversity. The effects of the production system on the fungal microflora are most likely due to the different plant protection strategies. The incidence of potential mycotoxin producers such as Penicillium and Alternaria species was not different between production systems. We suggest that higher fungal diversity may generally be associated with organic production and may increase the level of beneficial and antagonistically acting species known for their potential to suppress apple pathogens, which may be an advantage to organic apples, e.g., in respect to natural disease control.     

Production of ethanol by filamentous and yeast-like forms of <Emphasis Type="Italic">Mucor indicus</Emphasis> from fructose,glucose, sucrose,and molasses

The fungus Mucor indicus is found in this study able to consume glucose and fructose, but not sucrose in fermentation of sugarcane and sugar beet molasses. This might be an advantage in industries which want to selectively remove glucose and fructose for crystallisation of sucrose present in the molasses. On the other hand, the fungus assimilated sucrose after hydrolysis by the enzyme invertase. The fungus efficiently grew on glucose and fructose and produced ethanol in synthetic media or from molasses. The cultivations were carried out aerobically and anaerobically, and manipulated toward filamentous or yeast-like morphology. Ethanol was the major metabolite in all the experiments. The ethanol yield in anaerobic cultivations was between 0.35 and 0.48 g/g sugars consumed, depending on the carbon source and the growth morphology, while a yield of as low as 0.16 g/g was obtained during aerobic cultivation. The yeast-like form of the fungus showed faster ethanol production with an average productivity of 0.90 g/l h from glucose, fructose and inverted sucrose, than the filamentous form with an average productivity of 0.33 g/l h. The biomass of the fungus was also analyzed with respect to alkali-insoluble material (AIM), chitin, and chitosan. The biomass of the fungus contained per g maximum 0.217 g AIM and 0.042 g chitosan in yeast-like cultivation under aerobic conditions.     

The lipid content of cell walls obtained from juvenile,yeast-like and filamentous cells ofCandida albicans

Purified cell walls ofCandida albicans obtained from juvenile cells, mature yeast-like cells and filamentous cells were analyzed for their lipid components. Chloroform: methanol (2:1 v v) extraction of the acetone-treated dried cell walls indicated the total lipid content to be 2.1% of the dry weight of the juvenile cell walls, 1.8% of the mature yeast-like cell walls and 4.5% of the filamentous cell walls. Separation of the chloroform: methanol extractable fraction through a silicie acid column and quantitative determination of the fractions showed significant amounts of sterol esters, triglycerides, sterols, free fatty acids, and phospholipids in these extracts. Following acetone extraction sterols were shown to constitute a greater percentage of the cell wall of juvenile cells than mature cells. Thin-layer chromatography separated the acetone-extractable lipids into at least four components. Diethyl ether extracts of the cell walls indicated the presence of small amounts of glycerol phospholipids in the cell walls of juvenile and mature yeast cells. Boiling 95% ethanol also removed a small lipid fraction from the cell walls of both juvenile and mature yeast which could include sphingosine phosphatides or glycosides.     

Enhanced metabolism of phosphatidylinositol in Candida tropicalis in association with filamentous growth caused by ethanol

Candida tropicalis Pk 233 grows in filamentous form in ethanol-supplemented medium, and myo-inositol prevents the ethanol ettect [(1979) Bioch!em. Biophys. Res. Commun. 91, 351–355]. Cells, which were grown with ethanol to the log phase, exhibited an increased rate of phosphatidylinositol turnover as judged by pulse-chase experiments with ³²P_i. Such enhanced metabolism of phosphatidylinositol was not observed in fully developed filamentous cells at the stationary phase or in yeast-like cells which were grown without added ethanol or with ethanol plus myo-inositol. The metabolism of other phospholipids was not affected by ethanol.     

Lead biosorption by different morphologies of fungus Mucor indicus

Biosorption characteristics of Pb⁺² ions from aqueous solution were investigated using fungus Mucor indicus biomass treated with NaOH. Biosorption was measured as a function of biomass morphology, pH, biomass concentration, contact time, and metal concentration. The morphology of M. indicus biomass was manipulated towards filamentous or yeast-like forms. The highest and lowest biosorption capacities were observed for purely filamentous and yeast-like forms, respectively. Models of Langmuir, Freundlich, Temkin, and Scachard were applied to describe adsorption isotherm and fitted appropriately. Biosorption kinetics was successfully described using Ho’s pseudo-second-order model. Maximum and minimum values of biosorption capacity of Pb²⁺ were 22.1 and 12.1 mg g⁻¹ for purely filamentous and yeast-like morphologies, respectively. Increasing pH resulted in higher biosorption of Pb⁺² ions up to pH 5.5. Biosorption capacity of individual Pb⁺² ions was reduced in the presence of other metal ions in bi- or multi-metal ion experiments. Metal ions adsorption by the biomass could be eluted effectively with HNO₃.     

The machinery for cell polarity, cell morphogenesis, and the cytoskeleton in the Basidiomycete fungus Ustilago maydis-a survey of the genome sequence

Ustilago maydis, a Basidiomycete fungus that infects maize, exhibits two basic morphologies, a yeast-like and a filamentous form. The yeast-like cell is elongated, divides by budding, and the bud grows by tip extension. The filamentous form divides at the apical cell and grows by tip extension. The repertoire of morphologies is increased during interaction with its host, suggesting that plant signals play an important role in generation of additional morphologies. We have used Saccharomyces cerevisiae and Schizosaccharomyces pombe genes known to play a role in cell polarity and morphogenesis, and in the cytoskeleton as probes to survey the U. maydis genome. We have found that most of the yeast machinery is conserved in U. maydis, albeit the degree of similarity varies from strong to weak. The U. maydis genome contains the machinery for recognition and interpretation of the budding yeast axial and bipolar landmarks; however, genes coding for some of the landmark proteins are absent. Genes coding for cell polarity establishment, exocytosis, actin and microtubule organization, microtubule plus-end associated proteins, kinesins, and myosins are also present. Genes not present in S. cerevisiae and S. pombe include a homolog of mammalian Rac, a hybrid myosin-chitin synthase, and several kinesins that exhibit more similarity to their mammalian counterparts. We also used the U. maydis genes identified in this analysis to search other fungal and other eukaryotic genomes to identify the closest homologs. In most cases, not surprisingly, the closest homolog is among filamentous fungi, not the yeasts, and in some cases it is among mammals.     

Induction of alternative oxidase chain under salt stress conditions

This paper describes the effect of NaCl on the respiration of Citrus cell suspensions namely on the induction of the alternative oxidase. The exposure of two Citrus (cvs. Carvalhal tangor and Valencia late) cell suspensions to 200 or 400 mM NaCl lead to a reduction on cell respiration rates. Under these conditions, the respiration rate decreased less in the presence of KCN indicating a stimulation of the capacity of the alternative oxidase (AOX). In addition, immunoblots showed an increase on the amount of AOX protein. Antibodies raised against the Sauromatum guttatum enzyme recognized the reduced form of the enzyme near the 35 kDa band. The protein accumulation was correlated with the significantly higher AOX capacity observed for cv. Carvalhal tangor.     

Two alternative pathways for substrate assimilation byMucor circinelloides

Transition of an oleaginous strain ofMucor circinelloides from mycelial to yeast-like form was studied in conditions favoring lipogenesis. In culture media with citric acid as the sole carbon source at concentrations of 7.5 and 10 g/L (C/N ratio of 26 and 35, respectively), the mold accumulated significant quantities of lipid. At a higher citric acid concentration liposynthesis was inhibited and the fermentation mechanism decreased the high substrate concentration in the culture media. Under these conditions yeast-like morphogenesis was observed. In the yeast-like cells, biosynthesis of linoleic and γ-linolenic acid was inhibited. In contrast, no significant changes were observed in the biosynthesis of palmitoleic acid whereas the concentration of oleic acid was increased in the storage lipids of yeast-like cells.     

Optimization of conditions for the production of pullulan and high molecular weight pullulan by Aureobasidium pullulans

Aureobasidium pullulans had a maximum yield coefficient of pullulan (Y _p/s=0.24) with an initial pH of the culture broth of 6.5 in a shake-flask culture. In a batch culture, the maximum pullulan yield coefficient of 0.30 was obtained at the aeration rate of 0.5 vvm. A yeast-like form and mycelial form of cells were found at the culture broth with pH controlled at 4.5 with a maximum yield coefficient of pullulan of 0.27. However, a high portion (35%) of high molecular weight pullulan (M _w>2 000 000) was produced at pH 6.5 with a yeast-like morphology of the cells.