Proteomic study of the yeast Rhodotorula mucilaginosa RCL-11 under copper stress

In order to understand the mechanism involved in Rhodotorula mucilaginosa RCL-11 resistance to copper a proteomic study was conducted. Atomic absorption spectroscopy showed that the copper concentration in the medium decreased from 0.5 to 0.19 mM 48 h after inoculation of the yeast. Analysis of one-dimensional gel electrophoresis of crude cell extracts revealed expression of differential bands between cells with and without copper. In order to study this difference, two-dimensional electrophoresis of R. mucilaginosa RCL-11 exposed to Cu for 16, 24, and 48 h was carried out. Identification of differentially expressed proteins was performed by MALDI-TOF/TOF. Ten of the 16 spots identified belonged to heat shock proteins. Superoxide dismutase, methionine synthase and beta-glucosidase were also found over-expressed at high copper concentrations. The results obtained in the present work show that when R. mucilaginosa RCL-11 is exposed to 0.5 mM copper, differential proteins, involved in cell resistance mechanisms, are expressed.     

The combined effect of salt stress and heat shock on proteome profiling in Suaeda salsa

Under natural conditions or in the field, plants are often subjected to a combination of different stresses such as salt stress and heat shock. Although salt stress and heat shock have been extensively studied, little is known about how their combination affects plants. We used proteomics, coupled with physiological measurements, to investigate the effect of salt stress, heat shock, and their combination on Suaeda salsa plants. A combination of salt stress and heat shock resulted in suppression of CO₂ assimilation and the photosystem II efficiency. Approximately 440 protein spots changed their expression levels upon salt stress, heat shock and their combination, and 57 proteins were identified by MS. These proteins were classified into several categories including disease/defense, photosynthesis, energy production, material transport, and signal transduction. Some proteins induced during salt stress, e.g. choline monooxygenase, chloroplastic ATP synthase subunit beta, and V-type proton ATPase catalytic subunit A, and some proteins induced during heat shock, e.g. heat shock 70 kDa protein, probable ion channel DMI1, and two component sensor histidine kinase, were either unchanged or suppressed during a combination of salt stress and heat shock. In contrast, the expression of some proteins, including nucleoside diphosphate kinase 1, chlorophyll a/b binding protein, and ABC transporter I family member 1, was specifically induced during a combination of salt stress and heat shock. The potential roles of the stress-responsive proteins are discussed.     

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20°C, and the other psychrotrophic, capable of growth at temperatures above 20°C. In addition to species-specific heat shock protein (hsp) profiles, a heat shock (15°C–25°C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15°C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25°C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles. Received: August 7, 1997 / Accepted: October 22, 1997     

高氨氮利用酵母菌的筛选及相关酶活性

【背景】蛋白饲料的缺乏,促进了蛋白含量高、安全性能好的酵母类单细胞蛋白的研究与应用。【目的】筛选氨氮利用能力强的菌株,为单细胞蛋白的发酵提供优良菌株。【方法】从土壤、奶制品、水果采集样品分离酵母菌,根据形态学和分子生物学鉴定菌株,然后以硫酸铵为唯一氮源培养基,测定菌落大小、菌体干重、蛋白质含量,复筛氨氮利用率高的酵母菌,并对复筛菌株氨同化相关酶活性进行测定。【结果】经过形态学、分子生物学鉴定和氨氮利用能力评价,获得3株高氨氮利用的酵母菌,分别是胶红酵母(Rhodotorula mucilaginosa)、酿酒酵母(Saccharomyces cerevisiae)和戴尔有孢圆酵母(Torulaspora delbrueckii)。通过比较3株酵母菌的谷氨酸脱氢酶、谷氨酸合成酶、谷氨酰胺合成酶活性,酿酒酵母的3种酶活性最高,其次是胶红酵母。【结论】从奶酪和西瓜中分离的胶红酵母N5和酿酒酵母J1具有较强的氨氮利用能力以及酶活性,可为单细胞蛋白发酵提供优良菌株。     

Novel oxidative stress-responsive gene ERS25 functions as a regulator of the heat-shock and cell death response

Members of the yeast p24 family, including Emp24p and Erv25p, exist as heteromeric complexes that have been proposed to cycle between the endoplasmic reticulum (ER) and Golgi compartments. The specific functions and sites of action of p24 proteins are still unknown. Here we identified a human homolog of the yeast p24 family of proteins, named ERS25 (endoplasmic reticulum stress-response protein 25), and investigated its role in stress response. ERS25 is predicted to have an ER localization signal peptide, a GOLD (Golgi dynamics) domain, which is found in several eukaryotic Golgi and lipid-trafficking proteins, a coiled-coil region, and a transmembrane domain. We demonstrate that ERS25 is localized to the ER and is induced by ER-specific stress, heat shock, and oxidative stress. The selective induction of ERS25 by brefeldin A, but not tunicamycin, implicates the involvement of ERS25 in protein trafficking between the ER and the Golgi. Small interfering RNA-mediated inhibition of ERS25 results in a significant decrease in apoptosis as well as a reduction of reactive oxygen species induced by oxidative stress. Moreover, ERS25 depletion results in a significant increase in the levels of the ER chaperone HSP70 in response to heat-shock stress through increased levels of HSF-1. We also found that inhibition of ERS25 induction in response to heat shock enhanced the binding of HSP70 to Apaf-1, which is likely to interfere in stress-mediated apoptosis. Together, the data presented here demonstrate that ERS25 may play a critical role in regulation of heat-shock response and apoptosis.     

The role of carotenoids in preventing oxidative damage in the pigmented yeast, Rhodotorula mucilaginosa

Rhodotorula mucilaginosa is an obligate aerobic yeast which contains a high concentration of carotenoid pigment. To test whether carotenoids are able to protect R. mucilaginosa against oxidative injury, yeast cells in liquid culture were incubated with duroquinone (DQ) (100 microM), a redox-cycling quinone known to generate intracellular O2-. or were grown in a hyperoxic atmosphere (80% O2) under conditions where carotenoid concentrations were altered either intracellularly or extracellularly. Neither of these oxidative challenges affected cell growth unless carotenogenesis was blocked by the addition of diphenylamine (50 microM). In the diphenylamine-treated nonpigmented cells, growth was completely inhibited by DQ and by hyperoxia. In normoxia, however, diphenylamine alone reduced growth by only 30%. The growth inhibition observed in diphenylamine-treated cells exposed to hyperoxia was primarily mycocidal rather than mycostatic since plating of these cells onto solid media revealed that only 25% of the cells were viable after 50 h of incubation when compared to plated control cells. Addition of 10 microM beta-carotene to diphenylamine-treated cells completely prevented the growth inhibition caused by either hyperoxia or DQ. Carotenoids, therefore, are able to prevent oxidant-induced cytotoxicity in R. mucilaginosa. Analysis of the absorption spectra of chloroform extracts of beta-carotene-supplemented cells showed that beta-carotene, not the endogenous carotenoid, torularhodin, was the major carotenoid present in these cells. Superoxide dismutase (SOD) activity in R. mucilaginosa was compared with that of another yeast, Saccharomyces cerevisiae by two methods: (i) activity staining of proteins separated by gel electrophoresis and (ii) measurement of inhibition of ferricytochrome c reduction. By these techniques, the R. mucilaginosa SOD activity had the characteristics of Mn-SOD. No Cu/ZnSOD activity was detected. Thus, the apparent absence of Cu/ZnSOD may make the antioxidant capability of endogenous carotenoids even more critical in preventing oxidative damage in R. mucilaginosa.     

<Emphasis Type="Italic">Rhodotorula mucilaginosa</Emphasis>, a carotenoid producing yeast strain from a patagonian high-altitude lake

The red yeast Rhodotorula mucilaginosa strain CRUB 0138 (previously identified as R. lactosa) was isolated from a high-altitude Patagonian Lake Toncek (1700 m a.s.l.), and assigned with mucilaginosa species. Its biochemical, physiological and molecular features were assessed and compared to R. mucilaginosa PYCC 5166 type strain using a polyphasic approach; in addition, biomass and carotenoid pigment production at different C/N ratios were determined in an incubator shaker. Phenetic characterization by means of 70 current physiological tests including assimilation of aldaric acids and aromatic compounds, and also the ability to grow with amino acids as sole carbon sources, was carried out. According to numerical taxonomy calculations, similarity indexes between R. mucilaginosa CRUB 0138 and PYCC 5166 type strain were 0.86 and 0.77, corresponding to a complete set of physiological tests and MSP-PCR (Mini/Micro Satellite Primed PCR; (GTG)5, M13 and (GAC)5 primers were employed) fingerprinting. Killer activity against 2 native strains, Rhodosporidium kratochvilovae and R. mucilaginosa was detected. Maximum biomass-glucose conversion efficiency (87%) and maximum carotenoid yield (2.32 mg/L) were obtained at C/N = 5 in culture medium containing 10 and 40 g/L glucose, respectively. Different C/N ratios did not influence carotenoid pigment production but low C/N enhanced biomass yield.     

Influence of magnesium ions on heat shock and ethanol stress responses of Saccharomyces cerevisiae

This study has highlighted the role of magnesium ions in the amelioration of the detrimental effects of ethanol toxicity and temperature shock in a winemaking strain of Saccharomyces cerevisiae. Specifically, results based on measurements of cellular viability and heat shock protein synthesis together with scanning electron microscopy have shown that, by increasing the bioavailability of magnesium ions, physiological protection is conferred on yeast cells. Elevating magnesium levels in the growth medium from 2 to 20 mM results in repression of certain heat shock proteins following a typical heat shock regime (30–42°C shift). Seed inocula cultures prepropagated in elevated levels of magnesium (i.e. ‘preconditioned’) also conferred thermotolerance on cells and repressed the biosynthesis of heat shock proteins. Similar results were observed in response to ethanol stress. Extra- and intracellular magnesium may both act in the physiological stress protection of yeast cells and this approach offers potential benefits in alcoholic fermentation processes. The working hypothesis based on our findings is that magnesium protects yeast cells by preventing increases in cell membrane permeability elicited by ethanol and temperature-induced stress.     

Genome-wide screening of Escherichia coli genes involved in execution and promotion of cell-to-cell transfer of non-conjugative plasmids: rodZ (yfgA) is essential for plasmid acceptance in recipient cells

The HSP30 gene of the budding yeast Saccharomyces cerevisiae encodes a seven-transmembrane heat shock protein expressed in response to various types of stress including heat shock. Although Hsp30p contains a potential N-glycosylation consensus sequence (Asn(2)-Asp(3)-Thr(4)), whether it is actually N-glycosylated has not been verified. Here we demonstrate that N-glycosylation is induced at Asn(2) of Hsp30p by severe heat shock, ethanol stress, and acetic acid stress. Mild heat shock and glucose depletion induced the expression but not N-glycosylation of Hsp30p, indicating the N-glycosylation to be dependent on temperature and environmental conditions. N-glycosylation did not affect on the intracellular localization of Hsp30p but its physiological role under severe heat shock conditions. Since limited information is available on stress-responsive or condition-induced N-glycosylation, our findings provide new insight into the regulation of cellular stress response in yeast.     

Proteome analysis of tobacco leaves under salt stress

The mechanisms responsible for the effects of salt stress on tobacco plants were examined by means of proteomic analysis. Tobacco plants were exposed to 0, 150, 250, 300, or 400 mM NaCl. At 150 mM NaCl or above, the plants showed a reduction in fresh weight and an increase in proline levels. Proteins extracted from the leaves of tobacco plants exposed to 150 mM NaCl were separated by 2-DE. Of 205 protein spots that were detected reproducibly in each gel, 18 were differentially expressed under NaCl treatment. Up-regulated proteins belonged to the photosynthesis category, whereas down-regulated proteins correspond to defense-related functions. Dose- and time-dependent studies showed that a stromal 70-kDa heat shock-related protein was markedly down-regulated by NaCl. Thus, down-regulation of the stromal 70-kDa heat shock protein in response to salt stress is likely the cause of failure to protect cells against salt stress of tobacco plants.     

A Role for the DnaJ Homologue Scj1p in Protein Folding in the Yeast Endoplasmic Reticulum

Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants. Here, we show that the Δscj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Δscj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.     

Tolerance of Mesorhizobium type strains to different environmental stresses

The symbiosis between rhizobia and legumes is affected by different environmental conditions. Our aims were to evaluate stress tolerance of Mesorhizobium species and investigate species-specific stress response mechanisms. Tolerance of Mesorhizobium type strains to temperature, salt and pH stress was evaluated. Mesorhizobium thiogangeticum showed highest growth with 1.5% NaCl and Mesorhizobium ciceri at pH 5. Mesorhizobium plurifarium showed higher growth at 37°C. SDS-PAGE analysis revealed changes in the protein profiles, namely the overexpression of a 60 kDa protein, following heat stress. Under salt stress, five overexpressed proteins were identified in M. plurifarium and M. thiogangeticum. Northern analysis revealed an increase in groEL expression in Mesorhizobium huakuii and Mesorhizobium septentrionale after heat shock; by contrast, a decrease was detected in Mesorhizobium albiziae and M. thiogangeticum, upon salt shock. A high diversity in tolerance to temperature, salt and pH stress was detected among Mesorhizobium species. M. thiogangeticum and M. ciceri are moderately halophilic and acidophilic, respectively. Several proteins, overproduced in different strains, may be involved in stress tolerance. groEL expression increased upon heat and decreased upon salt shock. To our knowledge, this is the first study focusing tolerance to temperature, salt and pH stress, as well as groEL expression, in Mesorhizobium type strains.     

Heat Shock Response in the Thermophilic Enteric Yeast Arxiozyma telluris

Heat stress tolerance was examined in the thermophilic enteric yeast Arxiozyma telluris. Heat shock acquisition of thermotolerance and synthesis of heat shock proteins hsp 104, hsp 90, hsp 70, and hsp 60 were induced by a mild heat shock at temperatures from 35 to 40°C for 30 min. The results demonstrate that a yeast which occupies a specialized ecological niche exhibits a typical heat shock response.     

Histamine modulates the cellular stress response in yeast

The cellular stress response is a universal protective reaction to adverse environmental or microenvironmental conditions, such as heat and drugs, associated in part with the highly conserved heat shock proteins (HSPs). Histamine is a key inflammatory mediator derived from l-histidine that governs vital cellular processes beyond inflammation, while recent evidence implies additional actions in both prokaryotes and eukaryotes. This study explored the possible role of histamine in the heat shock response in yeast, an established experimental model for the pharmacological investigation of the cellular stress response. The response was evaluated by determining growth and viability of post-logarithmic phase grown yeast cultures after heat shock at 53°C for 30 min. Thermal preconditioning at 37°C for 2 h served as a positive control. The effect of histamine was investigated following long-term administration through the post-logarithmic phase of growth or short-term administration for 2 h prior to heat shock. Short-term treatment with 1 mM histamine resulted in de novo protein synthesis-dependent acquisition of thermotolerance, while lower doses or long-term administration of histamine failed to induce the heat-resistant phenotype. Preliminary investigation of HSP104, HSP70 and HSP60 expression by western blotting showed an increase of these proteins after thermal preconditioning. However, a differential HSP and tubulin expression appeared to underlie the response of yeast cells to histamine. In conclusion, histamine was capable of inducing the adaptive phenotype, while the contribution of HSPs and tubulin and the potential implications remain largely elusive.     

Initiation of Encystment in the Mycetozoan Protostelium mycophaga

SYNOPSIS. Protostelium was cultured monoxenically with the yeast Rhodotorula mucilaginosa on either corn meal agar or in liquid corn meal medium. Nearly synchronous encystment was induced in amoebae by washing them free of yeast and incubating them in an encystment medium devised by Neff et al. (1964) for Acanthamoeba.
The extrinsic requirements for encystment were investigated by replacing various components of the encystment medium with other substances. The results indicate that encystment depends on a high concentration of inorganic monovalent cations and that it occurs best at a basic pH. The effect of the ions does not appear to be strictly osmotic.