The relationship between freezing resistance and fatty acid composition of yeasts

Abstract The relationship between freezing resistance and cellular long-chain fatty acid composition of 18 selected yeast strains was studied. All strains produced a series of saturated and unsaturated evennumbered fatty acids ranging from 14 to 20 carbons in length. The majority of the freeze-resistant yeasts were found among fermentative species with a content of oleic acid above 40%.     

临床酵母样真菌的感染特点及耐药性研究

目的了解临床酵母样真菌的感染类型、分布以及耐药情况,为临床诊断治疗提供合理的用药依据.方法采用常规方法进行真菌培养,用科玛嘉显色培养基联合法国生物梅里埃API 20C AUX鉴定条进行鉴定,药敏试验采用微量稀释法.结果864株酵母样真菌中,白色念珠菌619株（71.6%）,其次为热带念珠菌116株（13.4%）和克柔念珠菌47株（5.4%）,非白色念珠菌感染的比例逐年上升（21.5%）.其中,呼吸道标本酵母样真菌检出率最高,达79.7%,其次是消化道为8.8%,泌尿道为4.7%.科室分布依次为干部科、呼吸科、急诊内科、血液科等;白色念珠菌对两性霉素B、5-氟胞嘧啶高度敏感,达90%以上,对氟康唑和伊曲康唑的敏感性有所降低.结论酵母样真菌的检出率与患者基础疾病密切相关;对氟康唑等药物的敏感性有下降的趋势,未发现对4种药物同时耐药的菌株,提示在治疗中,药敏监测是非常必要的.     

Copper resistance mechanisms in bacteria and fungi

Abstract: Copper is both an essential micronutrient and a toxic heavy metal for most living cells. The presence of high concentrations of cupric ions in the environment promotes the selection of microorganisms possessing genetic determinants for copper resistance. Several examples of chromosomal and plasmid copper-resistance systems in bacteria have been reported, and the mechanisms of resistance have started to be understood at the molecular level. Bacterial mechanisms of copper resistance are related to reduced copper transport, enhanced effiux of cupric ions, or copper complexation by cell components. Copper tolerance in fungi has also been ascribed to diverse mechanisms involving trapping of the metal by cell-wall components, altered uptake of copper, extracellular chelation or precipitation by secreted metabolites, and intracellular complexing by metallothioneins and phytochelatins; only the metallothionein chelation mechanism has been approached with molecular detail.     

A simple method for extraction of DNA from fungi and yeasts with anhydrous hydrogen fluoride

A novel method is described for the extraction of DNAs from fungi and yeasts. Anhydrous hydrogen fluoride (HF) selectively cleaves their cell walls under mild conditions (for 5 min at 0°C), enabling the effective extraction of DNAs from organisms with a cell wall. A possible mechanism for this method concerning the selective cleavage of O-glycosidic linkages in cell walls has been described previously [(1977) Anal. Biochem. 82, 289–309]. The extracted DNA is intact: in fact, the yeast DNA is directly applicable for restriction analysis and transformation of Escherichia coli.     

Nickel and cobalt resistance of various bacteria isolated from soil and highly polluted domestic and industrial wastes

Abstract From enrichment cultures in the presence of 1 mM NiCl₂ 200 strains of aerobic bacteria were isolated from 50 samples collected in the metal-processing industry, waste water treatment plants and from solid waste, highly polluted by heavy metals. The strains isolated were characterized with respect to their substrate spectrum and resistance to nickel, cobalt, zinc and cadmium salts and assigned to 21 groups. One representative of each group was described with respect to cell morphology. All strains were Gram-negative, non-sporing rods or cocci. The highest concentrations of nickel, cobalt, zinc, cadmium, copper, mercury, and silver allowing growth on solid media were estimated. Two strains were able to grow at 20 mM NiCl₂ and CoCl₂, one strain tolerated 12 mM and one 7.5 mM concentrations of these salts.
Fifteen out of 21 strains contained at least one plasmid two contained two plasmids. The plasmid sizes varied between 50 and 340 kbp, except strain 10A, which contained a miniplasmid (2.6 kbp). Attempts to cure four selected strains by exposure to mitomycin C or growth at elevated temperature failed.
By helper-assisted and unassisted conjugation the plasmids of strain 31A were shown to carry nickel and cobalt resistance determinants. Alcaligenes eutrophus strains H16 and N9A and denative of strain CH34 lacking one or both of its native metal resistance plasmids were used as recipients. Both plasmids, p TOM8 and pTOM9, of strain 31A carried resistance properties which were expressed in all recipients except. A. eutrophus H16, in which only nickel resistance was expressed.
Plasmid pTOM3 residing in strain 10A could not be transferred as such. However, transconjugants derived from helper (pULB113)-assisted matings carried co-integrates of various sizes and were resistant to nickel and cobalt.     

The Chitinase A from the baculovirus AcMNPV enhances resistance to both fungi and herbivorous pests in tobacco

Biotechnology has allowed the development of novel strategies to obtain plants that are more resistant to pests, fungal pathogens and other agents of biotic stress. The obvious advantages of having genotypes with multiple beneficial traits have recently fostered the development of gene pyramiding strategies, but less attention has been given to the study of genes that can increase resistance to different types of harmful organisms. Here we report that a recombinant Chitinase A protein of the Autographa californica nuclear polyhedrosis virus (AcMNPV) has both antifungal and insecticide properties in vitro. Transgenic tobacco plants expressing an active ChiA protein showed reduced damages caused by fungal pathogens and lepidopteran larvae, while did not have an effect on aphid populations. To our knowledge, this is the first report on the characterisation and expression in plants of a single gene that increases resistance against herbivorous pests and fungal pathogens and not affecting non-target insects. The implications and the potential of the ChiA gene for plant molecular breeding and biotechnology are discussed.     

Identification of fungi associated with municipal compost using DNA-based techniques

Fungi are important in terrestrial decay processes. However, fungi associated with organic decay during composting are still not well known. In this study culture-independent methods were used to identify fungi associated with composting organic municipal wastes to gain a better understanding of the diversity of fungi associated with this process. Fungal communities from 0, 210, and 410 day-old compost samples were assessed with DNA fingerprinting using denaturing gradient gel electrophoresis (DGGE) and by the analysis of DNA sequences from rDNA clone libraries. From 207 rDNA sequences, 82 fungal OTU’s were detected. A disproportionate number of yeast sequences were detected in Day 0 clone libraries, including the human pathogens Candida tropicalis and Candida krusei (Saccharomycetales). Basidiomycetes accounted for over half of the clones from the Day 210 sample. Clones of Cercophora and Neurospora species accounted for most of the fungal clones of the Day 410 sample. No Zygomycetes or Aspergillus species were detected in this study. These findings call for a reassessment of long held views about the organisms involved in the composting of organic municipal wastes.     

Ecology of siderophores with special reference to the fungi

Ecology of siderophores, as described in the present review, analyzes the factors that allow the production and function of siderophores under various environmental conditions. Microorganisms that excrete siderophores are able to grow in natural low-iron environments by extracting residual iron from insoluble iron hydroxides, protein-bound iron or from other iron chelates. Compared to the predominantly mobile bacteria, the fungi represent mostly immobile microorganisms that rely on local nutrient concentrations. Feeding the immobile is a general strategy of fungi and plants, which depend on the local nutrient resources. This also applies to iron nutrition, which can be improved by excretion of siderophores. Most fungi produce a variety of different siderophores, which cover a wide range of physico-chemical properties in order to overcome adverse local conditions of iron solubility. Resource zones will be temporally and spatially dynamic which eventually results in conidiospore production, transport to new places and outgrow of mycelia from conidiospores. Typically, extracellular and intracellular siderophores exist in fungi which function either in transport or storage of ferric iron. Consequently, extracelluar and intracellular reduction of siderophores may occur depending on the fungal strain, although in most fungi transport of the intact siderophore iron complex has been observed. Regulation of siderophore biosynthesis is essential in fungi and allows an economic use of siderophores and metabolic resources. Finally, the chemical stability of fungal siderophores is an important aspect of microbial life in soil and in the rhizosphere. Thus, insolubility of iron in the environment is counteracted by dissolution and chelation through organic acids and siderophores by various fungi.     

Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.     

Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.     

Adaptation of fungi,including yeasts,to cold environments

A wide range of cold environments exist, with an equally broad variety of fungi and yeasts that have adapted to such environments. These adaptations, which affect membranes, enzymes and other cellular components, such as radical scavenging molecules, display a great potential for exploitation in biotechnology. Alterations have been detected in membrane lipids, with an increase in fatty acid unsaturated bonds that enhance their fluidity. We report new data on the different phospholipid composition in membrane lipids in the same fungal species from both Antarctic and temperate regions. The decrease in temperature causes intracellular oxidative stress by inducing the generation of reactive oxygen species. We report the results of the first analysis of the non-enzymatic antioxidant response and phenolic compound production by an Antarctic strain of Geomyces pannorum. A survey on yeasts from the cryosphere is reported with a focus on their adaptation to a cold environment. Some studies have shown that the number of macrofungi in glacier forefronts rises as deglaciation increases. The survival success of many plants in such areas may be attributed to their mycorrhizal associations. We highlighted the macrofungal biodiversity of some Italian alpine habitats, in which we Inocybe microfastigiata, Laccaria montana and Lactarius salicis-herbaceae were recorded for the first time in Lombardy (Italy).     

Nickel uptake and utilization by microorganisms

Nickel is an essential nutrient for selected microorganisms where it participates in a variety of cellular processes. Many microbes are capable of sensing cellular nickel ion concentrations and taking up this nutrient via nickel-specific permeases or ATP-binding cassette-type transport systems. The metal ion is specifically incorporated into nickel-dependent enzymes, often via complex assembly processes requiring accessory proteins and additional non-protein components, in some cases accompanied by nucleotide triphosphate hydrolysis. To date, nine nickel-containing enzymes are known: urease, NiFe-hydrogenase, carbon monoxide dehydrogenase, acetyl-CoA decarbonylase/synthase, methyl coenzyme M reductase, certain superoxide dismutases, some glyoxylases, aci-reductone dioxygenase, and methylenediurease. Seven of these enzymes have been structurally characterized, revealing distinct metallocenter environments in each case.     

The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi

The view of the role of trehalose in yeast has changed in the last few years. For a long time considered a reserve carbohydrate, it gained new importance when its function in the acquisition of thermotolerance was demonstrated. More recently the cellular processes in which the trehalose biosynthetic pathway has been implicated range from the control of glycolysis to sporulation and infectivity by certain fungal pathogens. There is now enough experimental evidence to conclude that trehalose 6-phosphate, an intermediate of trehalose biosynthesis, is an important metabolic regulator in such different organisms as yeasts or plants. Its inhibition of hexokinase plays a key role in the control of the glycolytic flux in Saccharomyces cerevisiae but other, likely important, sites of action are still unknown. We present examples of the phenotypes produced by mutations in the two steps of the trehalose biosynthetic pathway in different yeasts and fungi, and whenever possible examine the molecular explanations advanced to interpret them.     

Overexpression of Bop3 confers resistance to methylmercury in Saccharomyces cerevisiae through interaction with other proteins such as Fkh1, Rts1, and Msn2

We found that overexpression of Bop3, a protein of unknown function, confers resistance to methylmercury in Saccharomyces cerevisiae. Bmh2, Fkh1, and Rts1 are proteins that have been previously shown to bind Bop3 by the two-hybrid method. Overexpression of Bmh2 and the homologous protein Bmh1 confers resistance to methylmercury in yeast, but overexpression of either Fkh1 or Rts1 has a minimal effect. However, the increased level of resistance to methylmercury produced by overexpression of Bop3 was smaller in Fhk1-deleted yeast as compared with that of the wild-type strain. In contrast, the degree of resistance was significantly elevated in Rts1-deleted yeast. Msn2 and Msn4 were previously reported as proteins that bind to Bmh1 and Bmh2. Overexpression of Msn2 conferred a much greater sensitivity to methylmercury in yeast, while deletion of the corresponding gene lowered the degree of resistance to methylmercury induced by overexpression of Bop3. These results suggest that multiple proteins are involved in minimizing the toxicity of methylmercury induced by overexpression of Bop3.     

Mechanisms of resistance to antifungal agents: yeasts and filamentous fungi

Failure to respond to antifungal therapy could be due to in vitro resistance (intrinsic or developed during therapy) or clinical resistance; the latter is associated with numerous factors related to the host, the antifungal agent, or the infecting isolate. Recently, a susceptible MIC breakpoint ( < or =2 microg/ml) was designed for Candida spp. to all three available echinocandins, anidulafungin (Pfizer), caspofungin (Merck) and micafungin (Astellas) and treatment failures have been associated with MICs > 2 microg/ml. In some of these cases, clinical failure was associated with the genetic mutations described below. Azole and flucytosine breakpoints, and the echinocandin susceptible breakpoint, are useful when isolates are tested by CLSI standardized methods; breakpoints are also available by the EUCAST method. More recently, in vitro resistant MIC breakpoints have been assigned for filamentous fungi (moulds) vs. five antifungal agents, but these categories are not based on correlations of in vitro with in vivo response to therapy. However, itraconazole (Janssen), amphotericin B (Bristol-Myers) and voriconazole (Pfizer) clinical failures in aspergillosis have been correlated with MICs > 2 microg/ml. This article provides a review of reported resistance molecular mechanisms to antifungal agents since 2005; previous related reviews are also listed.     

Identification of F-box proteins that are involved in resistance to methylmercury in Saccharomyces cerevisiae

We searched for F-box proteins that might be related to the mechanism that protects Saccharomyces cerevisiae against the toxic effects of methylmercury. We found that overexpression of Hrt3 and of Ylr224w rendered yeast cells resistant to methylmercury. Yeast cells that overexpressed Hrt3 and Ylr224w were barely resistant to methylmercury in the presence of a proteasome inhibitor. Our results suggest the existence of some protein(s) that enhances the toxicity of methylmercury in yeast cells and, also, that overexpression of Hrt3 or Ylr224w can confer resistance to methylmercury by enhancing the polyubiquitination of this protein(s) and its degradation in proteasomes.     

Malic enzyme: its purification and characterization fromMucor circinelloides and occurrence in other oleaginous fungi

Malic enzyme was purified 43-fold from Mucor circinelloides. The enzyme was dependent on Mg²⁺ or Mn²⁺ for activity, was not active with Dmalate and had a pH optimum at 7.8. The apparent K_m values for malate and NADP⁺ were 488 ΜM and 41 Μm respectively. The M_r of the native enzyme was 160 kDa. Five metabolic analogues of malate: oxaloacetate, tartronic acid, 1-methylenecyclopropane trans-2,3-dicarboxyIic acid, malonic acid and glutaric acid, were found to inhibit malic enzyme activity at 10 mM. Four oleaginous fungi, Mucor circinelloides, Mortierella alpina, Mortierella elongata and Pythium ultimum, were also examined, all possessed a soluble malic enzyme, two also possessed a microsomal malic enzyme.