A putative phospholipase C is involved in Pichia fermentans dimorphic transition

Background

Pichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi.

Methods

Pichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition.

Results

Five genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18.

Conclusion

Phospholipase C is part of a putative “methionine sensing machinery” that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition.

General significance

Phospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.     

The strange case of a biofilm-forming strain of Pichia fermentans, which controls Monilinia brown rot on apple but is pathogenic on peach fruit

A biofilm-forming strain of Pichia fermentans proved to be most effective in controlling brown rot on apple fruit when coinoculated into artificial wounds with a phytopathogenic isolate of Monilinia fructicola. Culture filtrates and autoclaved cells had no significant influence on the disease. When sprayed onto the apple fruit surface, this yeast formed a thin biofilm but failed to colonize the underlying tissues. When inoculated into wounds artificially inflicted to peach fruit or when sprayed onto the surface of peach fruit, the same strain showed an unexpected pathogenic behaviour, causing rapid decay of fruit tissues even in the absence of M. fructicola. Both optical and scanning electron microscopy were used to evaluate the pattern of fruit tissue colonization by P. fermentans. While on apple surface and within the apple wound the antagonist retained its yeast-like shape, colonization of peach fruit tissue was always characterized by a transition from budding growth to pseudohyphal growth. These results suggest that pseudohyphal growth plays a major role in governing the potential pathogenicity of P. fermentans, further emphasizing the importance of a thorough risk assessment for the safe use of any novel biocontrol agent.     

Influence of carbon and nitrogen sources on the transition from yeast-like cells to chlamydospores inAureobasidium pullulans

The transition from yeast-like cells to chlamydospores ofAureobasidium pullulans can be induced by growing the microorganism on a glucose medium with a limiting nitrogen source and a low buffer capacity. When glucose is used as the carbon source, a concentration higher than 3% (w/v) is required to induce the transition. On the other hand, growth limiting concentrations of the N source (ammonium sulphate) are not required, and higher concentrations actually stimulate the appearance of chlamydospores. Other N sources, such as glutamate or ammonium phosphate, do not induce the transition from yeast-like cells to chlamydospores.     

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.     

Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A.

The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.     

Medium pH, carbon and nitrogen concentrations modulate the phosphate solubilization efficiency of Penicillium purpurogenum through organic acid production

Aims: To study phosphate solubilization in Penicillium purpurogenum as function of medium pH, and carbon and nitrogen concentrations. Methods and Results: Tricalcium phosphate (CP) solubilization efficiency of P. purpurogenum was evaluated at acid or alkaline pH using different C and N sources. Glucose‐ and (NH₄)₂SO₄‐based media showed the highest P solubilization values followed by fructose. P. purpurogenum solubilizing ability was higher in cultures grown at pH 6·5 than cultures at pH 8·5. Organic acids were detected in both alkaline and neutral media, but the relative percentages of each organic acid differed. Highest P release coincided with the highest organic acids production peak, especially gluconic acid. When P. purpurogenum grew in alkaline media, the nature and concentration of organic acids changed at different N and C concentrations. A factorial categorical experimental design showed that the highest P‐solubilizing activity, coinciding with the highest organic acid production, corresponded to the highest C concentration and lowest N concentration. Conclusions: The results described in the present study show that medium pH and carbon and nitrogen concentrations modulate the P solubilization efficiency of P. purpurogenum through the production of organic acids and particularly that of gluconic acid. In the P solubilization optimization studies, glucose and (NH₄)₂SO₄ as C and N sources allowed a higher solubilization efficiency at high pH. Significance and Impact of the Study: This organism is a potentially proficient soil inoculant, especially in P‐poor alkaline soils where other P solubilizers fail to release soluble P. Further work is necessary to elucidate whether these results can be extrapolated to natural soil ecosystems, where different pH values are present. Penicillium purpurogenum could be used to develop a bioprocess for the manufacture of phosphatic fertilizer with phosphate calcium minerals.     

Increase in antioxidant enzyme activity,stress tolerance and biocontrol efficacy of Pichia kudriavzevii with the transition from a yeast-like to biofilm morphology

Morphological change, such as from yeast-like to biofilm, has been recently considered to be involved in the mode of action of some antagonistic yeasts used as postharvest biocontrol agents. In the present study, the biocontrol yeast, Pichia kudriavzevii, reversibly shifted from a yeast-like morphology on yeast peptone dextrose (YPD) medium with 2% agar to a biofilm morphology on YPD with 0.3% agar. The tolerance of P. kudriavzevii to heat and oxidative stresses, as well as the biocontrol efficacy against postharvest diseases on pear fruit, increased significantly from the yeast-like form to the biofilm form. The activity of antioxidant enzymes, including catalase and superoxidase dismutase, in the biofilm form was also significantly higher. The elevated activity of antioxidant enzymes was associated with less protein and lipid oxidation in the biofilm form, compared to the yeast-like form, under heat and oxidative stresses. These results suggest that activation of antioxidant system with the morphology shift contributes to the enhancement of abiotic stress tolerance and biocontrol performance of P. kudriavzevii. These findings provide new information on the biology of yeast antagonists that is essential for their potential application and development.     

模拟氮沉降对杉木幼苗细根的生理生态影响

细根对氮沉降的生理生态响应将显著影响森林生态系统的生产力和碳吸存。为了揭示氮沉降对杉木细根的生理生态影响,对一年生杉木(Cunninghamia lanceolata)幼苗进行了模拟氮沉降试验,并测定施氮1年后杉木幼苗细根生物量、细根形态学特征(比根长、比表面积)、元素化学计量学指标(C、N、P、C/N、C/P、N/P)、细根代谢特征(细根比呼吸速率、非结构性碳水化合物)。结果表明:(1)杉木细根生物量随氮添加水平的升高而显著降低,尤其是0—1 mm细根生物量;细根比根长和比表面积随氮添加水平升高而显著增大。(2)氮添加后杉木细根C含量、C/N、C/P显著降低,高氮添加导致1—2 mm细根N含量和N/P显著升高,而低氮添加导致1—2 mm细根P含量显著升高、N/P显著降低,而0—1 mm细根的N、P含量则保持相对稳定。(3)氮添加后杉木细根比呼吸速率无显著变化,细根可溶性糖含量随氮添加增加而显著增加,而淀粉含量和NSC显著降低。综合以上结果表明:氮添加后用于细根形态构建的碳分配减少,这可能会减少土壤中有机碳的保留,0—1 mm细根的形态更易发生变化,但是其内部N、P养分含量相对更稳定以维持生理活动,细根NSC对氮添加的响应表明施氮可能导致细根受光合产物的限制。     

Control of dimorphism in a biochemical variant of Candida albicans

The cellular morphology of a biochemical variant of Candida albicans could be controlled by the ratio of carbon dioxide to oxygen in the culture system or by individual amino acids. Predominantly pseudohyphal morphology was observed (i) at a CO(2) to O(2) ratio of 2:1 and (ii) without the addition of carbon dioxide, when either glycine, d- or l-ornithine, l-serine, l-methionine, l-phenylalanine, or l-tyrosine was the sole nitrogen source in the culture medium. When ammonium chloride, ammonium sulfate, l-glutamic acid, l-glutamine, or l-proline was the nitrogen source, yeastlike growth was observed in the presence or absence of CO(2). More adenosylmethionine was present in pseudohyphal than in yeastlike cells, and pseudohyphal cell wall preparations contained less methionine than cell walls from the yeastlike form. These results suggest a correlation between sulfur amino acid metabolism and dimorphism.     

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.     

Efficient lipid production with Trichosporon fermentans and its use for biodiesel preparation

Effects of medium components and culture conditions on biomass and lipid production of Trichosporon fermentans were studied. The optimal nitrogen source, carbon source and C/N molar ratio were peptone, glucose and 163, respectively. The favorable initial pH of the medium and temperature were 6.5 and 25 degrees C. Under the optimized conditions, a biomass of 28.1 g/l and a lipid content of 62.4% could be achieved after culture for 7 days, which were much higher than the original values (19.4 g/l and 50.8%) and the results reported by other groups. T. fermentans could grow well in pretreated waste molasses and a lipid yield of 12.8 g/l could be achieved with waste molasses of 15% total sugar concentration (w/v) at pH 6.0, representing the best result with oleaginous microorganisms on agro-industrial residues. Addition of various sugars to the pretreated molasses could efficiently enhance the accumulation of lipid and the lipid content reached as high as above 50%. Similar to vegetable oils, the lipid mainly contains palmitic acid, stearic acid, oleic acid and linoleic acid and the unsaturated fatty acids amount to about 64% of the total fatty acids. The microbial oil with an acid value of 5.6 mg KOH/g was transesterified to biodiesel by base catalysis after removal of free fatty acids and a high methyl ester yield of 92% was obtained.     

The production and growth characteristics of yeast and mycelial forms of Candida albicans in continuous culture.

The growth characteristics of Candida albicans CM145,348 have been examined under aerobic conditions in continuous culture. At different steady states the environment was controlled with respect to the concentrations of dissolved oxygen, carbon and nitrogen, the pH, and the temperature. Dry matter, substrate concentration, yield, specific oxygen uptake, specific carbon dioxide release and respiration quotient were examined as a function of the dilution rate. The morphology depended on the carbon source. Maltose produced a mycelial morphology, whereas with lactate a yeast culture was obtained. With fructose or glucose as a carbon source a mixed morphology of yeast, pseudo-mycelial and mycelial forms was produced. A larger number of different growth conditions were examined in batch culture but a mixed morphology was always obtained.     

Effect of culture medium composition on the activity of extracellular lectins of Lentinus edodes

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under the conditions of submerged culture was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and pH of culture medium. The activity of lectin in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C : N ratio, (9.5-12): 1)) on the day 15-18 of culturing at pH 8-9.     

Improving lipid production from bagasse hydrolysate with Trichosporon fermentans by response surface methodology

Oleaginous yeast Trichosporon fermentans was proved to be able to use sulphuric acid-treated sugar cane bagasse hydrolysate as substrate to grow and accumulate lipid. Activated charcoal was shown as effective as the more expensive resin Amberlite XAD-4 for removing the inhibitors from the hydrolysate. To further improve the lipid production, response surface methodology (RSM) was used and a 3-level 4-factor Box-Behnken design was adopted to evaluate the effects of C/N ratio, inoculum concentration, initial pH and fermentation time on the cell growth and lipid accumulation of T. fermentans. Under the optimum conditions (C/N ratio 165, inoculum concentration 11%, initial pH 7.6 and fermentation time 9 days), a lipid concentration of 15.8g/L, which is quite close to the predicted value of 15.6g/L, could be achieved after cultivation of T. fermentans at 25°C on the pretreated bagasse hydrolysate and the corresponding lipid coefficient (lipid yield per mass of sugar, %) was 14.2. These represent a 32.8% improvement in the lipid concentration and a 21.4% increase in the lipid coefficient compared with the original values before optimization (11.9g/L and 11.7). This work further demonstrates that T. fermentans is a promising strain for lipid production and thus biodiesel preparation from abundant and inexpensive lignocellulosic materials.     

Lipid accumulation and growth of Chlorella zofingiensis in flat plate photobioreactors outdoors

Culturing microalgae using natural sunlight is an effective way to reduce the cost of microalgae-based biodiesel production. In order to evaluate the feasibility of culturing Chlorella zofingiensis outdoors for biodiesel production, effects of nitrogen limitation and initial cell concentration on growth and lipid accumulation of this alga were investigated in 60 L flat plate photobioreactors outdoors. The highest μmax and biomass productivity obtained was 0.994 day(-1) and 58.4 mg L(-1)day(-1), respectively. The lipid content was much higher (54.5% of dry weight) under nitrogen limiting condition than under nitrogen sufficient condition (27.3%). With the increasing initial cell concentrations, the lipid contents declined, while lipid concentrations and productivities increased. The highest lipid content, lipid concentration, and lipid productivity obtained was 54.5%, 536 mg L(-1) and 22.3 mg L(-1)day(-1), respectively. This study demonstrated that it was possible to culture C. zofingiensis under outdoor conditions for producing biodiesel feedstock.     

出芽短梗霉的研究进展

出芽短梗霉是一类类酵母真菌,具有酵母样和真菌菌丝体两种形态,影响其形态的因素有碳源,氮源,离子种类及浓度和pH值等,出芽短梗霉的发酵产物多种多样,如多聚糖,酶,抗真菌素等,通过选育优良菌株可提高发酵产物的产量。     

The effect of culture conditions on the morphology of the dimorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415: a study incorporating image analysis

The effect of changing environmental conditions on the morphology of the yeast Kluyveromyces marxianus var. marxianus NRRLy2415 was investigated in batch and continuous culture, using a previously developed computer-aided image analysis protocol [O'Shea and Walsh (1996) Biotechnol Bioeng 51: 679–690]. The morphology of the organism is primarily controlled by the specific growth rate, μ. This finding was contrary to a previous investigation [Walker and O'Neill (1990) J Chem Tech Biotechnol 49: 75–89]. When the organism is cultured in batch with excess oxygen, μ can approach the maximum specific growth rate, μ_m, and the primary morphology of the culture is yeast-like. However, if the organism is cultured in a chemostat, thereby controlling the growth rate, the morphology reverts to a pseudohyphal form. This response is thought to be an adaptation by the organism to its environment, whereby it assumes a foraging form under adverse environmental conditions. The use of computer-aided image analysis made possible the discrimination of subtle morphological differences between samples and the determination of the relationship between morphology and growth rate. Received: 12 April 1999 / Accepted: 10 October 1999     

Physiological and biochemical response of seaweed Gracilaria lemaneiformis to concentration changes of N and P

This paper presents the studies of nitrogen and phosphorus (N/P) concentrations influence on the antioxidantivity of Gracilaria lemaneiformis and the physio-chemical features in an indoor seawater culture system. The results showed that the specific growth rate, the chemical composition (concentrations of chlorophyll a, phycoerythrin and soluble protein), nitrate reductase activity, antioxidantive defense system (the activities of superoxide dismutase, peroxidase, catalase) and propyldialdehyde content of G. lemaneiformis were affected by changes in concentrations of N and P, and in seaweed culture time. G. lemaneiformis showed increased growth rate when the N/P concentrations increased from 50/3.13 µmol/L to 400/25 µmol/L. But when the N/P concentrations exceeded 400/25 µmol/L, the growth rate dropped significantly. The trend became more obvious once the N/P concentrations reached 600 / 37.5 µmol/L. At these concentrations, the chloroplasts in G. lemaneiformis were damaged as evidences that 1) the number of thylakoids was increased, some of them were swollen up, irregularly aligned, ruptured and partly dissolved; 2) the chloroplast volume was increased; and 3) starch grains in chloroplasts accumulated significantly. On the basis of these results, a remediation critical point for the growth of G. lemaneiformis under high N/P concentrations was proposed.