Dimorphic Yeast Research Articles

Stable synthetic cationic bacteriochlorins as selective antimicrobial photosensitizers.

Photodynamic inactivation is a rapidly developing antimicrobial treatment that employs a nontoxic photoactivatable dye or photosensitizer in combination with harmless visible light to generate reactive oxygen species that are toxic to cells. Tetrapyrroles (e.g., porphyrins, chlorins, bacteriochlorins) are a class of photosensitizers that exhibit promising characteristics to serve as broad-spectrum antimicrobials. In order to bind to and efficiently penetrate into all classes of microbial cells, tetrapyrroles should have structures that contain (i) one or more cationic charge(s) or (ii) a basic group. In this report, we investigate the use of new stable synthetic bacteriochlorins that have a strong absorption band in the range 720 to 740 nm, which is in the near-infrared spectral region. Four bacteriochlorins with 2, 4, or 6 quaternized ammonium groups or 2 basic amine groups were compared for light-mediated killing against a gram-positive bacterium (Staphylococcus aureus), a gram-negative bacterium (Escherichia coli), and a dimorphic fungal yeast (Candida albicans). Selectivity was assessed by determining phototoxicity against human HeLa cancer cells under the same conditions. All four compounds were highly active (6 logs of killing at 1 microM or less) against S. aureus and showed selectivity for bacteria over human cells. Increasing the cationic charge increased activity against E. coli. Only the compound with basic groups was highly active against C. albicans. Supporting photochemical and theoretical characterization studies indicate that (i) the four bacteriochlorins have comparable photophysical features in homogeneous solution and (ii) the anticipated redox characteristics do not correlate with cell-killing ability. These results support the interpretation that the disparate biological activities observed stem from cellular binding and localization effects rather than intrinsic electronic properties. These findings further establish cationic bacteriochlorins as extremely active and selective near-infrared activated antimicrobial photosensitizers, and the results provide fundamental information on structure-activity relationships for antimicrobial photosensitizers.

Applying dimorphic yeasts as model organisms to study mycelial growth: part 2. Use of mathematical simulations to identify different construction principles in yeast colonies

The dimorphic yeasts Candida boidinii and Yarrowia lipolytica were applied as model organisms to study mycelial growth. A mathematical model of hybrid cellular automaton type was developed to analyze the impact of different biological assumptions on the predicted development of filamentous yeast colonies. The one-dimensional model described discrete cells and continuous distribution of nutrients. The simulation algorithm accounted for proliferation of cells, diffusion of nutrient, as well as biomass decay and recycling inside the mycelium. Simulations reproduced the spatio-temporal development of C. boidinii colonies when a diffusion-limited growth algorithm based on the growth of pseudohyphal cells was applied. Development of Y. lipolytica colonies could only be reproduced when proliferation was restricted to the colony boundary, and cell decay and biomass recycling were incorporated into the model. The results suggested that cytoplasm, which served as the secondary nutrient resource, had to be translocated inside the hyphal network.

Applying dimorphic yeasts as model organisms to study mycelial growth: Part 1. Experimental investigation of the spatio-temporal development of filamentous yeast colonies

Colony development of the dimorphic yeasts Yarrowia lipolytica and Candida boidinii on solid agar substrates under glucose limitation served as a model system for mycelial development of higher filamentous fungi. Strong differences were observed in the behaviour of both yeasts: C. boidinii colonies reached a final colony extension which was small compared to the size of the growth field. They formed cell-density profiles which steeply declined along the colony radius and no biomass decay processes could be detected. The stop of colony extension coincided with the depletion of glucose from the growth substrate. These findings supported the hypothesis that glucose-limited C. boidinii colonies can be regarded as populations of single cells which grow according to a diffusion-limited growth mechanism. Y. lipolytica colonies continued to extend after the depletion of the primary nutrient resource, glucose, until the populations covered the entire growth field which was accomplished by utilization of mycelial biomass.

The DNA Damage Checkpoint Regulates a Transition between Yeast and Hyphal Growth in Schizosaccharomyces japonicus

Dimorphic yeasts change between unicellular growth and filamentous growth. Many dimorphic yeasts species are pathogenic for humans and plants, being infectious as invasive hypha. We have studied the determinants of the dimorphic switch in the nonpathogenic fission yeast Schizosaccharomyces japonicus, which is evolutionarily close to the well-characterized fission yeast S. pombe. We report that camptothecin, an inhibitor of topoisomerase I, reversibly induced the unicellular to hyphal transition in S. japonicus at low concentrations of camptothecin that did not induce checkpoint arrest and the transition required the DNA checkpoint kinase Chk1. Furthermore, a mutation of chk1 induced hyphal transition without camptothecin. Thus, we identify a second function for Chk1 distinct from its role in checkpoint arrest. Activation of the switch from single cell bipolar growth to monopolar filamentous growth may assist cells to evade the source of DNA damage.

Association of Microascus cirrosus (Microascaceae, Ascomycetes) with Brown Leaf Spot of Pistachio in Iran.

Iran is the largest pistachio-producing and exporting country in the world with an annual production of approximately 200,000 t. In July 2009, brown spots were noticed on the leaves of an unknown cultivar of pistachio (Pistacia vera L.) in the Mazhan Region of Birjand, South Khorasan, Iran. Nearly 20% of the trees in the surveyed area (1 ha) showed irregularly shaped leaf spots that were mainly on the abaxial surface, measured 4 to 6 mm in diameter, and were initially pale brown but progressively became dark brown. Lesions were cut from diseased tissues, surface sterilized, and placed on acidified potato dextrose agar (APDA). An anamorphic fungus was consistently isolated from the tissues. The fungus was identified as Microascus cirrosus Curzi (1,4). Colonies on PDA at 25°C reached 9 mm in diameter in 7 days and appeared olivaceous brown and zonate, and perithecia developed in 14 days. Perithecia were black, superficial or partially immersed, spherical, 110 to 200 μm in diameter, with cylindrical necks measuring up to 60 μm long by 25 to 35 μm broad; asci were globose to ovate, eight spored, and 9 to 11 × 8 to 10.5 μm; ascospores were pale reddish brown, variable in shape, ranging from concavo-convex, sometimes plano-convex to oval, and 4.5 to 6 × 3 to 4 μm. The associated anamorph on PDA was a Scopulariopsis sp. with annellophores borne singly or in groups of two or three, 10 to 16 × 2.5 to 3.5 μm, with abruptly narrowed apices; conidia were produced in chains, globose to subglobose, basally truncate, pale olive to fuscous, and 4 to 5 × 3 to 4 μm; chlamydospores were globose to pyriform, terminal or intercalary, and 8.5 to 17.5 μm in long axis. Pathogenicity of two isolates was tested using the filter-paper method of Takahashi et al. (3). Mycelial plugs (5 mm in diameter) as well as filter papers containing conidia were placed on five needle-wounded or nonwounded attached leaves of pistachio cv. Kale-Ghuchi in triplicate. The leaves were covered with a translucent plastic bag for 4 days. Inoculated detached and inoculated wounded attached leaves showed symptoms after 6 and 9 days on 5-month-old and 2-month-old leaves, respectively. Control leaves (five in triplicate) remained symptomless. The pathogen was frequently reisolated from the margins of the lesions. To our knowledge, this is the first record of M. cirrosus occurring on pistachio as well as the first report of a member of this genus from Iran. The same fungus has been reported as the causal agent of stored corn deterioration in the United States (1). M. cirrosus also produced a dimorphic yeast phase on PDA but not on malt extract agar or water agar. Cultures with partial reversion developed chlamydospore-like cells. Higher temperatures (25 to 30°C) and surface-sterilized pistachio leaves placed on PDA accelerated the reversion of the yeast phase to hyphal phase. Although dimorphism has been reported in other plant pathogenic fungi (2), this is the first report of dimorphism in this fungus and of a dimorphic fungus attacking pistachio. Further investigations are needed to determine the relation between dimorphism and pathogenicity. A culture (IRAN 1497 C) of the fungus is preserved by R. Zare.

Oil phytoremediation potential of hypersaline coasts of the Arabian Gulf using rhizosphere technology

The rhizosphere and phyllosphere of the halophyte Halonemum strobilaceum naturally inhabiting hypersaline coastal areas of the Arabian Gulf harbor up to 8.1×104g−1 and 3×102g−1, respectively, of extremely halophilic oil-utilizing microorganisms. Such organisms were 14- to 38-fold more frequent in the rhizosphere than in the plant-free soil. Frequent genera in the rhizosphere were affiliated to the archaea Halobacterium sp. and Halococcus sp., the firmicute Brevibacillus borstenlensis, and the proteobacteria Pseudoalteromonas ruthenica and Halomonas sinaensis. The phyllospheric microflora consisted of the dimorphic yeast Candida utilis and the two proteobacteria Ochrobactrum sp. and Desulfovibrio sp. Individual strains grew on a range of pure aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy. All the strains, except C. utilis which could not tolerate salinities >2M NaCl, grew also in media with salinities ranging between 1 and 4M NaCl, with optimum growth between 1 and 2M NaCl. With the exception of the two archaeal genera, all isolates could grow in a nitrogen-free medium. The total rhizospheric and phyllospheric microbial consortia could attenuate crude oil in complete (nitrogen-containing) medium, but also equally well in a nitrogen-free medium. It was concluded that H. strobilaceum could be a valuable halophyte for phytoremediation of oil-polluted hypersaline environments via rhizosphere technology.

Flocculation of dimorphic yeast Benjaminiella poitrasii is altered by modulation of NAD-glutamate dehydrogenase

A strategy to control flocculation is investigated using dimorphic yeast, Benjaminiella poitrasii as a model. Parent form of this yeast (Y) exhibited faster flocculation (11.1 min) than the monomorphic yeast form mutant Y-5 (12.6 min). Atomic force microscopy revealed higher surface roughness of Y (439.34 rms) than Y-5 (52 rms). Also, the former had a zeta potential of −65.97 ± 3.45 as against −50.21 ± 2.49 for the latter. Flocculation of both Y and Y-5 could be altered by supplementing either substrates or inhibitor of NAD-glutamate dehydrogenase (NAD-GDH) in the growth media. The rate of flocculation was promoted by α-ketoglutarate or isophthalic acid and decelerated by glutamate with a statistically significant inverse correlation to corresponding NAD-GDH levels. These interesting findings open up new possibilities of using NAD-GDH modulating agents to control flocculation in fermentations for easier downstream processing.

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

The tannase-encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA by PCR, using as primers oligonucleotide sequences derived from peptides obtained after tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp, encoding a 587-amino acid protein, preceded by an N-terminal secretion sequence comprising 28 residues. The deduced amino acid sequence was similar to those of tannases from Aspergillus oryzae (50% identity), A. niger (48%) and putative tannases from A. fumigatus (52%) and A. nidulans (50%). The sequence contains the consensus pentapeptide motif (-Gly-X-Ser-X-Gly-) which forms part of the catalytic centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon source. Supplementation with tannic acid or gallic acid leads to induction of ATAN1, and accumulation of the native tannase enzyme in the medium. The enzymes recovered from both wild-type and recombinant strains were essentially indistinguishable. A molecular mass of approximately 320 kDa was determined, indicating that the native, glycosylated tannase consists of four identical subunits. The enzyme has a temperature optimum at 35-40 degrees C and a pH optimum at approximately 6.0. The enzyme is able to remove gallic acid from both condensed and hydrolysable tannins. The wild-type strain LS3 secreted amounts of tannase equivalent to 100 U/l under inducing conditions, while the transformant strain, which overexpresses the ATAN1 gene from the strong, constitutively active A. adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in glucose medium in shake flasks.

Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo- and thermotolerance can be addressed for the definition of culture conditions.

Actin and Phosphoinositide Recruitment to Fully Formed Candida albicans Phagosomes in Mouse Macrophages

Candida albicans is a dimorphic yeast that enters macrophages (Mφ) via the β-glucan receptor dectin-1. Phagocytosis of C. albicans is characterized by actin polymerization, Syk kinase activation and rapid acquisition of phagolysosomal markers. In mice, C. albicans are able to resist the harsh environment of the phagosome and form pseudohyphae inside the phagolysosomal compartment, eventually extending from the Mφ. In this study, we investigated these unique C. albicans phagosomes and found that actin localized dynamically around the phagosomes, before disintegrating. Membrane phosphoinositides, PI(4,5)P₂, PI(3,4,5)P₃, PI(3,4)P₂, and PI(3)P also localized to the phagosomes. Localization was not related to actin polymerization, and inhibitor studies showed that polymerization of actin on the C. albicans phagosome was independent of PI3K. The ability of mature C. albicans phagosomes to stimulate actin polymerization could facilitate the escape of the growing yeast from the Mφ.

Glycerol accumulation in the dimorphic yeast Saccharomycopsis fibuligera: cloning of two glycerol 3‐phosphate dehydrogenase genes, one of which is markedly induced by osmotic stress

Glycerol plays an important role in the osmoadaptation responses of Saccharomyces cerevisiae. However, there is no detailed investigation about the role of glycerol in the osmoadaptation responses of Saccharomycopsis fibuligera. Here we show that both intra- and extracellular glycerol concentrations in Sm. fibuligera cells responded very quickly when they were subjected to osmotic stress. We then cloned two isogenes encoding putative NAD(+)-dependent glycerol 3-phosphate dehydrogenase (GPD) from Sm. fibuligera PD70 by degenerate PCR and subsequent chromosome walking methods. Those two genes, designated SfGPD1 and SfGPD2, respectively, exhibited 86.6% pairwise identity in their encoding regions, while there was no obvious homology in their non-coding regions. Either SfGPD1 or SfGPD2 could complement the salt tolerance characteristics of the gpd1gpd2 double mutant strain of S. cerevisiae, further demonstrating that both of those genes are functional homologues of S. cerevisiae GPD1 and GPD2. Northern blot analysis revealed that SfGPD1 was induced markedly by osmotic stress, while SfGPD2 was not. In consistency with the observation that there was no obvious glycerol content change when the cells were transferred to anoxic conditions, neither SfGPD1 nor SfGPD2 was induced when the cells were transferred to anoxic conditions, thus suggesting a functional splitting of glycerol 3-phosphate dehydrogenase between S. cerevisiae and Sm. fibuligera.

Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission

In a dimorphic yeast, Yarrowia lipolytica, replicative plasmids can be established only in the coexistence of the replication origin (ORI) and centromere (CEN) from its own chromosomal DNA. Although six CEN sequences so far isolated from this yeast exhibit no similarity with conventional CEN DNA elements from other budding yeasts, they are confined within short regions (approximately 0.2 kb) and contain various conserved sequence blocks. We surveyed here a CEN1-1 sequence on an ORI-containing plasmid by deletion and site-directed mutagenesis, and found a partial palindrome, CCTAATTTGG designated DS9, to be an essential element for high-efficiency transformation. In particular, point mutations that alter symmetry and/or length of the palindrome abrogated the activity of CEN1-1. Gel mobility shift assay of CEN1-1 DNA fragments incubated with Y. lipolytica nuclear proteins revealed four bands corresponding to protein-DNA complexes, whereas the mutations within DS9 that disabled transformation also abolished the formation of part of these complexes, depending on particular mutations. These results demonstrate that the palindrome is a binding site for specific protein(s) necessary for plasmid transmission in Y. lipolytica.

Fungal Thigmotropism in Onychomycosis and in a Clear Hydrogel Pad Model

Background: Thigmotropism is a biological characteristic corresponding to the directional growth of cells following topographical guidance cues. This behavior has been shown experimentally with fungal hyphae of both dermatophytes and nondermatophyte molds, as well as with the mycelial phase of the dimorphic yeast Candida albicans. Objective and Methods: We presently document this phenomenon using histomycology in onychomycoses of various fungal origins. Results: This mechanism is involved in the invasive phase of the pathogen or opportunistic fungi, and it probably governs various clinical aspects of onychomycoses. We incidentally disclosed fungal invasions of hydrogel pads. Conclusion: Thigmotropism can in part explain the diversity of orientations and shapes of fungi invading nail plates. The same phenomenon was disclosed inside hydrogel pads. As this material is transparent and easy to cut for microscopic examination, fungal thigmotropism is conveniently explored by this way.

Peroxisomal Peripheral Membrane Protein YlInp1p Is Required for Peroxisome Inheritance and Influences the Dimorphic Transition in the Yeast Yarrowia lipolytica

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful inheritance of organelles by daughter cells in order to maintain the benefits afforded by the compartmentalization of biochemical functions. Little is known about the inheritance of peroxisomes, organelles of lipid metabolism. We have analyzed peroxisome dynamics and inheritance in the dimorphic yeast Yarrowia lipolytica. Most peroxisomes are anchored at the periphery of cells of Y. lipolytica. In vivo video microscopy showed that at cell division, approximately half of the anchored peroxisomes in the mother cell are dislodged individually from their static positions and transported to the bud. Peroxisome motility is dependent on the actin cytoskeleton. YlInp1p is a peripheral peroxisomal membrane protein that affects the partitioning of peroxisomes between mother cell and bud in Y. lipolytica. In cells lacking YlInp1p, most peroxisomes were transferred to the bud, with only a few remaining in the mother cell, while in cells overexpressing YlInp1p, peroxisomes were preferentially retained in the mother cell, resulting in buds nearly devoid of peroxisomes. Our results are consistent with a role for YlInp1p in anchoring peroxisomes in cells. YlInp1p has a role in the dimorphic transition in Y. lipolytica, as cells lacking the YlINP1 gene more readily convert from the yeast to the mycelial form in oleic acid-containing medium, the metabolism of which requires peroxisomal activity, than does the wild-type strain. This study reports the first analysis of organelle inheritance in a true dimorphic yeast and identifies the first protein required for peroxisome inheritance in Y. lipolytica.

Cloning and Expression of the XPR2 Gene from Yarrowia lipolytica in Pichia pastoris

Yarrowia lipolytica is a dimorphic yeast able to secrete different types of proteases depending on the pH of the environment. At neutral pH, the production of an extracellular alkaline protease (AEP) is induced. This protease could be useful in the leather, detergent, or food industries. The XPR2 gene, coding for AEP, was extracted from the pINA154 vector and cloned into the pHIL-D2 vector to obtain a new protease-producing recombinant Pichia pastoris strain. The gene was efficiently integrated in the P. pastoris genome and expressed from the AOX1 promoter actively induced by methanol. Finally, the protease was successfully secreted by P. pastoris GS115.

Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance

There is cumulative resistance against antibiotics of many bacteria. Therefore, the development of new antiseptics and antimicrobial agents for the treatment of skin infections is of increasing interest. We have screened six plant extracts and isolated compounds for antimicrobial effects on bacteria and yeasts with dermatological relevance. The following plant extracts have been tested: Gentiana lutea, Harpagophytum procumbens, Boswellia serrata (dry extracts), Usnea barbata, Rosmarinus officinalis and Salvia officinalis (supercritical carbon dioxide [CO2] extracts). Additionally, the following characteristic plant substances were tested: usnic acid, carnosol, carnosic acid, ursolic acid, oleanolic acid, harpagoside, boswellic acid and gentiopicroside. The extracts and compounds were tested against 29 aerobic and anaerobic bacteria and yeasts in the agar dilution test. U. barbata-extract and usnic acid were the most active compounds, especially in anaerobic bacteria. Usnea CO2-extract effectively inhibited the growth of several Gram-positive bacteria like Staphylococcus aureus (including methicillin-resistant strains - MRSA), Propionibacterium acnes and Corynebacterium species. Growth of the dimorphic yeast Malassezia furfur was also inhibited by Usnea-extract. Besides the Usnea-extract, Rosmarinus-, Salvia-, Boswellia- and Harpagophytum-extracts proved to be effective against a panel of bacteria. It is concluded that due to their antimicrobial effects some of the plant extracts may be used for the topical treatment of skin disorders like acne vulgaris and seborrhoic eczema.

Yarrowia lipolytica vesicle-mediated protein transport pathways

BackgroundProtein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway.ResultsWe identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y. lipolytica proteins are closer to animal ones, whereas they are only 13% in the case of S. cerevisiae.ConclusionThese results provide further support for the idea, previously noted about the endoplasmic reticulum translocation pathway, that Y. lipolytica is more representative of vesicular secretion of animals and other fungi than is S. cerevisiae.

Characteristics of copper tolerance in Yarrowia lipolytica

We discovered that a mutant strain of the dimorphic yeast Yarrowia lipolytica could grow in the yeast form in high concentrations of copper sulfate. The amount of metal accumulated by Y. lipolytica increased with increasing copper concentrations in the medium. Washing with 100 mM EDTA released at least 60% of the total metal from the cells, but about 20-25 micromol/g DW persisted, which represented about 30% of the soluble fraction of cultured cells. The soluble fraction (mainly cytosol) contained only about 10% of the total metal content within cells cultured in medium supplemented with 6 mM copper. We suggest that although a high copper concentration induces an efflux mechanism, the released copper becomes entrapped in the periplasm and in other parts of the cell wall. Washing with EDTA liberated not only copper ions, but also melanin, a brown pigment that can bind metal and which located at the cell wall. These findings indicated that melanin participates in the mechanism of metal accumulation. Culture in medium supplemented with copper obviously enhanced the activities of Cu, Zn-SOD, but not of Mn-SOD.

The Chemistry of Phospholipid Binding by the Saccharomyces cerevisiae Phosphatidylinositol Transfer Protein Sec14p as Determined by EPR Spectroscopy

The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.

Effect of essential oil of Citrus cinensis cv new hall – Citrus aurantium (indigenous in Greece) upon growth of Yarrowia lipolytica

The effect of essential oil from Citrus cinensis cv new hall – Citrus aurantium (indigenous in Greece) upon growth of the dimorphic non-conventional yeast Yarrowia lipolytica strain ACA-DC 50109 was studied. The microorganism was aerobically cultivated in batch mode in carbon-limited media. The essential oil was added into the culture medium in different quantities while the control experiment was carried out without addition. The essential oil caused a relatively important decrease of the highest concentration of biomass produced. Additionally, biomass yield on glucose consumed was significantly decreased with the addition of the oil on the cultivation medium. Moreover, the addition of the essential oil considerably increased the lag time of the culture. In all trials, a remarkable drop the pH value of the medium was observed due to the biosynthesis of small amounts of organic acids. Given that one principal component of this membrane is the one of cellular lipids, it was assumed that the extraction and the analysis of cellular lipids could provide information about the microbial behaviour. Total lipids were extracted, methanolyzed and analyzed with the aid of G.L.C. In the control experiment, the culture conditions did not favour accumulation of storage lipid inside the yeast cells and, hence, lipid produced corresponded to 5–9% (wt/wt) in dry cell mass. Similar concentrations of cellular lipids were produced when essential oil was added in various amounts. When essential oils were added, an increase of lower aliphatic chain saturated fatty acids was observed, suggesting an alteration in the membrane function.