Salineextracts(0.89%)oftissueswerepreparedinTeflonglasshomogenizer.ThecarbohydratecontentoftheextractwasdoneaccordingtothemethodofShibkoetal.(1967)

Carbohydrates

HaemoglobinwasdeterminedbySahlimethod.HaemoglobinisconvertedtoacidhaematinbytheactionofHC1.Theacidhaematinsolutionisfurtherdilutedwiththeaciduntilitscolormatchesexactlythatofthepermanentstandardofthecomparatorblock.TheHbconcentrationisreaddirectlyfromthecalibrationcurve.

Haemoglobin(Hb)determination

Theexperimentalfishwasacclimatedtoglassrespirometersforabout24hrandtheywerenotgivenanyfoodduringthisperiod.TheeffluentexposedfishesalongwiththecontrolsweresubjectedtoO2consumptionseparately.Theexperimentswereperformedinaninsulatedroombetween8to10AMwithlightson.TherateoftotalO2uptakethroughgillsfromflowingwaters(DO=7.2mg021'1)wasmeasuredinfishesofdifferent body weights.Forthis,acylindricalglassrespirometerof2litrecapacitywasused.Thefishwasintroducedintherespirometerwhichwasconnectedtoalargeconstantlevelwatertanktomaintaintheflowofwaterunderconstanthydrostaticpressure.Thewaterenteredtherespirometeratonesideanditsflowperminutewasmeasuredasitlefttheotherside.Theflowwasadjustedaccordingtothesizeofthefish.Thefishwasacclimatizedtotherespirometeratleast12hrbeforereadingswere taken.ConcentrationofdissolvedoxygeninthesampleswasmeasuredbyWinkler’svolumetricmethod(Welch,1948).ThedifferenceinO?levelsbetweentheambientwaterandthatsuppliedtotherespirometeraswellaswiththerateofwaterflowandtheweightofthefishwasusedtocalculatetherateof O2uptakeintermsoftime(ml02hr'1)withthehelpoftheequation:V02=Vw(Ci02CE02)Where,VO2=02uptake(ml02hr'1)Vw=water(mlm'1)andCi02-CE02respectivelythe02concentrationofinletandoutletwaters.Arespirometercontainingnoanimalsservedasacontrolforadjustingcalculationsfor02uptakeinthewater.Uponremoval,fisheswereblottedwithpapertoweling, andweighed

Aquaticrespiration

Theywereheld(28°±0.5°C;naturalphotoperiod)in240litretapwatertanks(1x1x0.3m)andacclimatedtothelaboratoryconditionsforaweekbeforeexperimentation.Agentlecontinuouswaterflowwasmaintainedthroughtheaquariaforconstantwaterrenewal.

Holdingtanks

Percentage lethality was calculated as:100×((number of non-CyO/ number CyO)×100)

Flies were maintained at 18°C or 25°C as appropriate. Through out this thesis, flies defined as wild-type were yellow white of the genotype: y67c23w118. BEAF32 null lines BEAF32AB-KO/CyOGFP, kindly provided by Craig Hart, University of Illinois (Roy et al., 2007a). Homozygous BEAF32AB-KOlines were obtained by selection against the CyOGFPmarker at the 3rdinstar larvae stage, using a Leica M165 FC with a GFP filter. Lethality of the BEAF32AB-KOallele was assessed against the dppHr27hypersensitive allele (genotype: dppHr27,cn1,bw1/CyO P{dpp-P23}). For this embryos were collected from the following crosses as set up by Catherine Sutcliffe:BEAF32AB-KO/+ ×dppHr27,cn1,bw1/CyO P{dpp-P23}and+/+ ×dppHr27,cn1,bw1/CyO P{dpp-P23}

Fly Stocks and Crosses

eppendorf tube had DNA of interest. The purified DNA fragments were checked on an agarose gel, with an appropriate marker, before setting up the ligation reaction

The plasmid DNAs for cloning, were digested with the respective enzymes, checked on an appropriate percentage of agarose gel along with 100 bp ladder or .A Hind ill marker (Promega, USA) and the required fragments were eluted from the gel using the Qiagel Gel Extraction kit (Qiagen, U.K.). According to the manufacturer's directions, the area of the gel containing the DNA fragment was excised using a clean and sharp blade, minimizing the amount of surrounding agarose excised with the fragment. The gel slice was weighed and placed in a microfuge tube. Three volumes of Gel Solubilization Buffer (QG) was added for every one volume of gel. The gel piece was then vortexed and incubated at 500C for 10 min. The contents were mixed in between, by inverting the tube few times, to ensure gel dissolution. It was then centrifuged at 13,000 rpm for 1min. The flow through in the discard column was carefully removed. Then 500 p.l of buffer PB was added to remove the traces of gel and the tube was centrifuged at 13,000 rpm for 1 min. The flow through in the discard column was removed and 750 p.l of Wash Buffer (PE) (containing ethanol) was added and the tube was centrifuged at 13,000 rpm for 1 min. The flow through was discarded and another spin at maximum for 2 min was given to remove the traces of wash buffer. The column was then put on a fresh tube and finally, to elute the DNA, 40 p.l of Tris-EDTA buffer (TE) or RNase-DNase-free water was added and then centrifuged at 13,000 rpm for 1 min. The flow through in the

Gel elution of DNA fragments:

was used to amplify the oligonucleotides (Promega Biotech, Madison, USA). pGEMT-Easy and p-TARGET cloning vectors were also obtained from Promega. In vitro transcription was carried out using Riboprobe transcription system (Promega Biotech, Madison, U.S.A.). BCA protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, U.S.A.). Reverse transcription was carried out using lmProm-TI™ Reverse Transcriptase kit from Promega. Luciferase activity in the cell extracts was measured using Luciferase assay System (Promega Biotech., U.S.A.).

Qiaprep spin mini kit and Qiagen plasmid midi kit (West Sussex, U.K.) were used for isolation of DNA. Isolation of DNA fragments from gel was carried out using QiaGel extraction kits or PCR products were purified using nucleotide removal kit from Qiagen (West Sussex, U.K.). PCR core system I

Kits

1X PBS diluted in distilled water 1X fixative solution diluted in distilled water 2.4.12.3 Staining Solution25 μl Solution A 25 μl Solution B 25 μl Solution C 125 μl 20 mg/ml X-gal in DMF

Working Solutions:

Procedure:

β- galactosidase assay was performed in a 96 well format. Briefly, 4000-5000 cells were plated in 96 well tissue culture coated plate. Cells were transfected with reporter plasmid after 18 -24 hrs and after 48 hrs the cells were washed once with D-PBS. 50μl of lysis buffer was added to the well and cells were lysed by freezing plate at -70°C and thawing at 37°C. Cells were pipette up and down and then the plate was centrifuged at 9000 X g for 5 minutes. The supernatant from each plate was transferred to clean eppendorf tube. Immediately prior to assay the ONPG cocktail was prepared as below: 47 μl 0.1 M sodium phosphate (pH 7.5)22 μl 4 mg/ml ONPG1 μl 100X Mg solution30μl of each well extract was added to microtitre well plate and70μl of ONPG cocktail was added to each well. The plate was kept on ice throughout the procedure. After addition of ONPG cocktail the plate was transferred to 37°C and the development of colour was monitored every 10 minutes for development of color. After development of yellow colour, the reaction was stopped by addition of 150μl of 1M sodium carbonate to each well

ethanol has dried. The pellet was resuspended in 20 μl of milliQ water and 20 μg/ml RNase added. The tube was incubated at 50°C for 45 min. the tube was vortexed for few seconds. Quality of the plasmid DNA was then accessed by running 1% agarose gel.

Overnight Grown culture was pelleted by centrifugation at 10,000g at 4°C for 3 min and the supernatant was discarded. Pellet was resuspended in 250 μl of ice-cold alkaline lysis solution 1. 300 μl of alkaline solution 2 was then added and the tube was inverted gently 3-4 times and incubated at room temperature for 5 min. 350 μl of ice cold solution 3 was added and mixed by inverting the tube rapidly for 3 or 5 times. Suspension was incubated on ice for 10 min. Bacterial lysate was spun at 10,000g for 12 min at 4°C. Supernatant was transferred to a fresh tube. 0.4 volume of phenol: chloroform was added to the supernatant and the contents mixed. It was then spun at 10,000g at 4°C for 12 min. Aqueous phase was taken out in a fresh tube and 0.6 volume of isopropanol was added, mixed properly and incubated at room temperature for half an hour followed by spinning at 10,000g at RT for 20 min. Supernatant was discarded. Pellet was washed with 70% ethanol. The tube was stored at room temperature until the

Preparation of Plasmid DNA by alkaline lysis

250 mM KCl 55 mM MnCl2.4H20 50 x TAE (1 litre): 242 g of tris base 57.1 ml of glacial acetic acid 100 ml of 0.5 M EDTA Alkaline Lysis Solution 1: 50mM tris-HCl (pH 8.0) 10.0 mM EDTA 50 mM glucose Alkaline Lysis Solution 2: 0.2M NaOH 1% SDS Alkaline Lysis Solution 3: 3.0M Potassium acetate

2.7 mM KCl 10 mM Na2HPO42 mM KH2PO4Tris Buffer Saline (TBS) (10X): 12.1gm Trizma Base 40.0gm NaCl Adjust PH to 7.6; make up the volume to 1 lit with milli Q water. HEPES Buffer Saline: 20 mM HEPES (pH 7.5) 150 mM NaCl Blocking Buffer: 5% fat free milk or 2% BSA in PBST or TBST. Stripping Buffer: 100 mM β-mercaptoethanol 2% (w/v) SDS 62.5 mM Tris-HCl (pH6.7) Luria Broth: 10g tryptone 10g NaCl 5g yeast extract, make up the volume to 1 lit with water. TB buffer for preparation of competent cells: 10 mM PIPES (free acid) 15 mM CaCl2.2H20

2.5 ml of 1.5 M Tris-Cl (pH 8.8) 4.0 ml of 30% acrylamide; bisacrylamide (29:1) mix 50.0 μl of 20% SDS 3.35 ml of milli-Q water 100 μl of 10% APS 10.0 μl of TEMED. 2X SDS loading Buffer: 130 mM Tris-Cl (pH 8.0) 20% (v/v) glycerol 4.6% (w/v) SDS 0.02% bromophenol blue 2% DTT SDS PAGE Running Buffer: 25mM Tris base, 0.2M glycine 1% SDS Western Blot: 1 x Blotting Buffer (2Litres): 25mM tris base, 0.2M glycine 20% methanol Phosphate Buffer saline (PBS): 137 mM NaCl

Whole cell lysis buffer: 20mM Tris (PH 7.5) 150mM NaCl 1mM EDTA 1mM EGTA 1 % triton X 100 2.5mM sodium pyrophosphate 1mM β-glycerophosphate 1mM Na3VO41μg/ml aprotinin, 1μg/ml leupeptin and 1μ.ml pepstatin SDS-PAGE: Stacking Gel Mix (4ml, 5%): 380μl of 1M Tris-Cl (pH 6.8) 500μl of 30% acrylamide ; bisacrylamide (29:1) Mix 15 μl of 20% SDS 2.1 ml of milli-Q water 30 μl of 10% APS 5 μl of TEMED. 12% Resolving Gel Mix (10ml):

Buffers and Media

Monoclonal antibody against KRAS were purchased from Merck Research Laboratories, phospho p44/42 (ERK1/2)and total p44/42 (ERK1/2)antibodies were purchased from Cell Signaling Technologies. Anti tubulin antibody was obtained Sgima-Aldrich Chemicals. HRP conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Bangalore Genei Pvt. Ltd.

Antibodies

1μl of the cell lysate was mixed with 200 μl of 5X Bradford reagent and 800 μl of water. O.D was measured at 595 nm. Standard curve of BSA was plotted using various dilutions of BSA protein by Bradford method. Protein estimation of the cell lysate samples was performed using the standard curve equation y=0.0695x + 0.0329 μg/μl

Protein estimation by Bradford method

Overnight cell culture raised in LB medium was subcultured 1:100 in LB with 20 mM MgCl2. When the A600 reached 0.4-0.6, the culture was centrifuged at 2800g for 5 min at 4 ̊C. To the cell pellet 0.4 volumes of ice-cold TBF-I buffer was added and incubated on ice for 15 min. The cell suspension was centrifuged at 2800g for 5 min at 4 ̊C and the cells recovered were dissolved in 0.04 volume of ice-cold TBF-II buffer and kept on ice for 45 min. 100 μl aliquots of these competent cells were used for transformation using the normal transformation protocol

TBF method for preparation of high competency cells

Quiagen/HiPura following the manufacturer's protocols

The rapid alkaline lysis method of plasmid isolation, as described by Sambrook and Russel (2001), was followed with minor modifications. Bacterial pellet from 3 ml of stationary-phase culture was resuspended in 200 μl of ice-cold solution I (50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA pH 8.0 containing 1 mg/ml lysozyme) by vortexing. After 5 min incubation at room temperature, 400 μl of freshly prepared solution II (0.2 N NaOH, 1% SDS) was added and the contents were mixed, by gently inverting the tube several times. This was followed by the addition of 300 μl of ice-cold solution III (5 M potassium acetate, pH 4.8) and gentle mixing. The tube was incubated on ice for 5 min and centrifuged at 20,0000g for 15 min at 4°C. The clear supernatant was removed into a fresh tube and, if required, was extracted with an equal volume of phenol:chloroform mixture. The supernatant was precipitated with either two volumes of cold 95% ethanol or 0.6 volumes of isopropanol at room temperature for 30 min. The nucleic acids were pelleted by centrifugation, washed with 70% ethanol, vacuum dried, and dissolved in appropriate volume of TE buffer. If required, the sample was treated for 30 min with DNase free RNase at a final concentration of 20 μg/ml. The plasmid DNA was checked on a 0.8% agarose gel and stored at −20°. The plasmid DNA thus isolated was suitable for procedures such as restriction digestion, ligation, and preparation of radiolabeled probes. Plasmid isolation was also done with any of the commercially available kits from

Isolation of plasmid DNA

dependent transcription termination within the untranslated region of trpE. Anthranilate is a precursor of tryptophan, which is the product of trpE-encoded anthranilate synthase. Therefore, in trpE(fs) strains, growth on minimal glucose plates supplemented with anthranilate (100 μg/ml) reflects transcriptional polarity relief at the trp locus, and the same was scored after streaking the relevant strains on such medium

The trpE9777 is a frameshift (fs) mutationconfers Trp auxotrophy and also polarity on the downstream trpDCBA genes in the operon due to premature Rho-

trpE(fs) assay

This test was therefore used for two purposes: (i) to distinguish relA+ from relA− strains, and (ii) as a qualitative measure of transcriptional polarity relief at the ilv locus. Growth in the presence of amino acids Serine, Methionine, and Glycine (SMG) was scored on glucose-minimal A plates supplemented with each of the amino acids at 100 μg/ml and compared with the growth on non-supplemented glucose-minimal A plates to score for SMG phenotype

The E. coli relA mutants exhibit SMG-sensitive (SMGS) phenotype i.e. growth-inhibition in the presence of Serine, Methionine and Glycine at 1 mM concentration each (Uzan and Danchin, 1978) and is proposed to be a consequence of transcriptional polarity exerted by a frameshift mutation in the ilvG gene on the expression of downstream genes of the ilvGMEDA operon (Lopes et al., 1989). It was observed in another study that the rho and nusG mutants that are defective for transcription termination conferred SMG-resistant (SMGR) phenotype in a relA1 strain (Harinarayanan and Gowrishankar, 2003)

SMG resistance

Plasmids constructed in this study

The bacteriophage P1kc was from our laboratory collection and is referred to as P1 throughout this thesis. Phage λcI857 was also from our laboratory collection. Other bacteriophages that were used in this study included the following: (i) λNK1098 carries a Tn10 transposon with a tertracycline (Tet) ressistance marker. (ii) λNK1324 carries a mini-Tn10 transposon Tn10dCm with a chloramphenicol (Cm)-resistance marker, Cmr. The lambda phage vectors above (Kleckner et al., 1991) were used to make random transposon insertions in the chromosome either for the purpose of insertional mutagenesis or for tagging antibiotic resistance markers to point mutations

Bacteriophages

Western blotting by adding 2X sample buffer as described in 3.2.B.19 and 3.2.B.20 respectively

Triton X-114 has a property of a low cloud point (23°C). At temperatures above the cloud point, detergent solutions separate into aqueous and detergent-enriched phases thus separating membrane proteins (detergent phase) from cytosolic ones (aqueous phase). Promastigotes in the logarithmic phase of growth were harvested and dead cells pelleted at 129 x g. 109 live promastigotes were resuspended into 10mL of 0.5% vI v Triton X-114 containing protease inhibitor cocktail. The cells were homogenized using a 30-40 strokes of dounce homogenizer. The homogenate was incubated on ice for 90 min with intermittent stirring. Following this insoluble material was pelleted by centrifugation at 3700 x g for 35 min at 4°C. The supernatant was incubated at 37°C for 2 hr., till layers separated well. The top aqueous layer was separated from the bottom detergent layer. Samples of both these were prepared for SD

Triton X-114 extraction of membrane proteins

First strand synthesis of mRNA into e-DNA was performed using First strand e-DNA synthesis kit from Invitrogen (Carlsbad, CA) following manufacturer's protocol. Briefly, 4 !lg of total RNA was denatured at 65°C for 5 min in the presence of Oligo dT12-18 and dNTPs and then cooled on ice for 1 min. DTT, MgCb and RNaseOUT in lOX reverse transcriptase buffer added to the above mixture and incubated at 42°C for 2 min. 1!!L/reaction of the Superscript Reverse Transcriptase enzyme was added to the denatured RNA and incubated at 42°C for 50 min. The enzyme was denatured by heating at 70°C for 15 min. The reaction was completed with degradation of the complementary RNA strand by incubating with RNase H for 20 min at 37°C. The DNA preparation was stored at -20°C.

First strand synthesis by reverse transcription

Murine macrophage cell line J774A.1 (ATCC no. TIB-67) was maintained in phenol red free DMEM supplemented with 10% heat inactivated (45 min at 65°C) foetal bovine serum at 37°C in 5% C02 and 95% air. The cultures were sub-cultured every three days or at the attainment of 80 % confluency.

In vitro J774A.l murine macrophage cultur

and a colourless upper aqueous phase. The upper aqueous phase in which RNA existsexclusively, was transferred to a fresh microfuge tube and RNA was precipitated byadding 0.5 ml of isopropyl alcohol for each ml of Trizol used. Samples were incubatedat 15 to 30ºC for 10-min and centrifuged at 12000 rpm for 10-min at 4ºC. RNA formeda gel like precipitate at the bottom of the tube. Supernatant was removed and RNA waswashed with 75% ethanol (by adding 1 ml of ethanol per ml of Trizolemployed). RNAcould be stored after this step in –20 or –70ºC for more than a year. RNA pellet was airdried for 15-to 30-min following which it was dissolved in nuclease free water. Theconcentrations and purity of RNA samples were determined spectroscopically as wellas by visual inspection on formaldehyde-agarose gel in MOPS buffer (Goodet al., 1996). Before loading onto the gel, RNA was mixed with loading buffer and heated at90ºC for 3-min

For isolation of RNA, cells were grown in minimal A medium supplemented with 0.2%glucose upto A600of 0.6. Cells were harvested by centrifugation and total RNA wasisolated by using Trizol (Invitrogen) according to manufacturer’s instructions. 1 ml ofTrizol was used to lyse cells equivalent of approximately 4 ml of overnight culture.Homogeneous lysis was achieved by gentle pipetting repeatedly. The homogenized samples were incubated at room temperature for 5-min to permit complete dissociationof nucleoprotein particles. Following homogenization, 0.2 ml of chloroform for each 1ml Trizol reagent was added and vigorously shaken with hand for 15-sec and incubatedfurther for 3-min at RT. It was then centrifuged at 12000 rpm for 10-min at 4ºC, whichseparates out the homogenate into lower phenol chloroform phase (red), an interphase

Isolation of total cellular RNA

Protein concentrations were estimated by the method of Bradford (1976). The A595wasmeasured after complexation with Bradford reagent. Bovine serum albumin was usedas standard against whichthe unknown protein concentrations were estimated

Protein estimation

Typically 200-300 ng of DNA was used in each ligation reaction. The ratio of vector toinsert was maintained between 1:3 to 1:5 for cohesive end ligation and 1:1 for blunt endligation. The reaction was generally performed in 10 μl volume containing ligationbuffer (provided by the manufacturer) and 0.05 Weiss unit of T4-DNA ligase, at 16ºCfor 14-to 16-hrs. On using the rapid ligation kitfrom Fermentas, incubation was at 22ºC for 1-2 hrs

Ligation of DNA

PCR products were purified using the PCR Purification Kit (Qiagen) as per the manufacturer's instructions

DNA fragments to be used for specific purposes like ligation or radioactive labeling were eluted from the agarose gel after electrophoresis. The gel piece containing thedesired band was sliced out from the gel and the DNA was purified using commerciallyavailable purification kits for this purpose. The efficiency of elution was determined bychecking a small aliquot of DNA sample on the gel

Purification of PCR products

Purification of DNA by gel elution

Around 0.5 to 1 μg of DNA was regularly used for each restriction digestion. 2to 5units of restriction enzyme were used in the total reaction volume of 20 μl containing 2μl of the corresponding buffer supplied at 10 X concentration by the manufacturer. Thereaction was incubated for 2 hrs at the temperature recommended by the manufacturer.The DNA fragments were visualised by ethidium bromide staining after electrophoresison a 0.8 to 1% agarose gels. Commercially available DNA size markers were run alongwith the digestion samples to compare with and to estimate the sizes of the restrictionfragments

Restriction enzyme digestion and analysis

TheDNA samples were mixed with appropriate volumes of 6 X loading dye (0.25%bromophenol blue and 0.25% xylene cyanol and 30% glycerol in water) and subjectedto electrophoresis through 0.8 to 1 % agarose gel in TAE buffer. The gel was stained in1 μg/ml ethidium bromide solution for 15-min at room temperature and visualised byfluorescence under UV-light in a UV-transilluminator

Agarose gel electrophoresis

werethen recovered by centrifugation at 12,000 rpm for 30-min. The pellet was washed oncewith 70% ethanol, air-dried and re-suspended in 100 μl of TE-buffer. It was treatedwith RNase at a concentration of 20 μg/ml by incubating at 37ºC for 1-hr. It was furtherextracted with an equal volume of phenol:chloroform mixture followed bychloroform:isoamyl alcohol (24:1) mixture. After centrifugation, the clear supernatantwas used for recovering the nucleic acids. The nucleic acids were precipitated with 200μl of alcohol in presence of 0.3 M sodium acetate (Sambrook and Russell, 2001). In casewhere high purity plasmid preparations are required (DNA sequencing) the plasmidisolation was carried out with the commercially available kits following themanufacturer’s instruction. Plasmids were observed on 1% agarose gel

1.5 ml of stationary phase culture wascentrifuged and cell pellet was re-suspended in 567 μl of TE buffer. To this 30 μl of10% SDS, and 3 μl of proteinase K (20 mg/ml) were added in that order and the cellsuspension was mixed and incubated at 37ºC for 1-hr. When the suspension was clear, 100 μl of 5 M NaCl was added and thoroughly mixed followed by the addition of 80 μlCTAB/NaCl (10% cetyl trimethyl ammonium bromide in 7 M NaCl). The suspensionwas incubated at 65ºC for 10-min, brought to room temperature and extracted with anequal volume (780 μl) of chloroform isoamyl alcohol (24:1), and aqueous phasetransferred to fresh tube. The aqueous phase was further extracted successively, firstwith phenol:chloroform:isoamyl alcohol (25:24:1) and then with chloroform isoamylalcohol (24:1). DNA was precipitated fromthe clear supernatant by the addition of 0.6volumes of iso-propanol. The chromosomal DNA was either spooled out or pelleted atthis stage and washed with 70% ethanol air dried and dissolved in 100 μl of TE-buffer

Isolation of chromosomal DNA

1.5 ml of cells from an overnight culture waspelleted by centrifuging in cold (4ºC) for10-min at 6000 rpm. The cells were re-suspended in 200 μl solution I (50 mM glucose; 25 mM Tris-Cl, pH-8; 10 mM EDTA, pH-8) with vortexing. 400 μl of freshly preparedsolution II (0.2% NaOH, 1% SDS) was added and mixed by gently inverting the tubes.Subsequently, 300 μl of solution III (prepared by mixing 60 ml of 5 M CH3COOK,11.5 ml glacial acetic acid, 28 ml water) was added and the tubes were invertedrepeatedly and gently for homogeneous mixing followed by incubation for 5-min onice. After centrifuging at 12,000 rpm for 15-min, supernatant wasdecanted into a freshtube, an equal volume of iso-propanol was added, the precipitated nucleic acids

Isolation of plasmid DNA

Recombinant DNA techniques

The colonies to be tested were streaked on the surface of minimal A-glucose plates containing either 0.4-0.7 M NaCl with 1 mM glycine betaine, and incubated at 37oC. NaCl-tolerant strains grew toform single colonies in 36-60 hrs whereas NaCl-sensitive ones did not. As controls, MC4100 (WT) and other previously identified NaCl sensitive mutants were streakedfor comparison

NaCl-sensitivity testing

Competent cells for high efficiency transformations were prepared by a method ofInoue et al. (1990) with few modifications. An overnight culture of the strain (routinelyDH5α) was sub-cultured into fresh sterile LB-brothin 1:100 dilutions and grown at 18ºC to an A600of 0.55. The cells were harvested by centrifugation at 2500 rpm for 10-min at 4ºC. This was re-suspended in 0.4 volumes of INOUE buffer and incubated inice for 10 min. The cells were recovered by centrifugation at 2500 rpm at 4ºC for 10-min and finally re-suspended in 0.01 volume of the same buffer. Sterile DMSO wasadded to a final concentration of 7%. After incubating for 10-min in ice, the cells werealiquoted in 100 μl volumes, snap frozen in liquid nitrogen and stored at –70ºC

Preparation of high efficiency competent cells

the infection mixture was centrifuged, washed in 5 ml of citratebuffer and plated without phenotypic expression

To 2 ml of fresh overnight culture of recipient strain, 108pfu equivalent of phage lysatewas added and incubated at 37ºC without shaking for 15-min to facilitate phageadsorption. The un-adsorbed phage particles were removed by centrifugation at 4000rpm for 5-min and pellet of bacterial cells was re-suspended in 5 ml of LB-brothcontaining 20 mM sodium citrate to prevent further phage adsorption. This wasincubated for 30-min at 37ºC without shaking to allow the phenotypic expression of theantibiotic resistance gene. The mixture was then centrifuged, and the pellet was resuspendedin 0.3 ml citrate buffer. 100 μl aliquots were plated on appropriate antibioticcontaining plates supplemented with 2.5 mM sodium citrate. A control tube withoutaddition of P1 lysate was also processed in the same way. In the case of selection ofnutritional requirement,

Phage P1 transduction

bindingsite lie upstream of the MCS to ensure the high level expression of any genecloned in MCS. A stretch of hexa-histidine (His6)-encoding codons followed by stopcodon is incorporated downstream of MCS to give a C-terminally His6-taggedrecombinant protein (EMD Biosciences).6. pBAD18:It is an expression vector with a pMB9derived origin of replication and allows for tightly regulated expression of genes cloned under the PBADpromoter of the araBADoperon (Guzman et al.,1995). The vector also carries thearaCgene, encoding the positive and negative regulator of this promoter.7. pCP20: pSC101-based Ts replicon, chloramphenicol resistant, ampicillin resistant, for in vivoexpression of Flp recombinase (Datsenko and Wanner, 2000)Plasmid DNA preparations were routinely made from recAstrainDH5αandwerestored in 10 mM Tris-Cl (pH-8.0) with 1 mM EDTA at –20ºC. The plasmid constructsused in this study are given in Table 2.2.Table 2.2Plasmid constructs

The plasmid vectors used in this study were as follows:1.pCU22: It is a derivative of pUC19 used to prepare supercoiled DNA for in vitrotranscription where two strong phage fdtranscription terminators flank MCS. This ensures that the transcripts originated from vector based promoters will not interferewith the transcription from the cloned promoter and that the transcript originated fromthe cloned promoter will be terminated after the MCS (Ueguchi and Mizuno,1993).2.pMU575: It is an IncW-based, single-copy, trimethoprim resistance bearingpromoter probe vector. It carries its MCS upstream of a promoterless galK’-lacZfusion. This fusion has the first 58 codons of galKfused to the 8th codon oflacZ, andthe resultant hybrid polypeptide possesses functional β-Galactosidase activity(afterassembly as a tetramer). Translation of the hybrid gene is controlled by the ribosomebinding site of galK. There are stop codons in all the three reading frames between MCS and initiation codon of galKso that there is no interference caused bytranslational read-through from inserts cloned into MCS region. A strong pheRterminator located upstream of the MCS prevents read through from any vector-basedpromoter into the lacZgene (Andrews et al.,1991).3. pTrc99A:It is an expression vector with ColE1 origin of replication and ampicillin resistance marker. It provides IPTG dependent induction of the cloned gene (Amann et al., 1988)4. pCL1920: It is a pSC101-based, low-copy-number vector with spectinomycin and streptomycin resistance marker carrying the MCS in lacZαregion and henceprovides the advantage of screening the insertions using α-complementation (Lernerand Inouye,1990).5. pET21b: It is a ColE1-based, high-copy-number expression vector bearing ampicillinresistance marker. A strong T7 RNAP-recognised promoter and an efficient ribosome

Plasmids

For TEM, C. glabrata cells were digested with zymolyase 20T for 3 h at 30◦C, centrifuged at 1,000 g and washed with YPD medium. Cell fixation was performed as described for SEM and dehydrated samples were embedded in araldite 6005 resin. After complete polymerization at 80 ̊C for 72 h, ultra-thin (50-70 nm) sections were preparedwith a glass knife on Leica Ultra cut (UCT-GA-D/E-1/00)microtomeand mounted on copper grids. Aqueous uranyl acetate-stained and Reynolds lead citrate-counterstained samples were viewed under Hitachi H-7500 transmission electron microscope

Transmission electron microscopy

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

Total β-glucan estimation

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

Purified vacuole membrane isolation

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

Estimation of glycogenlevels

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

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

Total membrane preparation

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

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE)

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=

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

Microarray analysis

.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.coliBW23473transformation by electroporationE

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

Electro-competentcell preparation

the disrupted gene, BLAST N of the sequences from rescued plasmids was performedagainstC. glabrataGenolevures database (http://www.genolevures.org/blast.html

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

Tn7insertion mutant rescueand gene identification

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.

Screening of C. glabrataTn7insertion mutants

and colony purified on CAA plate. 15% glycerol stocks were made for two independent transformants and stored at -80 ̊C

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

Cloning of CgYPS7gene

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

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

Quantitative real-time polymerase chain reaction (qRT-PCR)

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

Complementary DNA (cDNA) synthesis

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

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-

Total RNA isolation

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

Protocol III(Spheroplast lysis method

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

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

Protocol II (Glass bead lysis method)

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).

Protocol

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

Yeast genomic DNA isolation

Molecular biology tech

preparedin appropriate solvents, sterilizedby autoclaving or filtrationand stored at appropriate temperature

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

Growth assayand measurementof generation time

15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O pH was adjusted to 6.7 with 1 N KOH. MnCl2needsto beaddedseparately,drop by drop with stirring, tothe buffer. PIPES goes into solutionwhenpH is greater than 6.7. The solution, after pH adjustment to 6.7 was filter-sterilized and stored at -20ºC.Reagents for yeast transformation:1 M Lithium acetate (LiOAc)50% Polyethylene glycol10 mg/ml Carrier DNADimethylsulfoxide (DMSO)

INOUE transformation buffer:For bacterial DH5α ultra-competent cells preparation10 mM PIPES (free acid)

Transformation-related solutions

0.67% Yeast Nitrogen Base2% DextroseYeast Carbon Base (YCB):1.17% Yeast CarbonBase1% DextroseCAA:0.67% Yeast Nitrogen Base 2% Dextrose0.6% Casamino acids Plates weremade by adding 2% agar

Yeast Extract-Peptone-Dextrose (YPD):1% Yeast extract2% Peptone 2% DextroseYeast Nitrogen Base (YNB)

Yeast medium

antibodies,anti-mouse IgG andanti-rabbit IgG conjugated with horseradish peroxidase (HRP) were obtained from Cell Signaling Technology, USA

All chemicals were purchasedfrom commercial sources. Mediacomponents for bacterial and yeast growthwere obtained from BD (Becton, Dickinson and Company, USA). Other chemicals were purchased from Sigma-Aldrich Co., USA. Materials used in recombinant DNA experiments were primarily obtained from New England Biolabs, Invitrogen, Bangalore Genei and MBI Fermentas. SuperScript™ III first-strand synthesis system was purchased from Invitrogen.MESA GREEN qPCR MasterMix Plus for SYBR®Assay was purchased from Eurogenetec. Kits used for plasmid isolation, PCR product purificationand DNAgelextractionwerefrom Qiagen.Radioactive chemical, ortho-P32-phosphoric acid,wasprocured from BRIT-Jonaki, CCMB, Hyderabad.Anti-Pma1 polyclonal antibody raised against S. cerevisiaePma1 was purchased from Santa CruzInc.,USA. Anti-phospho-p44/42 MAPK (Thr202/Tyr204) was purchased from Cell Signaling Technology, USA. Anti-CPY polyclonal antibody raised against S. cerevisiaeCPY was procuredfrom Thermo Scientific. Anti-Gapdh antibody raised against human Gapdh was purchased from Abcam. Secondary

Chemicals and antibodies

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

Preparation and sonication of phosphatidylinositol-sodium salt solution

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

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

Mouse infection assay

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

Membrane preparation

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

Mutant rescue

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

Transformation of E. coliBW23473 cells by electroporation

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

Preparation of E. coliBW23473electrocompetent cells

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

Bacterial transformation

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

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

Preparation ofE. coli DH5α ultracompetent cells

A microtipful of cellsfor each yeaststrainfrom appropriate mediumwassuspended in 10μlzymolyase cocktailandincubated at 37ºC for 90 min. 2 μlof zymolyase-treated cell suspension was used as template in 25 μlPCR reaction

Yeast colony PCR

Alipophilic styryl dye,FM4-64,is a vital stain which istakenupby cells viaendocytosis through plasma membrane(Vida and Emr, 1995). Therefore, it fluorescesonly in live cells. Importantly, neitherfixed cells canbe stained with FM 4-64norcells canbe fixed afterFM 4-64staining. For vacuole staining, single colony of the test strain grown onYPD plate was inoculated in 10 ml YPD medium for overnight. 100 μlovernight culture was inoculated in fresh YPD medium and incubated at 30ºC for 3 hto obtain log-phase cells. C. glabratacells from 1 ml log-phase culture were harvested at 4,000 rpm for 5 minin a table top centrifuge. Supernatant was aspirated out,cells were resuspended in 50 μl YPD medium and 1 μl FM 4-64 (16 μM final concentration) was added.C. glabratacells were incubated in a 30ºC water bath for 30 min. 1 mlYPD medium was added and cells were harvested at 4,000 rpm for 5 minin a table-top centrifuge. After discarding supernatant,C. glabratacells were washed with fresh YPD medium and resuspended in 1 ml YPD medium. C. glabratacells were incubated at 30ºC for 90 min, washed with 1 mlsterile water and were resuspended in 50 μl YNB medium. Labeled C. glabratacells were observed underfluorescence microscope in red filter(730nm)

Stainingof C. glabratavacuoleswith FM4-64

centrifugation at 5,000 rpm for 4 minat room temperature. Harvested cells werewashed with PBS and treated with different compoundse.g.H2O2. After treatment,cells were harvested and further processed according to the type of experiments performed

For several experiments, log-phase C. glabratacells were harvested and treated with different compounds. For this, single colony of aC. glabratastrain was inoculated in YPD-liquid medium and grown for 14-16 h at 30ºC withcontinuous shaking at 200 rpm. Overnight cultures were reinoculated in YPD medium to an initial OD600of 0.1 andgrown for another 4 h. These log-phase cells were harvested by

Harvesting of and treatment to logarithmic phase C. glabratacells

For opsonization,C. glabratacells were incubatedwith 1 μg/μl human IgG for 30 min at 37°C and washed thrice with PBS. Alternatively, yeast cells were incubated with 25% human serum at 37°C for 30 min followed by threePBS washes

Opsonizationof C. glabratacells

C. glabratastrains were grown overnight in YPD medium. Cellswereharvested from 1 mlcultureandwashed withPBS.Cells were next washed with50mM NaH2PO4andresuspendedin 100 μlFITC-dextran(50mg/ml). After incubation at 37°Cfor 45 min, cells were washed thrice with PBS for complete removal of FITC-dextran.Yeast cells were resuspended in 1 ml PBS and used to infect PMA-treated THP-1 cells in 4-chambered glass slide

Fluorescein isothiocyanate(FITC)staining of C. glabratacells

24 h post infection, THP-1 macrophages were washed thrice with PBS, lysed in water and recovered yeast cells were used to infect THP-1 cells at a MOIof 1:10. Three rounds of macrophage infection foreach mutant pool were carried out to enrich for the desired mutants in the final population. The lysate of 3rdround infection was inoculated in YPD medium for overnight (output). Cells were harvested, genomic DNA isolated from each input and output cell pellet andunique signature tags were PCR-amplified with P32-labeledα-dCTP using primers complementary to theinvariant region flanking each unique tag sequence. LabeledPCR products were denatured at 95°C for 10 min, chilled on ice and were hybridized tonylon membranescarrying immobilized plasmid DNA containing 96 unique tagsfor 14-16 h at 42°C.Membranes were washed twicewith 0.1X SSC bufferand exposed to phosphorimager screen for 2-4 h. Radioactive counts for each spot were quantified using Image Quant and Fuji Multi Gauge V3.0 software. Relative percentage intensity for individual spot was calculated with respect to allspots present oneach hybridizedmembrane

YPD-grown cultures (0.05 OD600) of each mutant pool (96 mutants, each carrying a unique signature tag) were either inoculated in YPD medium for overnight (input) or used to infect differentiated THP-1 cells (1X106). After 2 h incubation, non-cell-associated yeastcellswere removed by washing THP-1 cellsthricewith PBS. At

Screening of C. glabrataTn7insertion mutant library

Single colony of C. glabratastrains wasinoculated in 10ml YPD-liquid medium and grown at 30°C with constant shaking at 200 rpm for 14-16 h. Overnight culture was used to inoculate 10 ml YPD broth to an initial OD600of 0.1 and culture was grown for 4-5 h to obtain log-phase culture. Log-phase C. glabratacells were harvested in 15 ml sterile polypropylene tubesby centrifugation at 4,000 rpm for 5 min. Harvested cells were washed with10ml sterile water,resuspendedin 1 ml sterile water and transferred to a 1.5 ml microcentrifuge tube. Cells were harvested at 4,000 rpm for 5 minand resuspended in 100 μl of100mM lithium acetate solution.Yeast transformation cocktail was prepared in a 1.5 ml microcentrifuge tube by mixing 240 μlpolyethylene glycol(50%), 36μl lithium acetate(1 M) and25μlheat-denatured single stranded carrier DNA(2 mg/ml). 50 μlC. glabratacell suspension and 50 μltransforming DNAwas added to the transformation cocktail, mixed well andincubatedat 30 ̊C for45 min. 43 μlDMSO was added and cells were subjected to heat shock at 42 ̊Cfor 15 min. After the heat shock, cells were transferred to ice for 10-15 seconds, centrifuged at 4,000 rpm for 5 min and supernatantwas removed.Cells wereresuspended in 200 μlsterile water andspread platedonappropriate selectionmedium. Plates wereincubatedat30 ̊Cfor 2-3 days

Yeast transformation

Identified mutants were phenotypically characterized in 96-well plate format. Mutant cultures were grown in YPD medium for overnight, diluted 150-fold in PBS and 5 μl of cell suspension was spotted on different plates with a 96-pin replicator. Growth was recorded after 1-2 daysof incubation at 30°C

Phenotypic profiling

at 30°C andimages were captured after 2-8daysof incubationdepending upon the medium used

Yeast strains were grown in YPD medium for 14-16 hat 30°Cunder continuous shaking at 200 rpm. Cells were harvested from 1 mlculture, washed with PBS and were diluted to an OD600of 1. Five ten-fold serial dilutions were preparedfrom aninitial culture of 1OD600.4 μl cultureof each dilution was spotted onYNB-agar plates containing different carbon sources. For spotting on YPD plates containing different compounds, 3 μl cultureof each dilution was spotted. Plates were incubated

Serial dilution spotting assay

For growth analysis of a C. glabratastrain,single colony wasinoculated in appropriate broth medium and grown for 14-16 h. Overnight grown culture was used to inoculate the test medium toan initial OD600of 0.1-0.3. Cultures were transferred to a shaker incubator set at 30°C and 200 rpm. Absorbance ofcultures was measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) at 600 nm at regular time-intervalstill 48h. Absorbancevalues were plotted with respect to time and generation time was determined from the logarithmic (log)phase of cell growth usingthefollowing formula.G = Generation time (h)T1= Initial time point taken for analysisT2= Final time point taken for analysisNf= Number of cells at time T2(1 OD600of C. glabrata corresponds to 2 X 107cells.)Ni= Number of cells at time T1(calculated from OD600value as mentioned above)

Growth analysis and determination of generation time

Bacterial strainEscherichia coli DH5αused for cloning purposewas revived on LB medium and grown at 37°C withcontinuous shaking at 200 rpm. LB medium was supplemented with appropriate antibiotics to growbacterial strains carrying plasmids. AnotherE. coli strain,BW23473,was used to rescue the Tn7transposon cassette from C. glabrataTn7insertion mutants. For plasmid DNA purification, bacterial strains were grown overnight in LB broth medium containingsuitable antibiotics

C. glabratastrains were routinely grown either in rich YPD medium or synthetically-defined YNB medium at 30°C withcontinuous shaking at 200 rpm unless otherwise stated. In general, C. glabratafrozen glycerol stocks wererevivedonYPD medium by streaking and allowed to grow for 1-2 days. C. glabratastrainsharboringthe plasmid with URA3as selectable marker were revived onCAA medium.To prepare liquid cell culture, single colony of eachC. glabratastrainwasinoculated either in YPD or YNB broth mediumand grown for 14-16 h. C. glabratastrains streaked on plates were storedat 4°C fora maximum period of2 weeks

Strains and culture conditions

Microbiological methods

To isolate primary peritoneal macrophages, 6-8 week old BALB/c mice were injected with 3% (w/v) thioglycollate broth (0.55% dextrose, 0.05% sodium thioglycollate, 0.5% sodium chloride, 0.05% agar)intraperitonealy (I.P. 50 μl/g body weight). After five days of injection, mice were euthanized by CO2inhalationand peritoneal macrophages were harvested byflushing the peritoneal cavity (lavage) with 10 mlDMEM medium(Zhang et al., 2008)

Isolation of primary (peritoneal) macrophages from BALB/c mice

150 mM NaCl1% Triton-X1% SDSBuffer B50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 MSorbitol50 mM β-mercaptoethanol (To be added just before use)Buffer C100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSAE buffer3 M Sodium acetate(pH 5.3)0.5 M EDTA (pH 8.0)Phenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated Phenol24 ml Chloroform1 ml Isoamyl alcholDNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol15% FicollDNA sample loading buffer was prepared in water

Buffer A50 mM Tris-HCl(pH 8)10 mM EDTA

Buffers for extraction and analysis of genomic DNA and RNA

10 mM NaCl2.5 mM KCl10 mM MgCl210 mM MgSO4LB-ampicillin and LB-kanamycin platesLBmedium50 μg/ml ampicillin30 μg/ml kanamycinMedia and solutions were sterilizedeither by routine autoclaving at 121°C and 15 psi for 20 minor by filtration through membrane of 0.22 μm porosity

Luria Bertani (LB)0.5% Yeast Extract1% Tryptone1% NaClSuper Optimal Broth (SOB)0.5% Yeast Extract2% Peptone

Bacterial media

All antibodies, their sources, clonality and dilutions used are listed in Table 2.2

Antibodies

For restriction digestion(either single or double), 0.5 to 1μg of DNA was used in a reaction containing2 to 5units of commercially available restriction enzyme(s)and 5μl of the recommended buffer (suppliedas 10X concentrationsby the vendor)in atotal reaction volume of 50μl. The reaction mixture was incubated for 2 h or overnight at 37°C. The digested DNA fragments were then visualised by ethidium bromide staining after electrophoresis on agarose gels. Commercially available DNA size markers were loaded along with the samples to ascertain or estimate the sizes of the digestedfragments

Restriction enzyme digestion