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In situ bioremediation processes are important for control of pollution and clean-up of contaminated sites. The study and implementation of such processes can be designed through investigations on natural mechanisms of absorption, biotransformation, bioaccumulation and toxicity of pollutants in plants and microorganisms. Here, the phytotoxic effects of Cr(VI) and Cd(II) on seed germination and plant growth of Lepidium sativum have been examined at various concentrations (30-300 mg/L) in single ion solutions. The studies also addressed the ecotoxicity of metal ions on Azotobacter chroococcum and Pichia sp. isolated from soil. Microbial growth was estimated by weighing the dry biomass and determining the enzymatic activities of dehydrogenase and catalase. The results showed that Cr(VI) and Cd(II) can inhibit L. sativum seed germination and root development, depending on the metal ion and its concentration. The phytotoxic effect of heavy metals was also confirmed by the reduced amounts of dried biomass. Toxicity assays demonstrated the adverse effect of Cr(VI) and Cd(II) on growth of Azotobacter sp. and Pichia sp., manifested by a biomass decrease of more than 50 % at heavy metal concentrations of 150-300 mg/L. The results confirmed close links between phytotoxicity of metals and their bioavailability for phytoextraction. Studies on the bioremediation potential of soils contaminated with Cr(VI) and Cd(II) using microbial strains focusing on Azotobacter sp. and Pichia sp. showed that the microbes can only tolerate heavy metal stress at low concentrations. These investigations on plants and microorganisms revealed their ability to withstand metal toxicity and develop tolerance to heavy metals., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study evaluated the efficacy of the essential oil from Mentha piperita L. (MPEO) to inactivate cells of the potentially spoilage yeasts Candida albicans, Candida tropicalis, Pichia anomala and Saccharomyces cerevisiae in cashew, guava, mango and pineapple juices during 72 h of refrigerated storage. Damage in different physiological functions caused by MPEO in S. cerevisiae in cashew and guava juices were investigated using flow cytometry (FC). The effects of the incorporation of an effective anti-yeast MPEO dose on sensory characteristics of juices were also evaluated. MPEO displayed minimum inhibitory concentration of 1.875 μL/mL against all tested yeasts. A >5 log reduction in counts of C. albicans, P. anomala and S. cerevisiae was observed in cashew and guava juices with 7.5 and 3.75 μL/mL MPEO. Tested MPEO concentrations (1.875, 3.75 and 7.5 μL/mL) were not effective to cause >5 log reduction in counts of target yeasts in mango and pineapple juices during 72 h of exposure. Incorporation of 1.875 μL/mL MPEO in cashew and guava juices strongly compromised membrane permeability, membrane potential, enzymatic activity and efflux pump activity in S. cerevisiae cells. This same MPEO concentration did not affect appearance, odor and viscosity in fruit juices, but negatively affected their taste and aftertaste. These results show the efficacy of MPEO to inactivate potentially spoilage yeasts in fruit juices through disturbance of different physiological functions in yeast cells. However, the combined use of MPEO with other technologies should be necessary to decrease its effective anti-yeast dose in fruit juices and, consequently, the possible negative impacts on specific sensory properties of these products., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The toxicity of cadmium (Cd) is the major limitation to its removal using microorganisms. The Cd tolerance of Pichia kudriavzevii was obviously enhanced by acid stress based on multi-stress cross-protection. RNA-Seq showed that most differentially expressed genes (DEGs) in the Pentose phosphate pathway, Citrate cycle (TCA cycle), Glycolysis/Gluconeogenesis, Peroxisome and Glutathione metabolism were up-regulated by acid stress. The up-regulated expression of genes related to ATP synthesis (GOR1, ALD5, ADH4, ADH6, MDH2, IDH1, IDH2, and ATP19) and Cd transport (GSTY2, GTO2, GLO2, and YOR1), and the improvement of intracellular GSH level and GST activity, reduced the Cd toxicity towards P. kudriavzevii. Cd efflux by YOR1 played a key role in the decline of intracellular Cd level. Acid stress obviously improved the gene expression levels and activities of antioxidant enzymes (SOD, POD, and CAT), which inhibited the Cd-induced ROS outburst and oxidative damage of proteins and membrane lipids. In addition, the enhanced expression of HSP12 protected P. kudriavzevii from the damage of Cd stress. These results provide some important clues to reconstruct robust strains using for Cd removal in aquatic environments., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Carbon dots (CDs) are promising nanomaterials for biosensing, bioimaging, and drug delivery due to their large surface area, excellent optical properties, and thermal and chemical stability. However, biosafety of CDs is still understudied, and there is not a generally accepted standard to evaluate the toxicity of CDs. We present a gradient network generator microfluidic device for dose-dependent testing of toxicity of CDs to a unicellular eukaryotic model organism, yeast Pichia pastoris. We fully characterized the microfluidic model and compare its performance with a conventional method. The gradient generator increased the contact area between the mixing species and enabled a high-throughput testing of CDs in a wide range of concentrations in cell chambers. Real time monitoring of yeast cell proliferation in the presence of CDs showed dose-dependent growth inhibition and various budding cell shape profiles. Comparing the result of microfluidic platform and conventional method revealed statistically significant differences in the proliferation rate of the cells between the two platforms. To understand the toxicity mechanism, we studied binding of CDs to P. pastoris and found increasing interactions of CDs with the cell surface at CDs larger concentrations. This study demonstrated the utility of the gradient generator microfluidic device as a convenient tool for toxicity assessment of nanomaterials at a cellular level., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Carbon dots (CDs) as a new fluorescent material with excellent water solubility, chemical inertness, and easy surface modification are a good candidate for bioimaging and biosensing due to their low toxicity and good biocompatibility. Although carbon is not an intrinsically toxic substance, carbon nanomaterials such as CDs may cause risks to human health and the potentially hazardous effects of CDs on various living systems must be completely determined. So far, cytotoxicity studies of CDs have focused on human cells and are mainly conducted on limited cell lines. In the present study, toxicity assessment of CDs was evaluated on yeast cells Pichia pastoris as a unicellular eukaryotic model. Results revealed dose-dependent toxicity of CDs on yeast cells and less relative cell growth in 25 mg/ml of CDs as compared to the control group. CDs binding curve confirmed the interaction between CDs and surface of yeast cells. SEM images showed that the CDs caused cell shrinkage and hole formation on the surface of yeast cells and also induced slightly cell deformation. It was demonstrated that CDs could generate the ROS dose-dependently. Finally, results showed the growth inhibition and ROS generation effects of CDs were enhanced at light exposure, as an important environmental factor. These findings could have important implications for applications of CDs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

To obtain a global view of the dynamic phospholipids in Pichia stipitis during the ethanol fermentation in the presence of three representative inhibitors (vanillin, 5-hydroxymethylfurfural, and acetic acid). Considerable efforts have been expended to elucidate the biochemical mechanisms of inhibitors interaction with phospholipids. In this study, a comparative lipidomic analysis was performed using liquid chromatography-mass/mass spectrometry (LC-MS/MS) on P. stipitis. Partial least squares-discriminate analysis (PLS-DA) was used to deal with the large quantity of data generated using the systematic methods. PLS-DA revealed that phosphatidylinositol (PI) (PI34:1, PI34:2 and PI34:6), phosphatidylserine (PS) (PS34:1 and PS34:2), phosphatidylethanolamine (PE) (PE34:1 and PE34:2), and phosphatidylcholine (PC) (PC34:1, PC34:2, and PC34:3) were the predominant biomarkers. Further analysis of different classes of phospholipids indicated that: (a) the samples from three combined inhibitors condition during the lag phase possessed the lowest PI/PS value 1.4%, (b) alterations in PC/PE ratios with changes in inhibitors were coincident with the changes in xylose utilization rates, and (c) the levels of unsaturated and the relatively long chain phospholipids increased in the inhibitor-plus condition. These findings suggest that regulation of membrane properties with inhibitors might offer a way of self-protection of yeast to inhibitors stress., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We investigated cell growth and activity of intra- and extracellular chitinase, β-1,3-glucanase, and chitin deacetylase with SDS-PAGE by incubating W. anomalus EG2 in PDB and YPD media for 24h in presence of different concentrations (0%, 0.1%, 0.3%, and 0.5%) of colloidal chitin. Maximum cell growth was observed in both PDB and YPD media without colloidal chitin. In the absence of colloidal chitin, maximum extracellular β-1,3-glucanase activity of 32.96 and 47.28 units/mL was reported at 18h in PDB medium and 6h in YPD medium, respectively. In addition, extracellular chitinase was unaffected by various concentrations of carboxymethyl chitin in both PDB and YPD media. In the absence of colloidal chitin, maximum intracellular chitinase activity was indicated to be 9.82 and 9.86 units/mg protein in PDB and YPD media, respectively. Maximum intracellular β-1,3-glucanase activity reported was 17.34 units/mg protein in PDB medium containing 0.5% colloidal chitin and 15.0 units/mg protein in YPD medium containing 0.3% colloidal chitin. Five major isozymes, GN1, GN2, GN3, GN4, and GN5, of intracellular β-1,3-glucanase were detected with glucan-containing high polymer complex as a substrate with or without colloidal chitin., (Copyright © 2017 Elsevier B.V. All rights reserved.)

During the past couple of years, graphene nanomaterials were extremely popular among the scientists due to the promising properties in many aspects. Before the materials being well applied, we should first focus on their biosafety and toxicity. In this study, we investigated the toxicity of synthesized graphene oxide (GO) against the model industrial organism Pichia pastoris. We found that the synthesized GO showed dose-dependent toxicity to P. pastoris, through cell membrane damage and intracellular reactive oxygen species (ROS) accumulation. In response to these cell stresses, cells had normal unsaturated fatty acid (UFA) levels but increased contents of polyunsaturated fatty acid (PUFA) with up-regulation of UFA synthesis-related genes on the transcriptional level, which made it overcome the stress under GO attack. Two UFA defective strains (spt23Δ and fad12Δ) were used to demonstrate the results above. Hence, this study suggested a close connection between PUFAs and cell survival against GO., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Maintaining cellular lipid composition is essential for many cell processes. Our previous study has demonstrated that Spt23 is an important transcription factor within the cell and responsible for the regulation of fatty acid desaturase genes. Disruption of SPT23 results in increased lipid saturation. In the present study, we found that lipid saturation caused by SPT23 deletion exhibited a growth defect under ethanol stress and increased chitin contents. Ergosterol synthesis-related genes were up-regulated to protect cells from plasma membrane damage in the presence of ethanol. The cell wall stress caused by increased chitin contents could not be attenuated by up-regulation of phospholipids synthesis-related genes in spt23Δ. Besides, lipid saturation induced expression of unfolded protein response (UPR) genes and reactive oxygen species (ROS) accumulation followed by activation of the cellular antioxidant system, which is associated with endoplasmic reticulum functions. Taken together, our data suggested that lipid homeostasis has a close connection with cell responses to both plasma membrane stress and endoplasmic reticulum stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study uses a mathematical approach to assessing the inhibitory effect of Zn(2)(+)(0-10 mM, obtained from ZnCl2 and ZnSO4) in presence of NaCl (0-8%) and hydroxytyrosol (0-2588 mg/L), on a yeast cocktail formed by species Pichia galeiformis, Pichia kudriavzevii, Pichia manshurica and Candida thaimueangensis obtained from spoilt green olive packages. The logistic/probabilistic models were built in laboratory medium using a total of 1980 responses (1188 for NaCl and 792 for hydroxytyrosol). ZnCl2 showed significantly higher inhibitory effect than ZnSO4 in the presence of both NaCl (p < 0.033) and hydroxytyrosol (p < 0.009). NaCl did not interfere the effect of Zn(2)(+)while hydroxytyrosol, at high levels, had a slight antagonistic effect. According to models, Zn(2)(+)inhibits (p = 0.01) the yeast cocktail in the range 4.5-5.0 mM for ZnCl2, or 8.5-9.5 mM for ZnSO4. Therefore, this work confirms the fungicidal activity of zinc compounds (mainly ZnCl2) in synthetic medium, and also shows that the loss of zinc effectiveness in real green Spanish-style olive packaging is not due to the presence of NaCl or hydroxytyrosol, two of the most abundant chemical compounds in the product., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The wide applications of titanium dioxide nanoparticles (TiO2 NPs) increase the possibility of their exposure to ecosystems, and therefore an improved understanding of their effects to organisms is required. However, their potential toxicity on eukaryotes, especially fungi, needs further detailed investigation. Here, we investigated the effects of anatase TiO2 NPs on the reactive oxygen species (ROS)-scavenging system in the model fungal organism, Pichia pastoris. Results showed that the NPs entered cells and had toxicity to this fungus, and their toxicity was attributed to cell wall damage, cell membrane damage, and ROS accumulation, but not apoptosis or autophagy. Interestingly, the synthesized TiO2 NPs impaired but not activated the ROS-scavenging system, which contributes to the cytotoxicity. Moreover, this impairment was associated with down-regulation of antioxidant-related genes, especially those genes involved in GSH regulation. Hence, GSH may play a key role in the interaction between TiO2 NPs and yeast cells., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Irisin is a novel hormone which is related to many metabolic diseases. In order to illuminate the function and therapeutic effect of irisin, gaining active irisin is necessary. In this work, a codon-optimized irisin gene was designed according to Pichia pastoris synonymous codon usage bias and cloned into the pPIC9K expression vector. Sequencing result indicating that the sequence of irisin was consistent with the modified irisin and the irisin was in frame with α-factor secretion signal ATG. The plasmid pPIC9K-irisin was transformed into GS115 P. pastoris cells through electroporation. The positive transformants were screened on MD medium and analyzed by PCR. Five recombinant GS115/pPIC9K-irisin strains were obtained, but only one strain expressed irisin successfully. SDS-PAGE and Western blot were used to assess the expression level and purity of irisin. The irisin was also simply purified and the effect of pH value, methanol concentration and induction time on the production of irisin was investigated. The results showed that the best conditions of irisin expression were as follows: pH 6.0, 2.0% methanol and induction for 96 h. This work laid the basis for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Protease inhibitors have been known to confer multiple stress tolerance in transgenic plants. We have assessed growth of yeast (Pichia pastoris GS115) strains expressing inhibitory repeat domains (PpIRD(+)) of previously characterized Capsicum annuum protease inhibitors under high salt, heavy metal and oxidative stress. PpIRD(+) strains exhibited multiple stress tolerance and showed differential molecular responses at transcriptional and translational level on exposure to stress inducing agents like heavy metal, high salt and H2O2. PpIRD(+) strains display significant reduction in metacaspase (Yca1) activity, the key enzyme in apoptosis, indicates the possibility of cross reactivity of IRDs (serine protease inhibitor) with cysteine proteases. PpIRD(+) and Saccharomyces cerevisiae knockout with Yca1 (ΔYca1) strain showed similar growth characteristics under stress, which indicated the delayed senescence due to cellular metacaspase inhibition. Molecular docking study showed a close proximity of IRDs reactive site and the active site of metacaspase in the complex that signified their strong interactions. Maintenance of GAPDH activity, primary target of metacaspase, in PpIRD(+) strain evidenced the inhibition of metacaspase activity and survival of these cells under stress. This report demonstrates a potential molecular mechanism of protease inhibitor-based multiple stress tolerance in yeast strains., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This work was conducted to determine the effect of the partial replacement of NaCl by KCl and CaCl2 (expressed as percentages, wt/vol) on the microbial stability and physicochemical characteristics of seasoned cracked olives using a simplex centroid mixture design. Neither Enterobacteriaceae nor lactic acid bacteria were found during the 50 days that olive packages were monitored. Therefore, microbial instability was considered due to the growth of yeasts, which were the only detected microorganisms; Saccharomyces cerevisiae and Pichia membranifaciens were the most relevant species. Yeasts decreased during the first 21 to 30 days after packing, but their populations rose to 3.5 log CFU/ml by the end of the storage period, clearly causing product deterioration. The partial substitution of NaCl with the other chloride salts slightly altered the phase of microbial inhibition and regrowth. Most of the quality characteristics were not affected by the use of the alternative salt mixtures, but the pH values and Cl(-) concentrations in brine decreased as the CaCl2 concentration increased. Hence, seasoned cracked table olives can be produced using a lower proportion of NaCl without causing significant changes in the shelf life and product quality, although further detailed studies are necessary to guarantee the stability of products packed with specific salt mixtures.

This article reports on the bioactivities of citrus extracts (citrus extract, lemon extract, and neroli) toward Saccharomyces cerevisiae, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Pichia membranifaciens, and Rhodotorula bacarum. The bioactivities of the extracts (from 10 to 100 ppm) were evaluated through a microdilution method; thereafter, citrus extracts (0 to 80 ppm) were tested in combination with either pH (3.0 to 5.0) or temperature (5 to 25°C). Finally, a confirmatory experiment was run in a commercial drink (referred to as red fruit juice) containing citrus extract (40 ppm) that was inoculated with either S. cerevisiae or Z. bailii (5 log CFU/ml) and stored at 4 and 25°C. Yeasts increased to 7 log CFU/ml (Z. bailii) or 8 log CFU/ml (S. cerevisiae) in the control at 25°C, but the citrus extract addition controlled yeast growth for at least 3 days; under refrigeration, the effect was significant for 10 days.

In the yeast Saccharomyces cerevisiae, the one-at-a-time deletions of either the high-affinity glucose sensor gene SNF3 or the low-affinity glucose sensor gene RGT2 only slightly reduced pexophagy; however, deleting both genes greatly reduced pexophagy, evincing interaction beyond the sum of the additive effects, as recently shown. The present study identifies the only ScSNF3/RGT2 ortholog in the methylotrophic yeast Pichia pastoris (designated as PpGSS1, from GlucoSe Sensor) and describes its roles in autophagic pathways (non-selective and selective). GSS1 knock-out strain has been constructed. The experiments support the hypothesis that Gss1 plays an important role in autophagic degradation of peroxisomes and glucose catabolite repression in P. pastoris., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This paper shows the ease of application and usefulness of mid-IR measurements for the investigation of orthogonal cell states on the example of the analysis of Pichia pastoris cells. A rapid method for the discrimination of entire yeast cells grown under carbon and nitrogen-limited conditions based on the direct acquisition of mid-IR spectra and partial least squares discriminant analysis (PLS-DA) is described. The obtained PLS-DA model was extensively validated employing two different validation strategies: (i) statistical validation employing a method based on permutation testing and (ii) external validation splitting the available data into two independent sub-sets. The Variable Importance in Projection scores of the PLS-DA model provided deeper insight into the differences between the two investigated states. Hence, we demonstrate the feasibility of a method which uses IR spectra from intact cells that may be employed in a second step as an in-line tool in process development and process control along Quality by Design principles., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Bioaccumulation of synthetic dyes viz. Acid Blue 93, Direct Red 28 and Basic Violet 3 by growing cells of yeast, Pichia fermentans MTCC 189 was investigated in growth media prepared from sugarcane bagasse extract. The maximum dye bioaccumulation was determined at pH 5.0 for all the dyes tested. Two kinetic models viz. Noncompetitive and Uncompetitive models were tested in order to determine the toxic effects of dyes on the specific growth rate of P. fermentans MTCC 189. Basic Violet 3 was found to be more toxic than the other two dyes. The combined effects of sugarcane bagasse extract and initial Basic Violet 3 dye concentrations on the specific growth rate and dye bioaccumulation efficiency of P. fermentans MTCC 189 was investigated and optimized using Response Surface Methodology (RSM). A 2(2) full factorial central composite design was successfully used for analysis of results. The optimum combination predicted via RSM confirmed that P. fermentans MTCC 189 was capable of bioaccumulating Basic Violet 3 dye upto 69.8% in the medium containing 10 mg/L of dye and 24 g/L sugar extracted from sugarcane bagasse., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO(2), ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO(2) in comparison to the other strains of Dekkera. These strains were inoculated (10(3)-10(4)cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 degrees C and 21 degrees C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10(5)cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 degrees C while slower growth was observed at 15 degrees C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 degrees C compared to 21 degrees C.

Kodamaea (Pichia) ohmeri was formerly considered a contaminant, but is now known to be a significant human pathogen that has been shown to cause fungemia, endocarditis, funguria, and peritonitis in immunocompromised patients. We report a case of fungemia caused by K. ohmeri in a 71-year-old man with cellulitis. The patient was sent to the emergency room due to leg edema, fever, and change of consciousness. During hospitalization, a series of examinations including blood cultures were performed. On hospital day 8, blood culture yielded a yeast colony. Fluconazole was given empirically, but had no effect. The pathogen was identified as K. ohmeri by Vitek YBC card, API 20C, sequencing of the 18S rRNA gene, and the D1/D2 domains of the 26S rRNA gene and the internally transcribed spacer (ITS) regions. Antifungal susceptibility testing was performed with the ATB-Fungus system, and a high minimum inhibitory concentration (level up to 64 mg/l) for fluconazole was found. Fluconazole was replaced with amphotericin B deoxylate, and the fever and cellulitis inflammation gradually subsided. The patient was discharged in a stable condition. This is the first case of K. ohmeri fungemia in Taiwan.

To improve the thermostability of Trichoderma reesei xylanase 2 (Xyn2), the thermostabilizing domain (A2) from Thermotoga maritima XynA were engineered into the N-terminal region of the Xyn2 protein. The xyn2 and hybrid genes were successfully expressed in Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from S. cerevisiae (alpha-factor). The transformants expressed the hybrid gene produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding strains expressed the native Xyn2 gene. The activity of the hybrid enzyme was highest at 65 degrees C that was 10 degrees C higher than the native Xyn2. The hybrid enzyme was stable at 60 degrees C and retained more than 85% of its activity after 30-min incubation at this temperature. The hybrid enzyme was highly specific toward xylan and analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products. These attributes should make it an attractive applicant for various applications. Our results also suggested that the N-terminal domain A2 is responsible for both the thermostability and substrate-binding capacity of T. maritima XynA.

Three benzoic acid derivatives (zinc p-iodobenzoate, zinc p-hydroxybenzoate, and zinc p-aminobenzoate) were synthesized and tested chemically and microbiologically in order to explain their mode of action against the yeast Pichia anomala. The yeast strains were cultivated in batch culture of chemically defined minimal medium (control) and with the addition of the studied compound at concentrations of 0.103 to 0.166% (wt/vol). The growth of microorganisms, H+ concentration, and the concentrations of both dissociated and undissociated forms of the appropriate weak acid in the medium were monitored at 1-h intervals during 24 h of incubation. The inhibitory effect of each compound on the growth of microorganisms was calculated based on measurement of optical density at 600 nm turbidity. The K parameter, defined as the ratio of the concentration of undissociated weak acid to the number of microorganisms in the medium, was determined. The K value is related to the degree of growth inhibition and provides new insight into the mode of action of weak organic acids against the studied yeasts. The buffering capacity of the chemicals studied was also found to be an inhibition parameter associated with microbial growth. Greater buffer capacity of a given compound reduced changes in the pH value of the medium, resulting in changes to antimicrobial effectiveness.

The single and combined effects, in a synthetic medium at selected pH values, of sorbic and benzoic acids on a yeast cocktail (Saccharomyces cerevisiae, Pichia anomala, Issatchenkia occidentalis, and Candida diddensiae, isolated from table olives) have been studied. Applying the checkerboard method the minimum inhibitory concentration (MIC) obtained for the respective individual preservatives (expressed as undissociated acid) were: sorbic acid, 5.94, 3.85 and 3.19 mM at pH of 4.5, 4.0, and 3.5, respectively; and benzoic acid, not detected (at total 20.5 mM), 10.40 and 6.83 mM, respectively, for the same pH levels. The estimated fractional inhibitory concentrations (FIC) indexes showed additive effects between inhibitors. Fractional area (fa), modelled by the (extended) Lambert and Lambert [2003. A model for the efficacy of combined inhibitors. J. Appl. Microbiol. 95, 734-743] equation (ELPM), also showed additives of both preservatives but different shapes in the dose-response curves; the individual MIC (as undissociated acid) deduced from this method were: 5.60, 3.31, and 3.26 mM for sorbic acid at pH of 4.5, 4.0, and 3.5, respectively; and 29.65 (extrapolated), 10.00, and 6.25 mM for benzoic acid at the same pH levels. Mixtures above the curves connecting the limits (MIC) at each pH were also inhibitory. There was agreement between MIC values from FIC and ELPM, although the last one provided further information on the inhibition behaviour. I. occidentalis was the most resistant yeast of the cocktail to sorbic and benzoic acids.

The time to detection (TTD) for Pichia anomala IG02 was defined, for inoculum sizes lower than 6 log(10)cfu/ml, as the time elapsed from inoculation to the moment at which an OD of 0.12 was reached. In other cases, TTD can be estimated by interpolation within the time elapsed from the previous readings below OD=0.12 and the next above it. A linear relationship, which depended on the inoculum size, between lnTTD with ln lambda and ln mu(m) was found. These relationships can be used to estimate the biological parameters of cultures with low inoculum levels. In addition, TTD for P. anomala IG02 could be modelled as a function of environmental conditions. The model can also be applied to lambda and mu(m) through their relationships with TTD. The effects of temperature, NaCl content and pH were quantified by the generalized z-values. An increase of 5.97 in NaCl concentration, a decrease of 1.97 units of pH, or a decrease of 6.08 degrees C doubled the TTD or caused a 2.53-fold increase in lambda and a 2.56-fold decrease in the mu(m).

Carnitine palmitoyltransferase I (CPT-I) and II (CPT-II) enzymes are components of the carnitine palmitoyltransferase shuttle system which allows entry of long-chain fatty acids into the mitochondrial matrix for subsequent oxidation. This system is tightly regulated by malonyl-CoA levels since this metabolite is a strong reversible inhibitor of the CPT-I enzyme. There are two distinct CPT-I isotypes (CPT-Ialpha and CPT-Ibeta), that exhibit different sensitivity to malonyl-CoA inhibition. Because of its ability to inhibit fatty acid synthase, C75 is able to increase malonyl-CoA intracellular levels. Paradoxically it also activates long-chain fatty acid oxidation. To identify the exact target of C75 within the CPT system, we expressed individually the different components of the system in the yeast Pichia pastoris. We show here that C75 acts on recombinant CPT-Ialpha, but also on the other CPT-I isotype (CPT-Ibeta) and the malonyl-CoA insensitive component of the CPT system, CPT-II.

A soluble derivative of Fet3 has been obtained from the methylotrophic yeast Pichia pastoris by limited proteolysis of membrane suspensions with trypsin. The soluble protein and the membrane-bound parent Fet3 have been purified to apparent homogeneity. Soluble Fet3 had molecular mass 100 kDa, while the full-length protein had molecular mass 110 kDa, in line with the expected decrease for cleavage and loss of a single transmembrane helix and a small cytoplasmic domain. The optical and EPR spectra of Fet3 were typical of the multicopper oxidases, indicating the presence of one type 1 blue copper site and a type 2/type 3 copper trinuclear cluster. V(max) values for iron oxidation by P. pastoris Fet3 were obtained similar to human ceruloplasmin and much higher than those reported for Saccharomyces cerevisiae Fet3., (Copyright 1999 Academic Press.)

Economical and thermostable phytase enzymes are needed to release phytate-phosphorus in plant foods for human and animal nutrition and to reduce phosphorus pollution of animal waste. Our objectives were to determine if a methylotrophic yeast, Pichia pastoris, was able to express a phytase gene (phyA) from Aspergillus niger efficiently and if suppression of glycosylation by tunicamycin affected its functional expression. The gene (1.4 kb) was inserted into an expression vector pPICZalphaA with a signal peptide alpha-factor, under the control of AOX1 promoter. The resulting plasmid was transformed into two P. pastoris strains: KM71 (methanol utilization slow) and X33 (wild-type). Both host strains produced high levels of active phytase (25-65 units/ml of medium) that were largely secreted into the medium. The expressed enzyme was cross-reacted with the polyclonal antibody raised against the wild-type enzyme and showed two pH optima, 2.5 and 5.5, and an optimal temperature at 60 degrees C. Compared with the phyA phytase overexpressed by A. niger, this phytase had identical capacity in hydrolyzing phytate-phosphorus from soybean meal and slightly better thermostability. Deglycosylation of the secreted phytase resulted in reduction in the size from 95 to 55 kDa and in thermostability by 34%. Tunicamycin (20 microg/ml of medium) resulted in significant reductions of both intracellular and extracellular phytase activity expression. Because there was no accumulation of intracellular phytase protein, the impairment did not seem to occur at the level of translocation of phytase. In conclusion, glycosylation was vital to the biosynthesis of the phyA phytase in P. pastoris and the thermostability of the expressed enzyme., (Copyright 1999 Academic Press.)

The purpose of this study was to examine the effect of preceding fluconazole treatment on the oral mycologic flora and on the sensitivity of oral Candida albicans isolates to fluconazole. Saline oral rinses were collected from 89 HIV-positive patients, of whom 48 had been exposed to fluconazole and 41 were fluconazole-naive. The rinses were cultured on Sabouraud's and Pagano Levin agars, and yeasts were identified by standard methods. Fluconazole sensitivity of C. albicans isolates was measured by disk diffusion assay. C. albicans was isolated from 69% of patients who had received fluconazole and from 93% of the patients who were fluconazole-naive (p < 0.05). Nine other species of yeasts were also isolated, most commonly C. glabrata. Five patients previously exposed to fluconazole harbored fluconazole-resistant C. albicans, whereas no resistance was detected among the patients who were fluconazole-naive (p < 0.01). Sixteen of the patients who were fluconazole-exposed carried yeasts other than C. albicans, compared with only five patients in the fluconazole-naive group (p < 0.01). All of the fluconazole-resistant strains were isolated from patients with low CD4 counts (less than 100 cells/ml) and after lengthy fluconazole exposures. Nevertheless, patients in Charlotte, N.C., who had a greater mean fluconazole exposure time (10.25 +/- 1.41 months) than patients in Glasgow, UK, (0.65 +/- 0.18 months; p < 0.005), did not develop significantly more in vitro resistance or species diversity. This study indicates that long-term fluconazole treatment can have significant effects on the yeast flora of the mouth, particularly in a patient with a CD4 count of less than 100 cells/ml.