The Yeasts || Chemotaxonomy of Yeasts

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<ul><li><p>Chapter 9</p><p>Chemotaxonomy of Yeasts</p><p>Hansjorg Prillinger, Ksenija Lopandic, Motofumi Suzuki, J. Lodewyk F. Kock and Teun Boekhout</p><p>1. INTRODUCTION</p><p>This chapter describes the use of some chemotaxonomic approachesused in yeast taxonomy. Emphasis is on cell wall carbohydrate com-position, coenzyme Q, electrophoresis of enzymes, and the analysisof fatty acids. For each of these approaches technical protocols areprovided.</p><p>2. CELL WALL CARBOHYDRATECOMPOSITION</p><p>2.1. Introduction</p><p>Cell wall composition is a useful marker to indicate taxonomic andphylogenetic affiliations among fungi (Bartnicki-Garcia 1968, 1970,Drfler 1990, Lopandic et al. 1996, Messner et al. 1994, Prillinger et al.1990a, b, 1991a, b, 1993a, 1997, 2002, von Wettstein 1921, Weijmanand Golubev 1987a). Bartnicki-Garcia (1970) divided the fungi intoeight groups using combinations of the two most dominant cell wallcarbohydrates present. Using qualitative and semi-quantitative anal-yses of cell walls, Weijman and Golubev (1987a) distinguished sixcategories of yeasts and yeast-like fungi based on these carbohy-drates. Prillinger et al. (1993a) differentiated seven cell wall typesamong yeasts and yeast-like fungi, using both quantitative and quali-tative analyses. Their typology is a refinement of that of Weijmanand Golubev (1987a).</p><p>Three cell wall types occur among the ascomycetous yeasts:</p><p>1. The mannose, glucose pattern.2. The glucose, mannose, galactose pattern.3. The glucose, mannose, rhamnose pattern, with galactose com-</p><p>monly present.</p><p>The first patterns are characteristic for the Saccharomycotina. Thesecond and third patterns occur within the Taphrinomycetes (i.e.,Taphrinomycotina) and Pezizomycotina (cited as Protomycetes andEuscomycetes, respectively, Prillinger et al. 2002). The presence of glu-cose, mannose and galactose is found in different orders ofAscomycota (namely some lineages within the Saccharomycetales[note: cited as Dipodascales, Lipomycetales, Stephanoascales(Prillinger et al. 1994)], Schizosaccharomycetales, Saitoella and differ-ent orders of the Euascomycetes) indicating that the phylogeneticvalue of the presence of galactose is low (Prillinger et al. 1994).</p><p>Among the basidiomycetous yeasts four cell wall types occur:</p><p>1. Microbotryum-type with mannose dominant, glucose present,fucose usually present and rhamnose sometimes present;</p><p>2. Ustilago-type with glucose dominant, and mannose and galactosepresent;</p><p>3. Dacrymyces-type with xylose present, and glucose and mannosepresent in equal amounts, traces of galactose may be present, butextracellular amyloid compounds are usually absent;</p><p>4. Tremella-type with glucose predominant, xylose, mannose andgalactose present, and extracellular amyloid compounds are usu-ally present.</p><p>The latter four types agree with the cell wall typology of theBasidiomycota given by Drfler (1990). The data can be usedto define classes within the Basidiomycota. The Microbotryum-typecorresponds with the Pucciniomycotina (cited as Urediniomycetes),the Ustilago-type with the Ustilaginomycotina (cited asUstilaginomycetes) and the Dacrymyces- and Tremella-types with theAgaricomycotina (cited as Hymenomycetes). From these data it isapparent that cell wall biochemistry is a useful tool in the taxonomyand phylogeny of yeasts and yeast-like organisms. Rhodotorula yarro-wii is a remarkable exception, having xylose in its cell wall, whichwould indicate placement in the Agaricomycotina, but dominantamounts of mannose and ribosomal DNA sequences, however, sug-gest a place within the Pucciniomycotina (Boekhout et al. 2000).</p><p>Four main methods have been applied to analyze the carbohy-drate composition of the yeast cell wall:</p><p>1. Gas chromatographic analysis of acid hydrolysates of whole cells,with derivatization using capillary columns (Weijman 1976,Weijman and Golubev 1987a) or packed columns (Sugiyamaet al. 1985);</p><p>2. Gas chromatographic analysis of acid hydrolysates of purified cellwalls, with derivatization (Drfler 1990, Lopandic et al. 1996,Messner et al. 1994, Prillinger et al. 1990a, b, 1991a, b, 1993a,1997b, 2002);</p><p>3. High performance liquid chromatographic (HPLC) analysis of acidhydrolysates of whole cells without derivatization (Suzuki andNakase 1988a);</p><p>4. High performance anion-exchange chromatography with pulsedamperometric detection (HPAE-PAD) of cell wall neutral sugarswithout derivatization (Prillinger et al. 1993a).</p><p>2.2. Methods</p><p>2.2.1. Analysis of Whole Cells</p><p>Analysis of whole cells has the advantage that the isolation of yeastcell walls is not needed. This method is sometimes preferred,because the taxonomic results of both methods are generally concor-dant. For the analysis of whole cell hydrolysates, Weijmans (1976)</p><p>129The Yeasts, a Taxonomic Study 2011 Elsevier B.V. All rights reserved.</p></li><li><p>method can be summarized as follows. Cells are hydrolyzed with 1 NHCl for 12 h at 100C. During hydrolysis monomeres are formed.Neutral polysaccharides are hydrolyzed completely at low concentra-tions of HCl (1 N), whereas chitin is converted to glucosamine athigh concentrations of HCl (5 N). After hydrolysis, the solubilizedcomponents are trimethylsilylated (TMS) prior to gas-liquid chroma-tography. Sugiyamas (1985) method differs in that dried cells arehydrolyzed in 2.5 N trifluoroacetic acid (TFA) at 100C for 15 h, fol-lowed by reduction of the neutral sugars to their corresponding aldi-tols by borohydride, and acetylation of the alditol derivatives byacetic acid anhydride. The final residues are trifluoroacetylated, andthen subjected to gas chromatographic analysis. In Prillingers(1993a) method, cell walls are isolated and purified before furtherprocessing. In order to accurately detect xylose, Suzuki and Nakase(1988a) developed a method using HPLC analysis of whole-cellhydrolysates without derivatization. In brief, whole cells are hydro-lyzed with TFA and directly analyzed using HPLC.</p><p> Analysis of Whole-Cell Hydrolysates UsingTrimethyl-Silylation (Weijman 1976, Weijman andGolubev 1987a)</p><p>Yeast cells are grown in 100 ml of 0.5% yeast extract, 1% peptone, 2%glucose (YPG) broth, in 300-ml Erlenmeyer flasks on a rotary shakerat 150 rpm and 24C (psychrophilic species at 17C). After 57 days,the cells are harvested by centrifugation (9000 3 g), washed with0.9% NaCl and washed again with deionized water. The resultant pel-let is freeze-dried and powdered. 15 mg of dried cells are hydrolyzedin 6 ml 1 N HCl or 5 N HCl, under nitrogen, in glass tubes with ascrewcap, for 12 h, at 100C in a sandbath. To detect xylose, the cellsare hydrolyzed with 2 N trifluoroacetic acid for 3 h at 100C. Aftercooling, the hydrolysates are filtered through Whatman No. 1 filterpaper, and 1 ml of the filtrate is dried in a rotary evaporator. An addi-tional 100 l Tri-Sil (Pierce) is used to silylate the sample. The reac-tion mixture is vigorously shaken and allowed to stand for 15 min.1 l is then injected into the gas chromatograph-mass spectrometer(GC-MS), which is equipped with a wall coated open tubular (WCOT)capillary column of 25 meters, coated with CP Sil 5CB with a filmthickness of 0.13 m and an inside diameter of 0.32 mm. The columnis programmed from 125 to 175C with a rate of 10C/min and anisothermal period of 5 min. Helium is used as the carrier gas at aflow rate of 30 ml/min. Electron Impact (EI) at 70 eV is used for ioni-zation and a quadrupole serves as a massfilter.</p><p> Analysis of Whole-Cell Hydrolysates UsingTrifluoroacetic Acid (TFA) and Reduction of Sugarsto Their Alditol Derivatives (Sugiyama et al. 1985)</p><p>Yeast cells are grown in liquid Wickerhams basal nitrogen medium,supplemented with 15 ml 1% glucose, at 25C, for 35 days on a testtube shaker. Cells are harvested by centrifugation, and washed withdeionized water. The pellet is freeze-dried and powdered. About30 mg of the dry cell powder is hydrolyzed in 5 ml 2.5 N trifluoroace-tic acid at 100C for 15 h in a sealed tube. The remaining acid isremoved by drying over a rotary evaporator, and 50 mg of sodiumborohydride in 10 ml distilled water is added to the residue. Thereaction mixture is allowed to stand overnight to reduce the sugarsto alditols. Excess sodium borohydride is removed by adding drop-wise 5% hydrochloric acid in methanol and by evaporating to dry-ness. Insoluble material and low-polar materials are removed bymembrane filtration (0.45 m, Gelman Sciences, Inc., Ann Arbor, MI,USA), followed by reversed-phase chromatography (Sep-Pak C,Waters Associates, Milford, MA, USA). After drying, 2 ml of methanolare added. The solution is dried in a rotary evaporator to removethe borate complex. This step is repeated several times. To 10 mg</p><p>of the residue, 0.1 ml of trifluoroacetic anhydride and 0.1 ml ofN-methyl-bis-trifluoroacetamide are added. The reaction mixtureis kept in a sealed tube and left overnight. 12.5 l of the sample isinjected in a gas chromatograph equipped with a hydrogen flameionization detector. The U-shaped glass column (4 m3 3 mm i.d.)is packed with Chromosorb W (HP) 80100 mesh coated with 2% sili-cone OV-105, 800 mesh. Nitrogen is used as the carrier gas at a flowrate of 35 ml/min. The column temperature is 140C, and the injectortemperature is 150C. Carbohydrates are identified on the basis ofsample coincidence with the relative retention times for the trifluoro-acetyl derivatives of the neutral monosaccharide standards.</p><p> Analysis of Whole-Cell Hydrolysates withoutDerivatization Using HPLC (Suzuki and Nakase 1988a)</p><p>Yeast cells are grown in a 500-ml Erlenmeyer flask containing200 ml YM broth supplemented with 2% glucose, on a rotary shaker,at 150 rpm and 25C (17C for psychrophilic species). After 45 daysthe cells are harvested by centrifugation (5,000 rpm) and washedtwice with deionized water. 50100 mg of acetone-dried cells aresuspended in 2 ml of 2 M trifluoroacetic acid in a test tube(133 100 mm) with a teflon-sealed screw cap, and kept at 100C for3 h in a metal block bath. After cooling, the hydrolysate is filteredthrough paper and evaporated to dryness. The residue is dissolved in0.5 ml water neutralized with small amounts of Amberlite IRA 410(OH form), filtered with a disposable filter unit (e.g., Shodex DT ED-13), and then subjected to HPLC. HPLC is performed using two differ-ent column systems. The two columns are:</p><p>1. Ligand exchange type column with water (HPLC grade) as themobile phase at a flow rate of 0.8 ml/min at 80C.</p><p>2. Sulfonated polymer type or amino type column, with acetonitrile-water (80:20, v/v, HPLC grade) as the mobile phase at a flow rateof 0.8 ml/min at 75C.</p><p>A refractive index detector is used to detect the carbohydrates.Neutral sugars and sugar alcohols are identified by comparing theirretention times with those of standard neutral sugars and sugaralcohols.</p><p>2.2.2. Analysis of Purified Cell Walls</p><p>An attempt has to be made to purify carbohydrates solely from thecell wall, however the results obtained by the analysis of whole cellhydrolysates and purified cell walls are usually concordant. For adetailed understanding of the taxonomic importance of cell wall car-bohydrates proper, as well as for a biochemical understanding ofthese important organelles, they need to be purified, and some pro-tocols are described below. For information on the biochemicalstructure of cell walls from various groups of yeasts the reader isreferred to Chapter 8.</p><p> Isolation and Purification of Cell Walls(Prillinger et al. 1993a)</p><p>Yeast cells are grown in 500 ml YPG broth on a rotary shaker at150 rpm for 35 days, harvested by centrifugation (10003 g),washed with deionized water until the supernatant is clear, and fro-zen at 220C until further use. For disruption, cells are suspended indistilled water (1:1, v/v), and disrupted in a French Press (20,000 PSI)until no intact yeast cells are present under the light microscope.Messner et al. (1994) have shown that disintegration of yeast cells bya Vibrogen Cell Mill (Tbingen, Germany) and 0.5 mm glass beads(yeast pellet/distilled water/glass beads51/1/3, w/w) is superior tothe disruption achieved with a French Press. Disrupted cells arewashed with ice-cold distilled water until the supernatant is clear.</p><p>130 PART | III Phenotypic, Ultrastructural, Biochemical and Molecular Properties Used for Yeast Classification</p></li><li><p>To remove cytoplasmic remnants, the cell walls are thoroughlywashed twice with 1% sodium desoxycholate (pH 7.8) with intensivestirring. After each sodium desoxycholate purification, the cell wallsare rinsed three times with distilled water. In the case of capsulatedyeasts, all the capsular material, which may form a second slimy layerabove the cell wall pellet, should be removed. Yeast cells without cap-sules (i.e., those not having a positive starch test with Lugols solu-tion) are lyophilized and powdered with a pestle and mortar andfurther processed.</p><p> High Performance Anion-Exchange Chromato-graphy with Pulsed Amperometric Detection (HPAE-PAD) of Cell Wall Neutral Sugars without Derivatization(Prillinger et al. 1993a) Acid hydrolysis of purified cell wallsand removal of TFA are performed according to the method ofSugiyama et al. (1985) (see above). Usually a mixture of 2 mg of pow-dered cell walls suspended in 2 ml 2 N TFA is hydrolyzed for 2 h at120C using teflon-sealed Pyrex test tubes. A standard mixture ofmonosaccharides containing 90 g of each neutral sugar is treated inthe same way. After evaporating the TFA in an airstream, samplesand standards are resolved in 10 ml distilled water. Monosaccharidesare separated on a Dionex CarboPac PA-1 column (4.6 3 250 mm),equipped with a guard column, using a flow rate of 1 ml/min at roomtemperature. They are eluted with NaOH as follows: 10 mM NaOHfor 3.9 min isocratic, followed by a step gradient to 100% deionizedwater for 30 min, and re-equilibration to the initial conditions for10 min. The system used for monosaccharide analysis consists of aDionex (Sunnyvale, CA) Gradient Pump Module GPM 2 and a PulsedAmperometric Detector PAD 2. A Dionex Eluant Degas Module isused to sparge and pressurize the elutants with helium. Eluant 1 is100 mM NaOH (preparation of a 50% NaOH stock solution with ultra-pure distilled water), and eluant 2 is 18 MOhm deionized water.Sample injection is via a Dionex High Pressure Injectio Valveequipped with a 10 l sample loop. To ensure a carbonate-free eluant,an anion trap column ATC-1 was installed before the injection valve.Detection of the separated monosaccharides is by a PAD, equippedwith a gold working electrode. The following pulse potentials areused: E150.1 V (t15300 ms); E250.6 V (t25120 ms); E3520.6 V(t3560 ms). The response time of the PAD 2 is set to 1 s. Resultingdata are integrated and plotted using Dionex A1-450 software.</p><p> Analysis of Purified Cell Walls Using Trifluoro-acetic Acid (TFA) and Reduction of Sugars to Their AlditolDerivatives (Lopandic et al. 1996) Approximately 2 mg ofpowdered cell walls were suspended in 0.5 ml of 2 M trifluoroaceticacid, overlaid with gaseous nitrogen, and hydrolyzed for 2...</p></li></ul>


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