1,581 Matching Annotations
  1. May 2019
    1. Cell wall β-glucan measurement was carried out as describedpreviously with some modifications(Kapteynet.al.,2001). Briefly, cell wall fractions were washed multiple times with 1 N NaCl. Washed cell walls were boiled twice in 50 mM Tris-HCl(pH 7.8) containing 2% SDS, 100 mM Na-EDTA and 40 mM β-mercaptoethanol for 5 min to remove non-covalently linked proteins and other contaminants. SDS-treated cell wall fraction was collected and rinsed thrice with water. For β-glucan isolation, cell wallswere extracted three times, each for 1 h, in 0.5 ml 3% NaOH at 75 ̊C and centrifuged at 1,200 g.All 3% NaOH supernatant fractions were saved for isolation of mannan as described below. 3% NaOH-extractable cell wall pelletwasneutralized twice in 100 mM Tris-HCl (pH 7.5) and once in 10 mM Tris-HCl (pH 7.5) and digested with 5 mg/ml zymolyase-20T in 10 mM Tris-HCl (pH 7.5) for 14-16 h at 37 ̊C. This treatment liberates approximately 90-95% glucose into the supernatant. Total glucan content in the cell wall was measured by estimating glucose from both the solubilised supernatant and zymolyase-20T insoluble pellet fractions with phenol-sulphuric acid carbohydrate estimation method using purified glucose as the standard
    2. Total β-glucan estimation
    3. min. Cells were normalized to equal OD600, resuspendedin 1 ml 50 mM Tris-HCl (pH 7.5) and transferred to 2 ml microcentrifuge tubes. Cells were lysed with glass beadsin a homogenizer (FastPrep®-24,MP Biomedicals)asdescribed earlier.Brokencells were washed from glass beadswith 500 μl Tris-HCl (50 mM, pH 7.5) and pelleteddown at 15,000 g for 10 minto obtainall cell wall and membrane content. Pellet was then boiled for 10 minin 1mlTris-HCl(50mM; pH 7.5)solutioncontaining 2%SDS. SDS-extractable material(mannoproteins)was savedand remaining pellet wasboiled again in 500 μl Tris-HCl(50 mM; pH 7.5)buffer containing 2%SDS. Cell wallwas collectedby centrifugation at 15,000 g for 10 min, washed twice with1 ml waterandresuspendedin 100 μl 67 mM potassium phosphatebuffer. This washed cell wall materialwas used for β-glucan estimation as described below
    4. Yeast cell wall was isolatedas describedpreviously(De Groot et al., 2004). Briefly, cells grown underdifferent environmental conditions were harvested at 5,000 g for 5
    5. Crude cell wall isolation
    6. Cell wall isolation, zymolyasedigestion assay and β-glucan estimation
    7. Cells grown to log-phase in YPD medium were spotted on CAAmedium and overlaid with a nitrocellulose filter. Cells were allowed to grow at 30 ̊C for 18-20 h. After incubation, the filter was washed with water to remove cells and membrane-bound CPY was detected by immunoblotting withpolyclonal anti-CPY antibody (Thermo Scientific) at a dilution of 1:15,000
    8. Carboxypeptidase Ysecretion assay
    9. CPY activity was measured as described previously (Jones,2002). A 2.5 mg/ml stock solution of CPY-specific substrate N-benzoyl-L-tyrosine p-nitroanilide(BTPNA, prepared in dimethyl formamide) was diluted 5 times with 0.1 M Tris-HCl (pH 7.5). 100 μl diluted substrate solution was added to a 96-well plate containing 25 μl cell suspension (5 x 107cells). After 18 h of incubation at 37 ̊C, plate contents were clarified by centrifugation and colour formation was quantified by absorbance at 405 nm. Background absorbance measured using BTPNA-free cell cultures was subtracted from BTPNA-loaded cell cultures and absorbancevalues were normalized to total number of viable cells to enumerate total cellular CPY activity
    10. Carboxypeptidase Y(CPY) activity assay
    11. ammonium molybdate, respectively, to the assay buffer.For specific inhibition of vacuolar membrane H+-ATPaseactivity, vacuolar membrane fractions were incubatedwith 1-2.5 μM bafilomycin for 5 minprior to the activity assay.ATPase activity was initiatedby adding ATP to the assay buffer to afinal concentration of 5 mM and incubating the reactionat 30 ̊C for 30-60 min.Reaction was stopped by adding an equal volumeof a stop-developing solution (1% (w/v)SDS, 0.6 M H2SO4, 1.2%(w/v)ammonium molybdate and 1.6%(w/v)ascorbic acid). Amount of inorganic phosphate (Pi) liberated was measured at A750nmafter 10 minincubation at room temperature. Standard curve prepared with 0-50 micromoles of KH2PO4 was used for the determination of total Pi. The ATPase activity of the vacuolarmembrane H+-ATPase was expressed in micromoles of Pireleased per milligram protein per min
    12. Vacuolar membrane H+-ATPase activitywas measured inbothcrude membrane fraction and purifiedvacuolar membrane fraction asdescribed previously(Woolfordet al.,1990).Activity inthe crude membrane fractions was carried out with 2.5-10 μgprotein in 50 μl assay buffer (5 mM MgCl2, 25 mM MES/Tris-HCl(pH 6.9)and 25 mM KCl). For activity inthe purified vacuolar membrane fraction, a totalof300 μl reactionmix was setup with of 2.5-10 μgprotein samples.Residual activities from other ATPases such as mitochondrial ATPases, plasma membrane H+-ATPase and phosphataseswere inhibited by adding 2 mM NaN3, 200 μM NaVO4and 0.2 mM
    13. Vacuolar H+-ATPase activity measurement
    14. Vacuole membraneswere isolatedwith slight modifications of Cabrera’s method(Cabrera et.al.,2008). Log-phase, YPD medium-grown cells wereinoculated in 1 lt YPDmedium to an initialOD600of 0.1. Cells were incubated at 30 ̊C with shaking at 200 rpm till the cell density reached to OD600of 0.8-1.0.Cells were harvested by centrifugation at 5,000 g and washed once with 30 ml 2% ice-cold glucose solution. Cells were incubated in 15 ml solution containingglycine-NaOH(50 mM; pH10)andDTT(2 mM) at 30 ̊C for 10 min. After incubation, cells were normalized to adensity of1000OD600and resuspendedin 15 ml spheroplasting buffer containing 10-15mg of zymolyase20T.Cells were incubated at 30 ̊C for 45-60 minor till the spheroplasting was completed.Spheroplasts werecollected by centrifugation at 4,500 rpmfor 5 minat 4 ̊C, washed gently with15 ml 1.2 M sorbitol solutionandresuspendedin 3.5 ml 15%ficoll solution made in PS buffercontaining 1X protease inhibitor cocktail. This suspension was homogenized on ice with 20-25 strokes in a loose-fitting Dounce homogenizer. Homogenate was transferred to an ice-cold,ultra-clear Beckman ultracentrifuge tube, overlaid witha gradient of3 ml 8%ficoll solution, 2.5 ml 4%ficoll solutionand 2.5 ml PS buffer lacking ficoll and centrifuged at 1,10,000g(30,000 rpm)for 90 minat 4 ̊Cin a pre-cooled Beckman ultracentrifuge with SW41-Ti swinging bucket rotor.Centrifugation was carried out with slow acceleration and deceleration settings.White creamy vacuole membrane layer wascollected from the interfaceof 0and4% ficoll gradientwithout mixing the layers.Total protein concentration in thevacuole fraction was estimated using BCAprotein assay kit as described earlier
    15. Purified vacuole membrane isolation
    16. Crude fractionation of total membraneswas carried outviadifferential centrifugation asdescribed previously (Moranoand Klionsky,1994)with slight modifications. Cells grown tolog-phase in YPDmedium werecollected, washed,normalizedto 10 OD600and resuspendedin 1 ml spheroplast buffer containing 1-2mg of zymolyase20T (MP Biomedicals).Following incubation at 30 ̊Cfor 30-45 min,spherolplastswerecollected by centrifugation at 800 g for 3 minat 4 ̊C and resuspendedin 1 mlice-cold Tris-EDTA (pH 7.5). Spheroplastswere lysed with 100 μl 0.5mm glass beads on a vortex mixer with 10 secpulsegiven thricewith intermittent ice-breaks.Cellsuspension was centrifuged at 800 g for 5 minat 4 ̊C to pellet unbrokenspheroplastsdown andthesupernatant was centrifuged at 15,000 g for 5 minat 4 ̊C to obtainthemembrane fraction pellet.Pellet was washed once with ice-cold Tris-EDTA (pH 7.5), resuspendedin 50 μl of the samebuffer and stored at -20 ̊Ctill further use. Protein concentration of pellet fraction was estimated using BCAprotein assay kit with BSA as thestandard
    17. Crude vacuolar membrane extraction
    18. Vacuolar H+-ATPase activity measurement
    19. A calibration curve of fluorescence intensity values versuspH was prepared for BCECF-AM-loaded wt cells by incubatingcellsin YPD medium containing 50 mM MES, 50 mM HEPES, 50 mM KCl, 50 mM NaCl, 0.2 M ammonium acetate, 10 mM NaN3, 10 mM 2-deoxyglucoseand5 μM carbonyl cyanide m-chlorophenylhydrazone, titrated to five different pH values in the range of 4.0-8.0. Fluorescence intensity values were measured by excitation at 440and 490 nm with emission at 535 nm and a graph was plotted between the ratio of intensity at 490 to 440 nm versuspH. Similar to pHi calibration curve, a polynomial distribution of fluorescent intensity signal and pH was observedfor BCECF-AMprobe
    20. In vivovacuolar pH calibration curve
    21. fluorescence by excitation at 440 (pH-independent) and 490 nm (pH-dependent) with emission at 535 nm. Ratio offluorescence intensity at 490 to440 nm was used tocalculatethe vacuolar pH. Background fluorescence was removed by subtracting the fluorescence intensity values of cells without BCECF-AM from the fluorescence intensity values of the probe-loaded cells
    22. Vacuole pH inyeast cells was determined asdescribed previously (Padilla-López and Pearce, 2006). Briefly, log-phase,YPD medium-grown yeast cells were harvested and suspended in 200 μl YPD medium containing 50 μM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Invitrogen # B1150) to the final cell density of 4 x 107 cells. Cells were incubated at 30 ̊C for 30 min at room temperaturefollowed by three washeswith YPD medium. Washed cells were resuspended in 1 ml YPD medium and 200 μl cell suspension was used for recording
    23. Measurement of vacuole pH
    24. Vacuolar morphology of C. glabratacells was examinedby staining vacuoleswith FM4-64 (Molecular Probes, Invitrogen). FM4-64 is a lipophilic dye that exhibits long wavelength red fluorescence when boundto lipids. FM4-64 binds to the plasma membrane and follows the endocytic pathway to reach the vacuole(Vida and Emr, 1995).Log-phase,YPDmedium-grown cells were harvested and washed with 1X PBS. 1 ODcells were resuspendedin 50 μl YPDmedium containing 30 μM FM4-64 andincubated at 30 ̊C for 30-45 min. After incubation, cells were washed thricewith YPD mediumand resuspendedin 100 μl of the samemedium. Cells were observed under confocal laser scanning microscope(Zeiss LSM 510 Meta)with 63X objective lens,2.5X final zoom, pinhole set at 108 μm and emission filterset to LP 565nmto capture fluorescence image.Along with the fluorescenceimage, aphase contrastimage was alsocaptured for each sample
    25. Staining of yeast vacuoleswith FM4-64
    26. cellswere collected and washed with chilled sterile water.1 OD600cells were resuspendedin 20 μl chilled10%TCA solution containing 8 mM EDTA (pH 8.0) and incubated at room temperature for 15-20 min.Followingincubation, cellsuspension was centrifuged at 12,000 rpm for 5 minat 4 ̊Cand supernatant was transferred to a fresh 1.5 ml microcentrifuge tube. 10 μl of this supernatant fraction was diluted 75-foldwith ATPassay mix dilution buffer provided with the kit. 50 μl of diluted suspension was added to anequal volume of ATPassay mix (Sigma # FLAAM) which containedfirefly luciferase and luciferin with MgSO4, EDTA, DTT and BSA inTricine buffer.Luminescence was measured inluminometer (Varioskan flash-3001,Thermo Scientific). Total ATP was quantified usingpurified ATP as the standardand expressed in moles/OD cells
    27. ATPconcentrationin yeast cells was measuredby luminometricluciferase-luciferinbased assayusingATPbioluminescent kit(Sigma # FLAA).Briefly, log-phase yeast
    28. Determination of intracellular ATPlevels
    29. Estimation of total glycogen in cells was performed asdescribed previously (Parrou et al., 1997) with slightmodifications.Briefly, YPD medium-grown C. glabratacells were harvested, washed once with 1 ml ice-cold waterandresuspendedin 250 μl sodium carbonate(0.25 M)solution. After incubation at95 ̊C for 4 hin water bath with occasional stirring, cell suspension was cooled and pH of the suspension was adjusted to 5.2 by adding 150 μl 1 M acetic acid. Tothis suspension,600 μl 0.2M sodium acetatewas added and cell suspension was incubated with 1-2 U/ml of α-amyloglucosidase from A.niger(Sigma #A7420)at 57 ̊C for overnight with constant agitation.Resultant glucose liberated by α-amyloglucosidase digestion was collected in the supernatant fraction and quantifiedby phenol-sulphuric acid methodof carbohydratedetermination.For quantification, commercially available purified glucose was used as a standard and total glycogen incells was expressed as μg/2 x 107cells tonormalizeagainstcell density
    30. Estimation of glycogenlevels
    31. Trehalose from C. glabratacells was extracted by trichloro acetic acid (TCA)solutionas described previously (Lillie et al.,1980). Cells grown in YPDmediumwere collected at different time pointsof growth and washed thrice with ice-cold sterile water. Cells were immediatelystored at-20 ̊Ctill further use.For trehalose isolation, 10-20 OD600cells were thawed in 500 μl TCA (0.5 M) solutionon ice and incubated at room temperaturefor 1 h.Supernatant fraction was collected by sedimenting cells at 14,000 rpm for 5 minat 4 ̊C.TCA extractionwas repeated withcells once more and the resultingsupernatant was mixed with the earlier fraction.Extractedtrehalose was measuredby phenol-sulphuric acid methodof carbohydratedeterminationwithcommercially available purified trehalose(Becton, Dickinson and Co.) as a standard.Total trehalosecontent was normalized to the cell densityand expressed as μg/2 x 107cells
    32. Estimation of trehalosecontent
    33. Estimation oftrehalose, glycogen and ATPlevels
    34. Quantitative measurement of periplasmic acid phosphatase activity in phosphate-starved C. glabratacells was performedas mentioned previously (Orkwis et al., 2010). A total of 0.5 OD600YNB-grown and phosphate-starved cells were collected, washed thrice with cold water and oncewith cold 0.1 M sodium acetatebuffer (pH 4.2). Washed cells were resuspendedin 500 μl sodium acetate (0.1 M)and incubated at 30 ̊C with constant stirring. After 10 min incubation, 500 μl freshly-prepared solution of 20 mM p-nitrophenyl phosphate in 0.1 M sodium acetate(pH 4.2) was added to the cell suspension. Enzymatic activity was stopped after incubation at 25 ̊C for 20 min by addition of 250 μl sodium carbonate (1 M)tothe reaction mix. Resultant colour change was measured by monitoring absorbance at 400 nm. Acid phosphatase activity was expressed as a ratio of OD400to OD600 to normalize against cell density
    35. Determination of acid phosphatase activity
    36. Cells grown overnight in YNBmedium wereinoculated in fresh YNB mediumand incubated at 30 ̊C with shaking at 200 rpm. Cells were harvested when the cell density reached to an OD600of 0.6-0.8.Cells were consecutively washed with sterile MQ water and YNB without phosphate (YNB-Pi) medium. Washed cells were inoculated either in YNB orYNB-Pimediumto the initial OD600of 0.1. Cells were incubated at 30 ̊C for 3-4 h, harvested and resuspendedin 100 μlYNB-Pimedium. Radioactive P32-labelled o-phosphoric acid(Jonaki# LCP 32)was added to the cell suspension to a final concentration of 1 μCi/mlandcells were incubated for 30 min.For determining phosphate uptake, a10-12 μl cell suspensionaliquot,after every5 min,was removed and kept on ice.To this cell suspension, 500 μl ice-cold YNB-Pimediumwas addedand cells were harvested by centrifugation at 5,000 g for 5 minat 4 ̊C.These cells were washed with ice-cold YNB-Pimedium thrice and resuspendedin 100 μlPBS(1X). 10-20 μl of this cell suspension was added to5 ml scintillation fluid and β-decay counts were measured in ascintillation counter(Tri-Carb 2910 TR Liquid Scintillation Analyzer, PerkinElmer).Scintillation counts were normalized to total cell number and plotted with respect to time. Total phosphateuptake was expressed as P32c.p.m/OD600cellswhere c.p.m refers tocounts per min
    37. Phosphate uptake assay
    38. Total cellular polyphosphates were quantified viapolyacrylamide-Tris borate gel electrophoresis (PAGE-TBE).Briefly, PAGEwas performed with Tris-borate buffer (pH 8.3) todeterminethe quantityand the typeof polyphosphatesextracted fromyeaststrains. Equal amount of total RNA (20 to 100 μg) was loadedon 34%PAGE-TBE gel (22cm long, 16 cm wide and 0.8 mm thick) and electrophoresedat 500Voltsfor 20-24 hin cold-roomtill the marker dye bromophenol blue(BPB)had migrated 15-16 cm awayfrom the well.After electrophoresis, total polyphosphates werevisualizedby staining the gel with 0.05%toluidine blue staining solutionfollowed by destaining. Polyphosphates wereobserved both as asmearin the top most portion of the gel as well asdiscreet bands of long chain polyphosphatesand shortchain polyphosphatesin middle andbottom half of the gel,respectively.Polyphosphateband intensity in the gelwas quantified using ImageJ software (http://rsbweb.nih.gov/ij/)and relative amountsof long chain and short chain polyphosphatesin C. glabratacells werecalculated
    39. Quantitative analysisof polyphosphates
    40. Polyphosphates from yeast cells were extracted with phenol-chloroform solutionas described previously(Neefand Kladde,2003). Cells grown eitherovernight or to logarithmic phase in YPDmediumwere harvested by centrifugation at 1,700 rpm for 5 min. Cells were washed with 10 ml sterile MQ water and resuspendedin ice-cold 500 μl20%trichloro acetic acid (TCA) solution to the final cell densityof 100 OD. Cell suspension was transferred toa1.5 ml microcentrifuge tube. After incubation at room temperature for 5 min, cells were harvested by centrifugation at 12,000 g for 10 minat 4 ̊C and resuspendedin 250-350 μlpolyphosphate extraction buffer.Equal volume of phenol-chloroform (25:24) was addedtothe microcentrifuge tube and aqueous phase was extracted by centrifugation at 12,000 g for 8 minat room temperature. Top aqueous layer was collected with a 200 μl tip. Aqueous layer extraction was repeated once more after removal of DNA with chloroform.After centrifugation,aqueous phasecontaining RNA and polyphosphates wascollected,RNA was quantified at A260nmandstored at -20 ̊C
    41. Polyphosphate extraction
    42. were maintained in log-phase by continuous passaging in fresh YNB medium every 4 h
    43. For phosphate starvation,yeastcells grown to log-phase in YNB medium were harvested,washed with water,transferred to either regular YNBor YNB medium lackingphosphate and were grown for 16 h at 30 ̊C. Cells cultured in YNB medium
    44. Phosphate starvationof yeast cells
    45. Plasma membrane H+-ATPase activitywas measured inthe total membrane fraction as described previously (Nakamura et al., 2001).5μg totalmembrane fraction was incubated at 30 ̊C in 120 μl reaction buffer containing 10 mM MgSO4and 50 mM KCl in 50 mM MES (pH 5.7) with 5mM adenosine tri-phosphate (ATP). To eliminate possible contribution of residual ATPases, viz.,vacuolar ATPases, mitochondrial ATPases or non-specific phosphatases, 50mM KNO3, 5mM NaN3and 0.2mM ammonium molybdate were used, respectively, in the assay mixture. Reaction was stoppedafter 30 minby adding 130μl stop-developing solution containing 1% (w/v) SDS, 0.6M H2SO4, 1.2%(w/v)ammonium molybdate and 1.6%(w/v) ascorbic acid. Amount of inorganic phosphate (Pi) liberated was measured at A750nmafter 10 minincubation at room temperature. A standard curve prepared with0-50 μmolesof KH2PO4 was used fordetermination of total Piamount.ATPase activity of the plasma membrane was expressed in micromoles of Pireleased per milligram protein per min. ATPase activity was also determined in the presence of plasma membrane H+-ATPase inhibitor diethylstilbestrol (DES,Sigma# D4628),wherein total membrane fraction was incubated with 0.2mM DES for 5 min, prior to the enzymatic measurement
    46. Plasma membrane H+-ATPase activity assay
    47. dithiothreitol and1X protease inhibitor cocktail. Cell suspension was rapidly frozen at -80 ̊C,thawed and lysed with 0.5mm acid-washed glass beadsin a homogenizer (FastPrep®-24,MP Biomedicals)at maximum speed of 60 secfive times. Homogenate wasdiluted with 5mlTris-HCl (0.1M; pH 8.0)solutioncontaining 0.33M sucrose, 5mM EDTAand 2mM dithiothreitoland centrifuged at 1,000g for 3 minat 4 ̊C. Supernatant was collected and centrifuged again at 3,000g for 5 minat 4 ̊C to remove unbrokencells. The resulting supernatant was centrifuged at 19,000g for 45 minat 4 ̊C to obtain total membrane fraction. Total membrane pellet was resuspendedin 100μl membrane suspension buffer and stored at -80 ̊Ctill further use. Total protein concentration in the membrane fraction was estimated using BCAprotein assay kit (Thermo Scientific, US) with bovine serum albumin (BSA) used as astandard
    48. Isolation of total membrane fractions from C. glabratastrains were carried out as described previously (Fernandes et al., 1998). Cells grown to log-phase under different environmental conditionswere harvested, washed and suspended to afinal density of 20 OD600cells in 1 ml solution containing100mM Tris (pH 10.7),5mM EDTA,2mM
    49. Total membrane preparation
    50. To assess the activity of plasma membrane proton pump, CgPma1, in cells grown in differentexternal pH environment,whole cell acidification assaywas carried out.This assay is a measurement of glucose-responsive proton pump activityin live cellsand is based on a decrease inthe pH of a weakly-buffered solutionupon extrusion of H+ions from thecell. The amount of change in the pH of the medium represents a crude measurement of the activity of functional plasma membrane proton pump in live cells. Whole cell acidification assay was conductedwithcellsgrown in YNB pH 5.5 and YNB pH 2.0medium as described previously (Martinez-Munoz and Kane, 2008) with slight modifications.After growth at30 ̊C for 2 h, cells were harvested, washed and resuspended(1.5-3.0 mg wet weight/ml) in 15ml MES/TEA (1mM; pH 5.0) buffer. Cell suspension was kept at 25 ̊C with continuousagitation. Extracellular pH of the buffer solution was recorded at 1 mininterval for 20 minwith the help of a pH meter(BT-600, BoecoGermany). To activate plasma membrane proton pumping, glucose and KCl were added to a final concentration of 40mM after 3 and 8 minincubation, respectively. Plasma membrane proton pump activitywas plotted as a change in the pH of the extracellular solutionversustime
    51. Whole cell acidification assay
    52. Measurement of plasma membrane H+-ATPase activity
    53. Log-phasecells grown in YPD medium containing or lacking CaCl2and FK506 were collected, PBS-washed and loaded with ratiometric, high affinity, membrane-permeable calcium indicator, Fura-2 AM (10 M; Sigma #47989). After 30 min incubation at 30◦C, labelled cells were washed thrice with cold PBS, suspended in PBS and fluorescence was recorded at 505 nm with dual excitation at 340 and 380 nm. The ratio of fluorescence intensities between 340 and 380 nm, representing Ca-bound and Ca-free Fura-2 molecules, respectively, reflected free intracellular calcium concentrations
    54. Measurement of intracellular calciumlevels
    55. Intracellular reactive oxygen species (ROS)levels in yeast cells weredetermined usingfluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA; Sigma# D6883). Cellular esterasesremove the diacetate groups ofthe DCFH-DAand produceDCFHwhich getsreadily oxidized to highly fluorescent product 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. The fluorescent intensity of DCF corresponds to the amount ofintracellular ROSpresent in the cell.Cells grown under different environmental conditions were harvested,washed once with tissue-culture grade phosphate-buffered saline (PBS) and resuspendedin PBS to the final cell density of 1 OD. Freshly-prepared DCFH-DA (0.01M stock in DMSO) was added to the cell suspension toafinal concentration of 100 μM. Cell suspension was mixed and incubated at 30 ̊C for 30 min. After incubation,cells were washed 2-3 times with 1 ml PBS and then resuspendedin 200 μlPBS. Fluorescence intensity values wererecorded usingspectrofluorophotometer (Varioskan flash-3001, Thermo Scientific) with excitation and emission at 488and 530 nm,respectively.Fluorescenceintensityvalues obtained from probe-loaded cells were subtracted from the fluorescence intensity values obtainedfrom cells-alone samplesto remove background fluorescence
    56. Determination of intracellular reactive oxygen species (ROS)levels
    57. For determiningthe intracellular pH from fluorescence intensity values of CFDA-SE-loaded cells, anin vivocalibration curve was prepared between fluorescent intensity and pre-adjusted environmental pH values. Briefly,CFDA-SE-loaded wild-typeC. glabratacellswere incubatedwith 0.5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma# C2759) at 30 ̊C for 10 min in 50 mM CP buffer adjusted to different pH values ranging from 4.0 to 7.5, with an interval of 0.5 unit.CCCP is an ionophore which dissipates the plasma membrane pH gradient, thus, rendering the intracellular pH similar to the extracellular pH. Fluorescent intensities were determinedand a calibration curvewas plotted between the ratio of intensity at 490 to430 nm versuspH.A polynomial distribution of fluorescent intensity signal and pH was observed for CFDA-SE probe(Figure2.1)and the graphequation was used todeterminethe intracellular pHof C. glabratacells
    58. In vivointracellular pH calibration curve
    59. OD600of 0.5and transferred to a 1.5 ml microcentrifuge tube. Probe loading was carried out by adding freshly-prepared CFDA-SE solution (0.01 M stock in DMSO) tocell suspension to a final concentration of 160 μM. Cell suspension was mixed on vortex mixerfor 10 secand incubated at 37 ̊C for 1 hwith shaking at 300 rpmon thermo mixer.Cells were harvested, washed twice with 1 ml 50 mM CP buffer to remove unloaded probe,resuspendedin 250 μl CP buffer andwereincubated at 30 ̊C for 30 minwith shaking to recover from the stress induced during probe loading. Afterincubation,fluorescent intensitywasdetermined with spectrofluorophotometer (Varioskan flash-3001, Thermo Scientific) by excitation at 430 nm (pH-independent) and 490 nm (pH-dependent) with emission at 525 nm. Background fluorescence of the probe was removed by subtracting the fluorescence intensity of the probe in CP buffer from the fluorescence intensity of the probe-loaded cells
    60. Intracellular pH(pHi)in yeast cells was determinedusing fluorescent 5,(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes) asdescribed previously (Bracey et al.1998). For pHiprobe estimation,YNB medium-grown log-phase cells were inoculatedin YNB, YNB-pH 2.0 or YNB medium supplemented with acetic acid and incubated at 30 ̊C for different time points.Log-phase C. glabratacells were harvested and washed twice with 50 mM citric-phosphate (CP) buffer (pH 4.0). Washed cells were resuspendedin 1ml 50 mM CP buffer to an
    61. Measurementof intracellular pH (pHi)
    62. Following transfer,membrane was either stained with Ponceau reagent for checking the efficiency of transfer or directly processed for proteindetectionusing protein-specificantibodies.For immunoblotting, membranes were blockedwith 5% (w/v)non-fat milk solutioneitherin PBS-T or TBS-T for 2 hat RTand probedwith primary antibodies against the target proteins.For detection of CgPma1, membranes were probed with1:1000 dilution of polyclonal anti-Pma1antibody raised against S. cerevisiaePma1 (Santa Cruz #sc-33735)in PBS-T with 5% (w/v)fat-free milk for overnight at 4°C.For detection of phosphorylated form of CgSlt2,immunoblotting analysis was done withan anti-phospho-p44/42 MAPK (Thr202/Tyr204) primary antibody raised against human p44 MAPK(Cell signalingtechnology# 4370S) at a dilution of 1:6000 in TBS-T with 5% (w/v)fat-free milk for overnight at 4°C.For detection of CgCPY, membrane was incubated with polyclonal anti-CPY antibody raised against S. cerevisiaeCPY(Thermo Scientific # PA 1-27244) at a dilution of 1:10,000 in TBS-T with 5% (w/v)fat-free milk for overnight at 4°C.For CgGapdhdetection,anti-Gapdh primary antibody raised againsthuman Gapdh(Abcam # ab22555) at a dilution of 1:7000was usedin TBS-T with 5% (w/v)fat-free milk.Secondary antibodies conjugated with horseradish peroxidase(HRP)enzymewereusedin 1:10,000 dilutionto detect the immune-reactivity of primary antibodieswith the help of ECL plus Western blotting system (GE Healthcare)as per manufacturer’sinstructions
    63. Total proteins resolved bySDS-PAGE were transferred to PVDFnylon membrane by Western blotting using a Bio-Rad Mini Trans-Blot electrophoretic transfer unit in Tris-glycinetransfer bufferat 4 ̊C either at 100 Voltsfor 3 hr or 30 Voltsfor overnight
    64. Western blot analysis
    65. SDS-PAGEwas performed as described previously (Laemilli, 1970).10-40 μg protein samples were mixed with 4X SDS loading buffer and either incubated at 50 ̊C or 90 ̊C for 10 min. Denatured samples were loaded either on8%or 10%SDS-PAGEgel and run in Tris-Glycine-SDSgel running buffer at 70-100 Volts for 2-3 hin a Mini-PROTEAN®3electrophoresis unit(Bio-Rad).After electrophoresis,gels were either visualized by coomassie brilliant blue (CBB) stainingor processedfor western blotting as described below
    66. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE)
    67. Log-phase yeast cell cultures were harvested and total protein was extracted by lysingyeast cells using glass beads. Briefly,10 mllog-phase yeast culturesgrownin appropriate medium were harvested,washed once with ice-cold water and suspended in 250 μl homogenizing buffer containing 1 mM phenylmethylsulfonylfluoride(inhibitsserine proteases), 10 mM sodium fluoride(inhibit Ser/Thr and acid phosphatases), 1 mM sodium orthovanadate (inhibits Tyr and alkaline phosphatases) and 1X concentration of protease inhibitor cocktail(RocheCat # 04693159001). Cells were lysedwith glass beads by vortexing five times at high speed for 1 min with intermittent 1 min ice breaks. Unbroken cells and cell debris were removed by centrifugation at 1,000 g for 5 min at 4 ̊C. Cell lysate was collected and protein was quantified using bicinchoninic acid (BCA)protein assay kit (Thermo Scientific # 23227) as per supplier’s instructions
    68. Protein extraction
    69. Biochemical techniques
    70. hybridizations from biological replicates for each sample. Data was extracted with Feature Extraction software v 10.5 (Agilent) and normalizedwith GeneSpring GX v 11.0.1 (Agilent) software using the recommended Percentile shift Normalization to 75th percentile. Raw Data sets for this study are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=

      GSE24267

    71. Log-phase wild-type and Cgyps1∆cells were grown in YNB and YNB-pH 2.0 medium. After 1 h incubation, yeast cells were collected, washed and were stored in RNAlater at -80°C. These frozen samples were sent to Genotypic Technology Ltd., Bangalore(http://www.genotypic.co.in) whichprovides services of global gene analysis on Agilent platform. A 8x15k GE array comprised of 60mer oligonucleotidesfor a total of C. glabrata5503 genes was used wherein average number of replicates for each probe was three. Labeling was done in single color and data is the average of two
    72. Microarray analysis
    73. .coliBW23473 electro-competent cell aliquots werethawed on ice and mixed with 1-2 lof plasmid DNA. Mixture was pulsed with the Gene Pulser®electroporation apparatus(Bio-Rad) at 1800 Volts with 25 μF and 200 Ωcurrentin a chilled0.1 cm electroporation cuvette(Bio-Rad). Immediately after successful pulsing, 1 ml LB medium was added to the cuvetteand suspension was transferred toa 1.5 ml sterile centrifuge tube. Cells wereincubated at 37°C for 1 hwith shaking and further plated onLB plates containing kanamycin(30μg/ml). Positive colonies were inoculated in LBliquid medium containing kanamycin(30μg/ml)for plasmid isolation

      E.

    74. E.coliBW23473transformation by electroporationE
    75. A single colony of E.coliBW23473strainfrom a freshly-streaked LBplate was inoculated in50ml LB medium. Culture was incubated overnight at 37°C with shaking at 200rpm. 25ml of the overnight-grown BW23473 culturewas transferred to500ml pre-warmed LB medium andincubated at 37°C till the OD600reached to 0.4. After incubation, cultureswere transferred to an ice-water bathandcentrifugeat 1,000g for 15 minat 4°C. Cells were washed twice with 500ml ice-coldwater, thrice with250ml ice-cold 10% glycerol solution and resuspendedin 1ml 10% glycerol solution. Cell suspension wasnormalized to final cell densityof 3x 1010cells/ml and dispensed in 50μl volume into sterile ice-cold microcentrifuge tubes. Aliquots weresnap frozen inliquid nitrogen and stored at -70 ̊C for further use
    76. Electro-competentcell preparation
    77. the disrupted gene, BLAST N of the sequences from rescued plasmids was performedagainstC. glabrataGenolevures database (http://www.genolevures.org/blast.html
    78. Identification of disrupted locus in Tn7insertion mutants was carried out as described previously(Kaur et al.,2004). Disrupted locus in each mutant is physically marked with a mini Tn7 transposon derivative containing conditional origin of replication R6K(facilitates Tn7recovery), S. cerevisiae URA3and Klebsiella pneumoniae hphgene (confers resistance to hygromycin B)(Castaño et al.,2003). Briefly,genomic DNA was isolated fromovernight grown Tn7insertion mutants using spheroplast lysis method. After RNAse treatment, 10μgDNA was either digested withMfe1or SpeIrestriction enzymeas the Tn7 cassette lacks these enzyme sites. Followingovernight digestion, DNA wasprecipitated with 1ml ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.2).DNApellet was washed twicewith ice-cold 70% ethanol, air driedand resuspendedin sterile MQ water. DNA was recircularized withT4 DNA ligase. Resultant circular plasmid contains the Tn7cassette flanked on either side by the gene,it has disruptedin the genome of C. glabrata.This circular plasmid DNA was transformedin E.coliBW23473 strain,which contains protein Π (the product of the pir gene)required by R6Korifor replication.Transformation of circularized DNA in E.coliBW23473 electrocompetent cells was performedas described below.Plasmids fromselected transformants were isolated and sequenced with outward primers from Tn7right and left ends to sequencethe disrupted gene fragment.For identification of
    79. Tn7insertion mutant rescueand gene identification
    80. C. glabrataTn7insertion mutantlibrary was screened for reduced growth in YNB-pH 2.0 medium. Thismutant library,composed of 9,134 Tn7insertion mutants, isarrayed in 96-well microtitre plates(Castaño et al.,2003). 2 μlof each mutant strain was inoculated in 120μl YNB medium and grown overnight at 30 ̊C in an incubator with constant shakingat 120 rpm. Overnight grown cultures were 120-folddiluted with 1X PBS in a 96 well block and transferred, using a 96-well pin replicator, to YNB and YNB-pH 2.0 medium. Plates were incubated at 30°C and mutant phenotypes were recorded after 3-4days.
    81. Screening of C. glabrataTn7insertion mutants
    82. and colony purified on CAA plate. 15% glycerol stocks were made for two independent transformants and stored at -80 ̊C
    83. C. glabrataCgYPS7ORFwas cloned in a self-replicating pGRB2.2plasmidwhich contains C. glabrata CEN-ARS, S. cerevisiaeURA3gene, S. cerevisiaePGK1promoter and C. glabrataHIS3-3′ untranslated region. For cloning CgYPS7in pGRB2.2,CgYPS7ORF (1.764 kb) was PCR-amplified from the wild-type genomic DNA with high fidelity Platinum PfxDNA polymeraseusing primers carrying restriction sites for XbaIand XhoI. The1.764 kb amplifiedPCR product waspurified with QIAquick PCR purification kit (Qiagen # 28104),digested with XbaI and XhoI and cloned in the pGRB2.2plasmid at XbaI–XhoI sites in the multiple cloning site (MCS)region downstream of the PGK1promoter.Positiveclones were verified by PCR, sequencing and complementation analysesofCgyps7∆mutant. Yeast transformantsobtained by lithiumacetate methodwere selected on plates lacking uracil
    84. Cloning of CgYPS7gene
    85. stranded DNA. Final reaction volume was adjusted to 20 μl with DEPC-treated waterandamplificationreaction was carried out usingthese parameters: initial denaturation at 95 ̊C for 5 min followed by 40 cycles of denaturationat 95 ̊C for 30 sec, annealing at 55 ̊C-57 ̊C for 30 sec, elongation at 72 ̊C for 40 sec and final extension at 72 ̊C for 10 min. Transcript levelswerequantified with an end-point value known as Ct (cycle threshold). The Ctdefines the number of PCR cycles required forthe fluorescent signal of SYBR green dye to cross more than the background level. The Ctvalue isinversely proportional to the amount of nucleic acid product. Ctvalues were obtained during exponential phase of amplification and used forcalculation of relative-fold change in gene expression after normalization to Ctvalues ofeither housekeeping gene ACT1 (gene encoding actin)orTDH3 (gene encoding Gapdh)with the help of the following formula. Fold change in expression = 2-∆∆Ct∆∆Ct= ∆Cttreated -∆Ctuntreated∆Cttreated = Ctvalue forgene of interest under test/treatedcondition -Ctvalue forinternal controlgene(ACT1/TDH3) under test/treatedcondition∆Ctuntreated = Ctvalue forgene of interest under untreatedcondition -Ctvalue forinternal control (ACT1/TDH3)gene under untreatedcondition
    86. Todeterminethe expression level of a specific gene, quantitative real-time polymerase chain reaction (qRT-PCR/qPCR)was performed oncDNA usinggene specific primers. Primers for qPCR weredesigned in such a way so as to get amplification products in a size range of 150 to 300 bp. Optimalprimer and cDNA concentrationswere standardized and qPCR was performed in ABI Prism 7000/7500 Real time PCR Machine (Applied Biosystems). Briefly, 0.4 μl cDNA was mixed with 0.1 to 0.2 picomolesof gene specific forward and reverse primers and 10 μl 2X MESA GREEN qPCR™Mastermix Plus containing SYBR green dye (Eurogentec) in awell of a96-well PCR plate (Axygen). SYBR green is a dye that specifically binds to double
    87. Quantitative real-time polymerase chain reaction (qRT-PCR)
    88. 1 μg good quality RNA was treated with DNase I (amplification grade, Invitrogen) to remove DNA contamination and used for complementary DNA (cDNA) synthesis using reverse transcriptase enzyme and oligo-dT primers.SuperScript®III First-Strand Synthesis System (Invitrogen) was used to carry out cDNA synthesis reaction according to the manufacturer’s instructions. cDNA was stored at -20 ̊C
    89. Complementary DNA (cDNA) synthesis
    90. autoclavable plastic items to removeRNase contamination. RNA was isolated from C. glabratacells using hot phenol extraction strategy.Log-phase cells well harvested at 5,000 g for 5 min at 4 ̊C, resuspended in 1 ml ice-cold DEPCwater and transferred to a 2 ml microcentrifuge tube. Cells were spun down at 6,000 g for 3 min at 4 ̊C and resuspended in 350μl AEsolution. 50 μlSDS and 400 μl acid phenol wereadded tothe above tubeand mixed well by vortexing. Tubes were incubated at 65 ̊C for 15minwith continuousmixing. After incubation, tubes were kept on ice for 5 min and centrifuged at 12,000 rpm for 5 min at 4 ̊C. Aqueous phase was collected and re-extracted with an equal volume of cholroform. Total RNA was precipitated at -20 ̊C with1/10thvolume of 3 M sodium acetate (pH 5.2) and 2.5 volume of ice-cold 100% ethanol and collected by centrifugation at 12,000 rpm for 5 min at 4 ̊C. RNA pellet was washed with ice-cold 70% ethanol and resuspendedin 100 μl commercially available DEPC-treated water (Sigma # 95284). RNA concentration was measured byrecordingabsorbance at 260 nm. Purity of RNA sample was checked by A260nm/A280nmratio where ratio of >1.8 was considered as good quality RNA. RNA integrity was checked by gel electrophoresis on 8% agarose gel made in DEPC-treated TAE buffer
    91. All solutions were made in RNase free diethylpyrocarbonate(DEPC)water. Microcentrifuge tubes and tips employed for RNA workwere autoclaved twice and kept at 70 ̊C for overnight before use. RNaseZap®(Ambion) was sprayed on non-
    92. Total RNA isolation
    93. Themethod was used for isolation of good quality genomic DNA that wasused to map Tn7insertionin C. glabratamutants.Briefly,10 mlsaturated yeast culturewasharvested, resuspendedin 1 ml sterile water and transferred toa2 ml microcentrifuge tube. Cells were pelleteddown by centrifugation at 4,000 rpm for 5 min. Supernatant was discarded and the pellet was resuspendedin 500 μl freshly prepared solutioncontaining100mM EDTAand 5% β-mercaptoethanol andincubated at 42 ̊C for 10 min. After incubation,cells were spun down at 5,000 rpm for 1 minand resuspendedin 500μl freshly-prepared BufferB. One tip full of lyticase(Sigma # L4025) was added and cellsuspension was incubated at 37 ̊C for 1 h. Following incubation,cell suspension was spun down at 6,000 rpm to recover spheroplasts.Spheroplasts weregently resuspendedin 500μl BufferCand DNA was twice extracted with 500μl phenol:chloroform:isoamyl alcohol (25:24:1)solution.Aqueous layer was collected in a new 2ml microcentrifuge tube and DNA was precipitated with 1ml ethanol and 1/10thvolume of 3M sodium acetate (pH 5.2)by centrifugation at 13,000 rpm for 5 min. Pellet was resuspendedin 200 μl TE containing 0.3 μl of RNase Cocktail™and incubated at 37 ̊C for 30 min.After incubation, 300 μl additional TE was added and DNAwas re-precipitated withethanol and 3 M sodium acetateas described above. Pellet was washed with 70% ethanol anddried under air. DNA pellet was finally suspended in 100 μl TE and stored at -20 ̊C
    94. Protocol III(Spheroplast lysis method
    95. phenol:chloroform:isoamyl alcohol (25:24:1)was added to the tube and mixed thoroughly.Aqueous phase was collected after centrifugationat 12,000 rpm for 3 minand was transferred toanew 2 ml microcentrifuge tube.1 ml absoluteethanol was added to the aqueous phase and DNA was precipitated by centrifugation at 12,000 rpm for 8 minat 4 ̊C.DNA pellet was washed with chilled 70%ethanol and dried under air. DNA pellet was resuspendedin 50 μl TE containing 0.3 μl of RNase Cocktail™(Ambion®# AM2286)and incubated at 50 ̊C for 20 min. 200 μl additional TE was added to the above suspension and DNA was stored at -20 ̊C
    96. In this method of genomic DNA extraction,yeast cells werelysed by mechanical disruption with glass beads. Briefly, yeast cells were harvested after overnight growth in YPD medium, resuspendedin 500 μl waterand transferred toa2 ml microcentrifuge tube.Cells were pelleteddown at 10,000rpm for 1 min. Resulting supernatant was discarded and the pellet was resuspendedin 500 μl Buffer A. The tube was incubated at 65 ̊C for 15 min. After incubation, 500 μl ofphenol:chloroform:isoamyl alcohol (25:24:1) and 0.5 gm of acid-washed glass beads (Sigma # G8772) were addedto the tube. Cells were lysed by three cycles of high speed vortexing withintermittent ice breaksfor 45 secand pelleteddown at 12,000 rpm for 3 minat 4 ̊C.Uppermost aqueous phase was transferred to a 2 ml microcentrifuge tube,500 μl of
    97. Protocol II (Glass bead lysis method)
    98. This quick extraction method was used to isolate genomic DNA which was used as templateto amplify gene of interestor toverify the knock-out. C. glabratacells were grownovernight to saturation in 10 mlYPD medium at 30 ̊C.Cells were harvested at 4,000 rpm for 5 min, resuspendedin 400 μl Buffer Acontaining 50 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1%Triton X-100 and 1%SDSand weretransferred to a2 ml microcentrifuge tube. Equal volume ofphenol-chloroform solution was added to the abovesuspensionfollowed byvortexingfor 2-3 minand incubationat 42 ̊C for 30 minwithcontinuous agitation at 800 rpm on thermomixer (Eppendorf). Cell debris was removed bycentrifugation at 12,000 rpm for 5 minand aqueous fraction(~ 350 μl)was transferred to a new 2 ml microcentrifuge tube.0.3 μl RNaseCocktail™(Ambion® # AM2286) containing RNase A (500 U⁄ml) and RNase T1 (20,000 U⁄ml) was added and tubes were incubated at 37 ̊C for 30 min. DNA was precipitated with 2.5 volumesof chilled ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.2).DNA pellet was washed with chilled 70%ethanol and semi-dried under air.Pellet was suspendedin 100μlTE (10 mM Tris-HCland 1 mM EDTA; pH 8.0)and stored at -20 ̊C.DNA concentration was determined by recordingabsorbance at 280 nmin Nanodrop (Nanodrop ND-1000, Thermo Scientific).
    99. Protocol

      I (Quick genomic DNA isolation)

    100. Based on the subsequent use, DNA from C. glabratacells was extracted using three different methodologie

      s

    101. Yeast genomic DNA isolation
    102. Molecular biology tech

      -niques

    103. C. glabratayeast cells were grown overnight in 5 ml YPD medium at 30 ̊C. An aliquot from the overnight culture was inoculated in 10 ml fresh YPD medium to an initial OD of 0.1. Cells were incubated at 30 ̊C till the cultureOD600was between 0.4 and 0.6. Cells were harvested in a sterile 50 ml centrifuge tube and washed twice with sterile Milli-Q(MQ)water. Washed cells were suspended in 100 μl of 100 mM LiOAc, mixed thoroughly and transferred to a sterile 1.5 ml microcentrifuge tube. A transformation mix containing 240 μlpolyethylene glycol(PEG) (50% (w/v)), 36 μl LiOAc(1 M), 25μl ultrapure single-stranded salmon sperm DNA (2 mg/ml) (Clonetech) was added to 50 μl cell suspension. 50 μltransforming DNA (1μg circular plasmid DNA) was added to the above suspension. Whole mixture was vortexed gently and incubated at 30 ̊C for 45 min. 43 μl DMSO was added to the tubeand incubated at 42 ̊C for 15 min. Cells were collected after centrifugation at 5,000 rpm for 1 min and suspended in minimal medium containing 0.6% Bacto-Casamino acid. Transformation mixture was plated on CAA plates and transformants were selected for uracil prototrophy
    104. Yeast transformation usinglithium acetate (LiOAc) strategy
    105. 5-10 ml saturated bacterial culture harboring the desired plasmid was harvested at 5,000 g for 3 min. Plasmid DNAwas isolated using QIAprep Spin Miniprep Kit (Qiagen, USA) or GenElute™ HP Plasmid Miniprep kit (Sigma-Aldrich, USA) as per manufacturer’s instructions
    106. Bacterial plasmid isolation
    107. E. coli DH5α ultra-competent cells were transformed with plasmid DNA by heat shock at 42 ̊C for 90 sec as described previously in Molecular Cloning-A Laboratory Manual (Sambrook and Russell,2001). Bacterial transformants were selected on LB agarmediumcontaining appropriate antibiotics. Transformants obtainedwere colony purified on LB plates containing antibiotics.Presence of the desired insertwas first verified by colony PCR followed by PCRusing extracted plasmid DNA as template
    108. Bacterial transformation
    109. suspension was kept on ice for 10 min and 50 μl volume was aliquoted to chilled sterile microcentrifuge tubes. Cellswere immediately snap-frozen in liquid nitrogen and stored at -80 ̊C
    110. A single colony of E.coli DH5-α strain was inoculated in 10ml LB medium and incubated at37 ̊C for overnight. 4 ml of thisovernight culture was inoculated in 2 lt SOB medium and incubated at 18 ̊C till theOD600reaches to 0.5. Cells were harvested by centrifugation at 2,500 g for 10 min at 4 ̊C and washed gently in 80 ml ice-cold Inoue transformation buffer. Cells were collectedby centrifugation at 2,500 g for 10 min at 4 ̊C and gently resuspended in 20 mlice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. Cell
    111. E. coli DH5α ultra-competent cells preparation
    112. All experiments in this studywere performed with log-phase cellsunless otherwise mentioned. For obtaining log-phase cells, overnight YNB-or YPD medium-grown yeast cellswerere-inoculated in fresh YNB or YPD medium to an initial OD600of 0.1-0.2.Cells were incubated at 30 ̊C with shaking at 200 rpmtill the OD600reached to 0.4-0.6 OD. After incubation, log-phase cellswere collected bycentrifugation at 4,000 rpm for 3 min,washed once with the same medium and usedforfurtheranalysis
    113. Cultivation of logarithmic-phase cell culture
    114. C. glabratastrains were grown overnighteither in YPDor YNBliquid mediumat 30 ̊C with shaking at 200 rpm. Cells were harvested and suspended in 1X PBS to a final OD600of 1.0.Five 10-fold serial dilutions of cell suspension wereprepared in PBS and3-4μlwasspotted on YPD/YNBplates containing various test compoundsusing a multi-channel pipette.Plates were incubated at 30 ̊C and growth profileswererecorded after2-4days
    115. Serial dilution spot assay
    116. Yeast cell viability was measured by plating appropriate dilutions of cell cultureonYPD plates at various time intervalsduringgrowth.Cell suspension was diluted in1X PBS. YPD plates were incubated at 30 ̊C for 2-3 daysand total colony forming units(CFUs)were calculated by counting the number of coloniesthat appeared onYPDplatesand dividing that number by anappropriate dilution factor
    117. Yeast cell viability assessment viacolony forming unit (CFU) assay
    118. preparedin appropriate solvents, sterilizedby autoclaving or filtrationand stored at appropriate temperature
    119. For growth analysisof C. glabratastrains, a single colony from YPD or YNBagar mediumwas inoculated in appropriate liquid medium and incubated at 30 ̊C with shaking at 200 rpmfor 14-16 h. This overnight grown culture was used toinoculatetest medium to an initial OD600of 0.1to 0.3.Optical density/Absorbance of the cell suspensionwas measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) at600nmat regular time-intervals up to a period of 96 h.Absorbance values were plotted with respect to time. Generation time of yeast strains wascalculated fromthe logarithmic (log) phase of cellgrowth. Growth profilesbetween 4 (t1)and 8 h(t2)time interval wereconsideredfor calculationof generation time usingfollowing formula. Generationtime(G)= (t2-t1) x {log (2)/ [log (Bf/Bi)]}G= Generation time in ht1=Initial timepoint taken for analysist2 = Final timepoint taken for analysisBf= Number of cells at time t2(calculated on the basis of OD600values, wherein1 OD600of C. glabratacorresponds to 2 X 107cells.)Bi= Number of cells at time t1(calculatedas mentioned above)Severalyeast strains used in this study were analysed for their susceptibility to variouschemical compounds,drugsand metal ions. For this purpose, stock solutions were
    120. Growth assayand measurementof generation time
    121. mM final concentration) and pH was adjustedto the desired valueby addition of HCl or NaOH. Medium was sterilized by autoclaving.YNBagar plates ofdifferent pHwereprepared by mixing equal volume of separately autoclaved 4% bacto-agar solution and2X varied pH-adjusted-YNB liquidmedium.All routine sterilization of mediumand solutionswas either carried outby autoclaving at 121 ̊C for 15-20 minat highpressure condition(15 psi)or filtration with 0.2 μmpolyvinylidene fluoride(PVDF) membranefilter unit (Millex®-GV, Millipore).Both yeast and bacterial strains were stored as frozen 15% glycerol stock at -80 ̊Cfor extendedlifetime
    122. C.glabratastrains were maintainedeither on rich YPDor synthetically-defined YNB medium. C.glabratacells were routinely culturedat 30 ̊Cwith shaking at 200 revolutions per min(rpm)unless otherwise mentioned. Forgrowthexperiments, C. glabratastrains were freshly revived on YPDmediumfrom glycerol stocks.Escherichia coliDH5α bacterial strainwasused for plasmid transformation and propagationpurposes and maintained on LB medium.E.coliBW23473 bacterialstrainwas used to rescue Tn7transposon cassette from C. glabrataTn7insertion mutantsand maintained on LB medium. Bacterial strainsharboring plasmids were maintained on LBagar plates supplemented withappropriate antibiotics.For plasmid isolationpurpose,bacterial strains were grown overnight in liquid LB brothcontainingappropriate antibiotics at 37 ̊C with shaking at 200 rpm. Forpreparation of the solid medium, 2%bacto-agar was added to the mediumand autoclaved. To prepare medium of different pH, YNB mediumwas either buffered with citrateor HEPESbuffer (100
    123. Strainsand culture conditions
    124. Microbiological techniques
  2. sg.inflibnet.ac.in sg.inflibnet.ac.in
    1. activated TLC silicagel-60plate and transferred to theTLC chamber. After the solvent had migratedupwards (1.5 cm fromthetop), TLC plate was removed, air dried behind perspex shield, wrapped with cling plastic wrap and was exposed to phophorimager screenfor 2 h. Phosphorimager screen was scanned usingaFugi-FLA 9000 scanner
    2. To resolvephospholipids,a TLC chamber was prepared by pouring50 ml developing solution and sealing the chamber with aluminium foil so that developing solution can generate vapor. TLC silicagel-60plate(Merck)was incubated at 80ºCfor 4 h for activation. After 30 min of TLC chamber preparation, phospholipidsextracted from C. glabratacells werespotted at thebottom (1.5 cm fromthelowerend) of the
    3. Seperation of phospholipids by thin layer chromatography(TLC)
    4. PI-3kinase reaction was set up ina total volume of50μlin a 1.5 ml microcentrifuge tube as described below.PI-3 kinase reaction buffer = 25 μlSpheroplast lysate = 20 μl (equivalent to 10 μg protein)Sonicated phosphatidyl inositol = 5 μlReaction mix was incubated at 25ºC for 20 min and enzyme reaction was stopped by adding 80μlHCL (1N) solution. To extract phospholipids, 160 μl chloroform:methanol (1:1) was added to the reaction mix withcontinuous mixing. Organic phase containing phopholipidswas separated fromaqueous phase by centrifugation at 7,500g for 4 min at 4ºC and transferred to a new vial. Using vacuum evaporator apparatus, solvent was evaporated and phospholipidsweredissolved in 10 μl chloroform
    5. PI-3 kinase reaction set up and phopsholipid extraction
    6. 10 mg phosphatidylinositol-sodium salt(from Glycine max)was dissolved in 2 ml chloroform to prepare a 5 mg/ml stock solution. This solution was prepared in a small glass vial aschloroformis known to reactwith polypropylene. Small aliquots of stock solution were madeand stored at -20ºC till further use. To avoid spillage due to vapor pressure, vials containing phosphatidylinositol-sodium salt solutionwereopened very carefully.To prepare sonicated phosphatidylinositolfor one PI-3 kinase reaction, 2 μlof the stock phosphatidylinositolsolution (10 μg) wastransferredtoanew1.5 ml microcentrifuge tube. Using vacuum evaporator apparatus, chloroformwas evaporated from the solution and phosphatidylinositol-sodium saltwas resuspended in 5 μl sonication buffer.For sonication, a total of 20 pulses, each of 30 sec with30 sec resting time weregiven on ice
    7. Preparation and sonication of phosphatidylinositol-sodium salt solution
    8. A single colonyof desired C. glabratastrainwas inoculated in YPD-liquid mediumand grown for 14-16 h. 50 μl overnight culture was inoculated inYPD-liquid mediumfor 4 h. Log-phase-grownyeast cells were harvested,washedwith PBSandwereinoculated atinitial OD600of 2 and 4,into YNB-dextrose and YNB-sodium acetate liquid medium,respectively.After 4 hincubation,yeast cells were harvested by centrifugation at 2,500g for 5 minand treated with 1.2 M zymolyasefor 1 hto obtain spheroplasts.Post zymolyase treatment, spheroplasts were resuspended in 100 μl resuspension bufferandanequal amount of 0.25 mm glass beadswasadded to lyse the spheroplasts. Using bead beater apparatus, spheroplasts were lysed and protein concentration in spheroplast lysateswas determined usingbicinchoninic acid assay (BCA) method and samples were stored at -20ºC till further use
    9. Preparation of cell lysate
    10. In vitroPI-3 kinase reactions wereset up to measure PI-3P synthesized as described earlier(Whitman et al., 1988)
    11. Phosphatidyl inositol-3 kinase (PI-3 kinase assay)
    12. Colony blot assay was performed to analyse secretion of carboxypeptidase Y(CPY)as described previously (Roberts et al., 1991). Single colony of a C. glabratastrain was inoculated in YPD medium andculture was grown till stationary phase. 0.1 OD600equivalent cellsfrom this culture were spotted on CAA medium,overlaidwith a nitrocellulose membrane and plate was incubated at30 ̊C for 18-20 h.Afterincubation, nitrocellulose membranewas washed with water to remove cells and membrane-bound CPYwas detected by immunoblotting with polyclonal anti-CPY antibody at a dilution of 1:10,000
    13. Colonyblot assay
    14. was washed three times with PBS to remove non-adherantC. glabratacellsand Lec-2 cells were lysed in 5% SDS. Lysates were transferred totubes containing scintillation fluidand radioactive counts obtained were considered as ‘output values’. Percentage adherence wasdetermined using following formula
    15. Adherence of C. glabratacells toLec-2 epithelial cells wasmeasured as described previously(Cormack et al., 1999).Lec2cells were seeded ina 24-well tissue culture plate at a seeding density of 5X105cells per well and allowed to adhere for 12 h. After 12 h,medium supernatant was discarded by inverting the plate in a reservoir and cells were washed thrice with PBS. Lec2 cells were fixed in 3.7% para-formaldehyde for 15 minfollowed by 2 PBS washes. PBS containing antibiotics, penicillin and streptomycin,was added toeach well of the 24-well plate and Lec-2 cellswere stored at 4°C.For adherence measurement,strains were taken out either on YPD or CAA mediumandgrown at 30°C for 2 days. Single colony of a C. glabratastrain wasinoculated in 10 ml CAA medium ina 100 ml culture flaskand allowed to grow at 30°C for 16-20 h. 100 μlyeast culture wasreinoculated in fresh 5 ml CAA liquid medium in a 15 ml polypropylene tube. 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN labelmix(JONAKI, India) was added to thetube and cultures were grown at 30°C for 16-20 h for radiolabeling of C. glabratacells. C. glabratacells from 1 ml culture were harvested and washed threetimes with PBS to remove residual S35(Met:Cys-65:25) labeling mix from medium supernatant. Next,cells were resuspended in 1 ml PBS. OD600was measured and cell suspensions of 0.5 OD600were prepared. PBS was aspirated out of the wells of 24-well plate containing fixed Lec-2 cells. 200 μl of S35(Met:Cys-65:25)-labeled C. glabratacell suspensions were added to each well. To determine the total amount of radioactivity present in labeled C. glabratacell suspension, 200 μl of S35(Met:Cys-65:25)-labeledC. glabratacell suspensions were transferred to a scintillation vial containing scintillation fluid. Radioactive counts present in this fraction were considered as ‘input values’. For measurement of yeast adherence to Lec-2 cells, plates were centrifuged at 1,000g for 5 min and incubated for 30 min at room temperature. Following incubation, each wel
    16. Adherence assay
    17. 20 mg protein samples, isolated from RPMI-grown and macrophage-internalized yeast, were usedto measure KDAC activityusing HDAC Fluorimetric Assay/Drug Discovery Kit (EnzoLifeScience) as per manufacturer’s instructions
    18. Lysine deacetylase (KDAC) activity measurement
    19. For protein extraction, yeast cells were suspended in 50-100μl protein extraction buffer containing 320 mM (NH4)2SO4, 200 mM Tris-Cl (pH 8), 20 mM EDTA (pH 8), 10 mM EGTA (pH 8), 5 mM MgCl2, 1 mMDTT, 10% glycerol and protease inhibitorsand disrupted using glass beads.Cell lysate was centrifuged at 7,500g and4oC for 15 min. 30 μg of total protein was resolved on a 15% SDS-PAGE gelat 32 mA till the dye front reachedthe bottom. Resolved proteins were transferred to Hybond-P membrane at 350 mA for 1.5 h in the cold room.Transfer of the proteins was visually confirmed by examining marker’s lane and membranes wereincubated in a small box for 2 h in 5% fat free milkprepared in 1X TBST for blocking. Blocking solutions were discarded and primary antibody, appropriately diluted in 5% fat free milkprepared in 1X TBST,was added to the box containing membrane. After overnight incubation in primary antibody, membranes were washed thrice with 1X TBST for 10 min. Membranes wereincubated for 2 h inappropriate secondary antibodydiluted in 5% fat free milkprepared in 1X TBST. Blots were either developedby chemiluminescence based ECL-Plus western detection system orChemidocTMgel imagingsystem. CgGapdhwas used as a loading control. To exclude the possibility of any contribution of THP-1 proteins tocell extracts prepared frommacrophage-internalized yeast, two control experiments wereperformed. First, we probedthe blots with antibodies specific for mammalian tubulin and actin.As expected, we neitherdetectedanysignal for mammalian actin nor formammalian tubulin. In the second control experiment, we treated macrophage lysates with proteinase-K prior to the yeast pellet disruptionand probed yeast lysates for different histone modifications.This proteinase-K treatmentdid not alter the epigenetic signature of C. glabratacells.Together, these data indicate that yeast protein samples were devoid ofany mammalian protein contamination
    20. Protein extraction and immunoblotting
    21. homogenizedin 1 ml PBS and fungal burden was assessed by plating appropriate dilutions of tissue homogenate on YPD plates containing penicillin and streptomycinantibiotics (100units/mlpenicillin and 100μg/mlstreptomycin). All mice experiments were repeated twice with a set of 7-8 mice per strain in each experiment
    22. Experiments involving mice were conducted at VIMTA Labs, Hyderabad.100 l YPD-grown C.glabratacellsuspension(4 X 107cells)was injected into female BALB/c mice (6-8 weeks old) through tail vein. Seven dayspost infection, mice weresacrificedand kidneys, liver,spleenand brainwere harvested. Organs were
    23. Mouse infection assay
    24. Experiments involving mice were conducted at VIMTA Labs Limited, Hyderabad in strict accordance withguidelines of The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the Vimta Labs Ltd. (IAEC protocol approval number: PCD/OS/05). Procedures used in this protocol were designed to minimizeanimalsuffering
    25. Ethics statement
    26. Othermethods
    27. CgRTT107(3.3 kb),CgRTT109(1.3 kb),CgVPS15(3.4kb) and CgVPS34(2.4kb) ORFs were PCR amplified from genomic DNA of the wild-type strain using high fidelity Platinum Pfx DNA polymerase with primers carrying restriction sites for SmaI-SalI,BamHI-SalI,XmaI-XhoIandSalI-XmaI,respectively.Amplified fragments werecloneddownstream of the PGK1promoterin the pGRB2.2 plasmid. Clones were verified by bacterial colony PCR, sequencing and complementation analysis
    28. Cloningof C. glabrataORFs
    29. After restriction enzyme digestion, digested products wereresolved on agarose gels and desired DNA fragmentswereextracted from the gel. Concentration of gel-extracted DNA fragments was determined usingspectrophotometerand ligation reactions were set up using a molar ratio of vector to insert of 1:3 and 1:1 for sticky and blunt end ligations, respectively. Ligation mixwas incubatedeither at 22ºC for 4 hor at 16°Cfor 14-16 h. After incubation,T4DNA ligase was inactivatedat 65ºC for 20 min
    30. Ligation
    31. QIAGEN QIAquick PCR purification kit containing buffers, spin columns and collection tubes wasused topurify DNA fragments from PCR andenzymatic digestion reactions as per the kit manufacturer’s instructions
    32. Purification of restriction enzyme-digestedand PCR amplifiedproducts
    33. QIAGEN QIAquick Gel extraction kit containing required buffers, spin columns and collection tubes was used to extract and purify DNA from agarose gels. Digested DNA sample was resolved on 1-1.2% agarose gel and gel piece containing desired fragment was cut ona UV-transilluminator. DNA fragment was purified as per the kit manufacturer’s instructions
    34. Gel extraction of DNA
    35. C. glabratacells grown either in RPMI medium or harvested from THP-1 macrophages were collected, washed with DEPC treated water and were disrupted with glass beads in trizol. Total RNA was isolated using acid phenol extraction method and frozen at -80C. Quality of RNA was examined by determiningtheRNAintegrity number (RIN) before microarrayanalysis.Microarray experiments wereperformed atOcimum Biosolutions Ltd., Hyderabad (http://www.ocimumbio.com). Briefly, a4x44K GE Agilent array comprised of 10,408 probes representing 5,205 ORFs of C. glabratawas used wherein average number of replicates for each probe was four to five. Feature Extraction software version 10.7.3.1. (Agilent) and Quantile normalization was used for data analysis. Hierarchical clustering was performed using Complete Linkage methodwith Euclidean Distance as distance measure. Data arethe average of two hybridizations from biological replicates ofeach sample and raw data sets for this study areavailable at the Gene Expression Omnibus database(Accession number -GSE38953)
    36. Microarray Analysis
    37. To preclude the possibility of human RNA contamination, cDNA prepared from internalized yeast was examined for the presence of human transcripts encoding Ccl5 and histone H3. However,no amplification forhuman genes was observed, thus, eliminating any possiblecontamination of THP-1 RNA with yeast RNA
    38. Primersfor real-timePCR analysisweredesigned using Primer3 plus software and are listed in Table 4. To extractRNA from macrophage-ingested C. glabratacells, infected THP-1cells were washed twice with PBS and lysed in 1 ml ice-cold water. Lysate was centrifuged followed by two quick washes with DEPC-treated water andwashed yeast cell pellets were frozen on dry ice.For RNA extraction, yeast cells were disrupted with glass beads in trizol and total RNAwas isolated usingacid phenol extraction method described above.Optimal primer and cDNA concentrations were standardizedand qRT-PCR was performedusing ABI 7500 Fast Real-Time PCR System (Applied Biosystems).In brief, 0.5 μl cDNA,0.1 to 0.2picomoles of gene specific primers and 10 μl 2X MESA GREENqPCR™ Mastermix Plus containing SYBR green dye (Eurogentec)were mixed in thewellsof a 96-well PCRplate (Axygen). Final reaction volume was adjusted to 20 μl with DEPC-treated water. Transcript levels were quantified with an end-point value known as Ct(cyclethreshold). Expression of TDH3, which encodes CgGapdh,was used asaninternal control. The Ct defines the number of PCR cycles required for the fluorescent signalof SYBR green dye to cross beyondthe background level.Fold-change in transcript expression was determined usingfollowing formula.Fold change in expression = 2-ΔΔCtΔΔCt= ΔCttreated -ΔCtuntreatedΔCttreated = Ctvalue forthe gene of interest under treated condition -Ctvalue forthe internal control gene (TDH3) under treated conditionΔCt untreated = Ctvalue forthegene of interest under untreated condition -Ctvalue forthe internal control (TDH3) gene under untreated condition
    39. Quantitative real-timePCR
    40. Complementary-DNA synthesis was doneusing reverse transcriptase enzyme and oligo-dT primers. For this, 1 μg good quality RNA was treated with1μl(1 unit) DNase I for 15 min to remove DNA contamination. Next, SuperScript III First-Strand Synthesis System kit (Invitrogen) was used to synthesize cDNA according to the manufacturer’s instructions. cDNA synthesized was stored at -20 ̊C till further use
    41. Synthesis of complementary DNA (cDNA)
    42. For RNA experiments, all solutions were prepared in RNase free diethylpyrocarbonate (DEPC) water. Microcentrifuge tubes and tips used for RNAworkwere autoclaved twice and driedat 70 ̊C for overnight before use. Non-autoclavable plastic items were wiped with Ambion RNAseZap to remove RNAse contamination, if any. RNA was extracted from C.glabrata cells usingacid phenol extraction method. C. glabratacells were harvested at 2,500g for 5 minat 4 ̊C, resuspended in 1 ml ice-cold DEPC water and were transferred toa2 ml microcentrifuge tube. Cells were spun down at 6,000g for 3 minat 4 ̊C and resuspended in 350 μl AE solution. Next, 50 μl SDS and 400 μl acid phenol(pH 4.5)solutions were added to thetube and mixed well by vortexing. The tube wasincubated at 65 ̊C for 15 min with continuous mixing. After incubation, tube was kept on ice for 5 minand centrifuged at 7,500g for 5 minat 4 ̊C. Aqueous phase was transferred toa new 1.5 ml microcentrifuge tube and RNAwasextracted with an equal volume of chloroform. Total RNA was precipitated at room temperature with 1/10thvolume of 3 M sodium acetate (pH 5.2) and 2.5 volumesof chilledabsolute ethanol for 20 min. Precipitated RNA was collected by centrifugation at 7,500g for 5 minat 4 ̊C. RNA pellet was washed with chilled 70% ethanol and resuspended in 50 μl nuclease-free water. RNA concentration was determined by measuring absorbance at 260 nm. Quality of extracted RNA was examined by gel electrophoresis on 0.8% agarose gel prepared in DEPC-treated TAE buffer
    43. RNA extraction
    44. Nucleosomal-associated DNA was extracted from RPMI-grownand macrophage-internalized C. glabratacells using EZ NucleosomalDNA prep kit (ZYMO Research),treated withmicrococcal nuclease digestionfor 2.5, 5, 7.5 and 10 min at 25ºC andwasresolved on 2% agarose gel
    45. Micrococcal nuclease digestion assay
    46. temperature. Genomic DNA pellet was dissolvedeitherin 50 μl 0.1X TE or molecular biology grade water containing 0.3 μlAmbion RNAse cocktail and incubated at 37ºC for 30 min.After RNA digestion, 100 μl of 0.1X TE or nuclease-free water was added to the tube and stored at -20ºC. Quality of extracted genomic DNA was checkedon 0.6% agarosegel by electrophoresis
    47. Desired C. glabratastrain wasgrownovernight in YPD liquid medium and yeast cells were harvested by centrifugation at 2,500g in 15 ml polypropylene tube.Yeast cells were washed with PBS, resuspendedin 500μl lysis buffer (Buffer A) andwere transferred toa2ml microcentrifuge tube. Yeast cells were incubated for 15 minon a thermomixer set at 65 ̊C and 750 rpm. After incubation, 0.5 gm glass beads (0.5 mm) and 500 μl PCI solution were added to thetube. Yeast cells were lysedthree times for 45 seconds each on a bead beating apparatus with intermittent cooling on ice to prevent overheating. Cell lysates were centrifugedat 7,500gfor 5 minandupperaqueous phase (300-350 μl) wastransferred carefully to a new 1.5 ml microcentrifuge tube. 1 mlabsolute ethanol was added andmixedwellby inverting the tube3-4 times. To precipitate genomic DNA, suspension was centrifuged at 7,500g for 10min.Precipitated genomic DNA was washedwith 70% ethanolanddried at room
    48. Genomic DNA isolationby glass bead lysis method
    49. C. glabrata cells were grown overnight in 10 ml YPD liquid medium. Cells were harvested at 2,500g for 5 min, resuspended in 400 μl Buffer A (50 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 1% SDS) and were transferred to a 2 ml microcentrifuge tube. Equal volume of phenol-chloroform-isoamyl alcohol (PCI) solution was added tocell suspension and tubes were vortexed for 2-3 min. After incubation at 42 ̊C for 30 minon a thermomixer set at 800 rpm (Eppendorf), cell debris was removed by centrifugation at 7,500g for 10 minand aqueous phase (300-350 μl) was carefully transferred to a new 2 ml microcentrifuge tube. Genomic DNA was precipitated with800 μl chilled absolute ethanol and 35 μl sodium acetate (3 M, pH 5.2). DNA pellet was washed with chilled 70% ethanol and dried at room temperature for 5-10 min. Genomic DNA pellet was dissolved either in 50 μl 0.1X TE or molecular biology grade water containing 0.3 μl Ambion RNase cocktail and incubated at 37 ̊C for 30 minfor digestionof RNA. After RNA degradation, 100 μl of 0.1X TE or nuclease-free water was added to the tube and stored at -20ºC. Quality of extracted genomic DNA was checkedon 0.6% agarosegel by electrophoresis
    50. Genomic DNA isolationby quick genomic DNA extraction method
    51. Stripping of membranes in buffer containing 0.4 M NaCl yielded slightly better results. Hybond membranes were reused for 5-10 times after stripping
    52. Radiolabeled-bound probes were stripped from the membrane by boiling in 1% SDS containing 0.1X SSC for 15 min. Alternatively, membraneswereincubatedtwicein stripping solution (0.4 M NaOH)at 45°C for 30 minto remove the bound probes
    53. Stripping of probes from hybridized membranes
    54. After 2-3 h exposure,phosphorimagerscreenwas scannedon Fuji FLA-9000 to acquire hybridization images. Next,signal intensity for each spot on the membrane for both input and outputsampleswas quantifiedusing Fuji Multi Gauge V3.0 software andpercentage intensity foreach spot relative tothe whole signal intensity ofthe membranewas determined.To identify mutants with altered survival profiles,ratio of output (Op) to input (Ip) signal for each spot (oligonucleotide tag)present on the membranewas calculated.Mutantsdisplaying at least 6-fold higher and 10-fold lower survival were selectedas “up’ (Op/Ip= 6.0) and ‘down’ (Op/Ip = 0.1) mutants, respectively
    55. Data analysis
    56. After14-16 hincubation, hybridization buffer was decanted to a radioactive liquid waste container.Membraneswere washedtwice with 2X SSC (saline-sodium citrate) containing 0.1% SDS for 15 min at 55°C followed by two washes with 1X SSC containing 0.1% SDS for 15 min at room temperature. Post washes,membranes were rinsed with 1XSSC buffer at room temperature and exposed to phosphorimager screen for 2-3 h
    57. Post-hybridization washes
    58. Primers (OgRK 25 and OgRK 26, 200 pM ) –1 μl, eachTaq DNA polymerase –0.5 μlαP32-dCTP( specific activity-3,000 Ci/mMol)–2.5 μlWater –38.5 μlαP32-dCTPlabeled PCR productswere denatured by incubatingtubesat100°C for 5 min followed by immediatechilling on ice. After 2 h prehybridization, radiolabeleddenatured input and output probes were added to respective bottles and bottles were transferred to a hybridization oven set at 42ºC and 6 rpm
    59. Two Hybond-N membranes,each carrying 96 immobilized DNA with unique signature sequences,were transferred to 15 cm long hybridization bottlesandlabeledas input and output. 10 mlprehybridization buffer was added to each bottle and bottles were transferred to a hybridization oven setat 42ºC and 6 rpm. To prepare input and output probes,genomic DNAsfrom input and output C. glabratacell pelletswere extracted using glass bead lysismethodandunique signature tags were PCR amplifiedwith nucleotide mix containing αP32-dCTP using primerscomplementary to the invariant region flanking each unique oligonucleotide sequence.Following is the composition of50 μlPCR cocktail used to prepare radiolabeledinput and output probes.10X PCR buffer–5 μldNTP mix (without dCTP, 2mM) –0.5 μldCTP (0.05 mM) –1 μl
    60. Southern hybridization
    61. E. colistrains containing plasmids with unique oligonucleotide signature sequences were inoculated in LBmedium containing ampicillin and grown overnight at 37°Cand 200 rpm. Plasmids were extracted, quantitated anddenatured in alkaline denaturing solution. Approximately, 200 ng of each plasmid DNA was transferred to theHybond-Nmembraneusing96-well Dot Blot apparatus. Membranes were neutralized in 2X SSC and denatured plasmids were cross-linked to Hybond-N membranes usingUV cross linker
    62. Membrane preparation
    63. E. colistrains carrying plasmids were inoculated and grown overnight at 37ºC and 200 rpm in LB-liquid medium supplemented with either 50 μg/ml ampicillinor 30 μg/ml kanamycin. Cells were harvested by centrifugation at 2,500g for 5 min. Plasmids were extracted using Qiagen plasmid miniprep kit following the manufacturer’s instructions. Concentration of the extracted plasmid DNAs was measured using spectrophotometerat 280 nmandstored at -20ºC
    64. Plasmid DNA purification
    65. Molecular biology methods
    66. Genomic mapping of disrupted locusin Tn7insertion mutants was carried out as describedpreviously(Kaur et al., 2004).C. glabratamutants carrying Tn7insertionswere grown in YPD-liquid medium and genomic DNA was isolated fromovernight cultures. 10 μg genomic DNA was digested either with restriction enzyme MfeIor SpeI.Restriction enzyme-digestedDNA was precipitated with 1 ml ethanol and 1/10thvolume of sodiumacetate (3 M,pH 5.2). DNA pellet was washed twice with ice-cold 70% ethanol, air driedand was resuspended in sterilewater. DNA was recircularized with T4 DNA ligase.Resultant circular DNA carriedTn7cassette flanked on bothsidesby the disrupted locus oftheC. glabratagenome. CircularDNA wastransformed in E. coliBW23473 strainwhich contains protein Π (the product of the pirgene) required by R6Kγorifor replication.Twoverified transformants were grown overnight in LB-kanamycin medium and plasmids were extracted. Purified plasmids were sequenced withprimers reading outwards (OgRK 183 and OgRK 184) from both ends ofTn7cassette.Sequences obtained were compared,usingBLAST,against C. glabratagenome sequence database and regionsof Tn7insertions in C. glabratawere mapped
    67. Mutant rescue
    68. E. coli BW23473 electro-competent cell aliquots were taken out from -70ºC freezer, thawed on ice and were mixed with 1-2 lplasmid DNA. Mixture was pulsed with the Gene Pulser® electroporation apparatus (Bio-Rad),set at 1800 Volts, 25 μF and 200 Ω,in a chilled 0.1 cm electroporation cuvette. After electric pulse, 1 ml LB medium was immediately added to the cuvette and suspension was transferred to a 1.5 ml sterile microcentrifuge tube. Cells were incubatedat 37°C and 200 rpm for 1 h, centrifuged and were plated on LB-agar plates containing kanamycin (30 μg/ml). Transformants were colony purifiedon LB-kanamycin plates. Positive clones were verified by colony PCR and inoculated in LB-liquid medium containing kanamycin (30 μg/ml) for plasmid isolation
    69. Transformation of E. coliBW23473 cells by electroporation
    70. To prepareelectrocompetent cells, a single colony of E. coli BW23473 strain from a freshly-streaked LB agar plate was inoculated in 50 ml LB liquid medium. Culture was incubated at 37°C for 14 h with continuous shaking at 200 rpm. 25 mlovernight-grown E. coliBW23473 culture was transferred to 500 ml LB liquid medium and incubated at 37°C till the OD600 reached to 0.4. Post incubation, cultures were transferredto ice and centrifuged at 1,000g for 15 minat 4°C. Cells were washed twice with 500 ml ice-cold sterile water, three times with 250 ml ice-cold 10% glycerol solution and resuspended in 1 ml 10% glycerol solution. After absorbance measurement, cell suspension was normalized to final cell density of 3X1010cells/ml and dispensed in 50 μl aliquots to sterile ice-cold 1.5 ml microcentrifuge tubes. Aliquots were snap frozen in liquid nitrogen and stored at -70ºC
    71. Preparation of E. coliBW23473electrocompetent cells
    72. E. coliDH5α strain was transformed with plasmids carrying appropriate inserts to clone and generatedeletion strains of C. glabrataORFs(Sambrook, 2001). Ultracompetentcells stored at -70⁰C were thawed on icefor 5-10 min. 5 μlligated plasmid was added to100 μlultracompetent cells andcells were incubatedon ice. After 30 min, competent cells were subjected to heat shock at 42⁰C for 90 seconds. Cells were immediately transferredtoicefor 2-3min. Next, 800 μlSOC (or LB) medium was added and cells were allowed to recover for 45 minon a shaker incubator set at 37⁰C.After the recovery, cells were centrifuged at 2,500g for 4 min. Medium supernatant was discarded and cells were resuspended in 200 μlfresh sterile LBmedium. Cells were plated on LB agar medium containing appropriate antibiotics. Plates wereincubatedat37⁰C for 12-16 h
    73. Bacterial transformation
    74. 10 min at 4 ̊C and gently resuspended in 20 ml ice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. Cell suspension was kept on ice for 10 min and 50 μl volume was aliquoted to chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80 ̊C
    75. A single colony of E. coli DH5α strain was inoculated in 10 ml LB medium and incubated at 37 ̊C for overnight. 4 ml overnight culture was inoculated in 2 lt SOB medium and incubated at 18 ̊C till the OD600 reached to 0.5. Cultures were centrifugedat 2,500 g for 10 min at 4 ̊C and harvested cells were washed gently with80 ml ice-cold Inoue transformation buffer. Cells were collected by centrifugation at 2,500 g for
    76. Preparation ofE. coli DH5α ultracompetent cells