Reverse engineering a bronze cannon from theLaBelleshipwreck

The a-PMB chain was subjected to acid-acetone treatment to separate the heme from the a globin. Briefly, a solution of concentrated a-PMB chain (5 ml; 30 mg/ml) was added dropwise to I 00 ml of thoroughly chilled acid-acetone solution (0.5% v/v HCI in acetone) with constant shaking, and then incubated at -20°C for 30 min to allow complete precipitation of the globin. The precipitated globin was isolated by centrifugation at 7000 rpm (4°C) for 15 min and the supernatant containing soluble heme was discarded

Cells growing in culture medium were harvested by trypsinization and washed twice with ice cold PBS. Cells were fixed by adding ice cold 70% ethanol and stored at 4°C. Before harvesting cells were washed twice with PBS and re-suspended in adequate amount of PBS containing Propidium Iodide (PI) to a final concentration of 50μg/ml and RNase to a final concentration of 10μg/ml. Thereby the cell suspension was incubated at 37°C for 30 minutes in dark. Analysis was done by running the samples in BD FACS Vantage System according to the standard procedures after calibration of instrument with Calibrite beads

The plates were kept in incubator gently and the colony formation was monitored every week. Media (500μl) was added to the plates every 4th-5th day to avoid drying. Colonies formed in soft agar photographed were taken without staining, under a microscope in light field

Agar solution was prepared in a sterile 50ml Schott Duran Bottle and boiled in microwave until fully dissolved and kept at 55°C to 65°C. Master Mix with the rest of the components of bottom agar was made in a sterile corning 50ml tube prewarmed at 55°C and agar solution was added. The solution was once vortex briefly and then added (2ml) carefully to each well avoiding air bubbles. The plates were left undisturbed in laminar flow hood until the agar set fully. Two days before final assay, the bottom agar plates were kept in tissue culture incubator for equilibration. On the day of assay the following mix was prepared for Top Agar 4 dishes 5 dishes1.media with FBS, L-glutamine and Pen-Strep 4.8 ml 6 ml 2.fetal bovine serum 1.8 ml 2.5 ml 3.sterile water 1.8 ml 2.5 ml 4.agar 1.8% (1.8 g/100mLs) 1.8 ml 2.5 ml 5. cell suspension 1.0 X 105/ dish 100 to 350 μl 100 to 350 μl 6. Total 10.2 ml 13.5 ml Top agar mix without cells was first prepared and kept at 42°C. The cells were then trypsinized and re-suspended after counting in final volume of 100μl to 200 μl. Cells were then mixed with top agar and solution was quickly poured over the bottom agar.

For soft agar assays 2x104, (A549) or 1x105 cells (E-10) were used in 1.5ml top agar. For preparing bottom agar plates (0.64% final con. of agar), a following mix was prepared for five dishes. 1.2X media with FBS, L-glutamine and Pen-Strep 10 ml 2.fetal bovine serum 5 ml 3.sterile water 1 ml 4.noble agar 1.8% (1.8 g/100mLs) 9 ml 5.Total 25 m

For clonogenic assays, 1x103 (A549) or 2x103 (E-10) cells were seeded per well of a six well tissue culture plate and grown for 15 days. For identification of signaling pathways various inhibitors were used viz, PI3K inhibitor LY294002 (10μM), MEK inhibitor PD98059 (10μM) or p38 inhibitor SB203580 (10μM). Cells were grown in the presence of inhibitor for seven days following which fresh medium was added. For staining, cells were washed twice with PBS and fixed in 10% formalin for 10 minutes, washed extensively with water and stained with 0.25% crystal violet prepared in 25% methanol for 4hrs at 4°C. Plates were then washed with milli Q water and dried before scanning

and fixed with 100μl of fixative solution per well, for 10 minutes at room temperature. The cells were then washed twice with PBS and 100μl of staining solution was added to each well. The plate was kept at 37° C, until the color development.

4x103-5x103 cells were plated in 96 well plate, well. Cells were transfected with reporter plasmid 18 -24 hrs after plating. After 48 hrs, cells were washed once with PBS

This protocol is for the detection of β-gal expression in fixed cells. It was performed on 96-well plates for initial screening of tTA transfected clone, and is a modification of Sanes et al., 1986

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

Lysis Buffer: 0.1% Triton X-100/0.1 M Tris-HCl (pH 8.0). 450 ml distilled water 50 ml 1M Tris-HCl (pH 8.0) 0.5 ml Triton X-100 detergent • 100X Mg++ solution: 0.1 M magnesium chloride 4.5 M 2-mercaptoethanol Stored at 4°C. • 0.1 M sodium phosphate (pH 7.5)41 ml 0.2 M Na2HPO4 9 ml 0.2 M Na H2PO4 50 ml distilled water • 4 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside) in 0.1 M sodium phosphate (pH 7.5) containing 2 mM β-mercaptoethanol, Stored at –20°C. • 0.1 mg/ml β-gal standard: 0.1 mg/ml β-gal in 0.1 M sodium phosphate (pH 7.5) containing 2 mM 2-mercaptoethanol Stored at 4°C. • 1 M sodium carbonate in water

normalized to the optical density at day 0 for the appropriate cell type. Growth curve was determined twice

Growth curves were prepared for various cell lines using the modified method adopted by Serrano et al, 1997. Briefly, 10, 000 cells were seeded in a 24 well plate in quadruples. At the indicated times, cells were washed once with PBS and fixed in 10% formalin for 20 minutes and rinsed with distilled water. Cells were stained with 0.05% crystal violet for 30 minutes, rinsed extensively and dried. Cell associated dye was extracted with 1.0ml acetic acid. Aliquots were diluted 1:4 with water and transferred to 96 well microtitre plates and the optical density at 590nm determined. Values were

After PCR, 1 μl of Dpn1 enzyme (10U/μl) was added to the amplification mix and incubated at 37°C for 6hours. After that, 10ml of the amplification mix was taken to transform Dh5a cells. Positive clones were selected after confirming the sequence of plasmid DNA

The PCR parameters were as follows

The reaction mix included 2ml of PSKll(39+) (50ng) containing wild type K-Ras cDNA , 5ml 10x buffer, 20pmoles of primers , 1ml of 10mM dNTP mix and 1ml of deep vent polymerase (NEB).

appropriate secondary antibody (conjugated with horse-radish peroxidase) diluted in 5% fat free milk solution (in PBST) and incubated for 45 minutes at room temperature. After incubation the membrane was washed and processed for the detection of protein bands using ECL-plus detection reagent (Amersham Biosciences) followed by detection of signal on X-ray film (Hyperfilm-ECL, Amersham Biosciences)

The proteins were resolved using denaturing SDS-PAGE gel and after completion of the run, the gel was over laid on a nitrocellulose paper cut to the size of gel and kept in the blotting cassette in the presence of blotting buffer. Finally the cassette was put in the mini transblot apparatus (Bio Rad) and blotting was done for 4 hours at a constant voltage of 60 V. Then the membrane was taken out and rinsed in PBS containing 0.1% Tween - 20 (PBST) for 5 minutes by gentle shaking. Later the membrane was immersed in 5% non-fat milk solution in PBST with gentle shaking for 1 hour at 37°C. The membrane was washed off from the traces of the fat free milk with PBST and the membrane was over laid with primary antibody diluted in PBST for 3 hours at 4°C with shaking. After incubation the membrane was washed with PBST and layered with

Immunobloting

For adherent cells from which lysates have to be prepared , culture medium was removed and cells were washed with ice cold 1X PBS twice and then scraped with cell scraper in Cell Lysis buffer. Cells were rotated at 4°C for 30min at cold room and centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected and protein concentration was estimated using BCA assay. For standard western, 50-70μg of protein was loaded on to the gel

Lysate Preparation for Immuno- blotting

DTT. Then contents were then mixed and 1μl (200 units) of M-MLV was added. The mixture was then incubated at 37°C for 50 minutes. The reaction was stopped by incubating the mixture at 70°C for 15 minutes. The cDNA thus prepared was then used as a template for PCR. The expression of the investigated genes was determined by normalizing their expression against the expression of actin or GAPDH gene

Semi-quantitative RT-PCR

μg of total RNA was reverse-transcribed using poly-T oligonucleotide and M-MLV reverse transcriptase (Invitrogen) according to manufacturer’s protocol. Briefly, a 20μl reaction volume was made for 1μg of RNA. In a microcentrifuge tube, 1μl oligo (dT)(500μg/ml) , 1μg total RNA, 1μl 10mM dNTP mix and sterile water was added to afinal volume of 13μl. The mixture was then incubated at 65°C for 5 minutes ad quickly chilled on ice. To this mixture were added 4μl of 5X first strand buffer and 2μl of 0.1M

Total RNA was isolated by TRIzol method using the manufacturer’s protocol. Briefly, medium was removed, from 35mm dish and 1ml to TRIzol was added directly to the dish and kept at room temperature for 5 minutes. The cells were harvested by pipetting up and down three four times and transferred to a 1.5ml microfuge tube. For each 1mlTRIzol, 200μl of chloroform was added and tubes were shaken vigorously for 10 seconds to completely dissociate the nucleoprotein complexes, followed by vortexing for about 30 seconds. The mixture was kept for 3-5 minutes at room temperature and then centrifuged at maximum speed for 10 minutes. The upper aqueous phase was transferred into a fresh microcentrifuge tube and 500 μl of isopropanol was added and this was incubated at -20°C for 1 hour. The RNA was pelleted by centrifugation at maximum speed for 30 minutes at 4°C. The supernatant was decanted and the pellet washed with 1ml of 70% ethanol followed by a second wash with 1ml of 90% ethanol and centrifugation at maximum speed for 10 minutes. The supernatant was removed and the pellet air-dried for about 5 minutes and re-solubilized in 30-50 μl RNase free deionized (DEPC-treated Milli-Q) water and aliquots were stored at -70°C

Transient transfection of plasmid DNA in culture cells was performed using Lipofectamine 2000 according to manufacturer’s protocol. Briefly, forty million cells were seeded in a 35mm tissue culture dish, one day before transfection. Transfection was performed 18-24 hrs after seeding the cells. 4μg DNA was mixed in 50μl of Opti-MEM in one eppendorf tube. In another tube, 5μl of Lipofectamine 2000 was diluted in 50μl Opti-MEM and incubated at room temperature for 5 minutes. After five minutes, DNA and Lipofectamine 2000 were mixed together and complexes, incubated for 30 minutes at room temperature. Meanwhile, the adherent cells were washed twice with PBS and 1ml of Opti-MEM was added. 100μl of complexes were then added to each dish containing cells and medium. After 6hrs, the medium containing complexes was removed and complete medium was added and transgene expression was accessed 24-48 hrs after transfection

Transient transfections in adherent cells

The quantity and purity of nucleic acids was determined by measuring the absorbance at 260 and 280 nm. The concentration of nucleic acids was calculated by taking 1 OD 260= 50 μg/ml for DNA, 40 μg/ml for RNA and 33 μg/ml for single stranded oligonucleotides. The purity of nucleic acids was checked by their A260/A280 ratio

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.

stored. To prepare competent cells pre-inoculum was prepared. A single bacterial colony was picked from LB agar plate, inoculated into 3 ml LB medium, and incubated overnight at 37°C temperature with shaking at 200 rpm. 1% of this pre-inoculums was sub cultured in 100 ml LB-broth and incubated at 18°C with shaking until OD at 600nm reached 0.5 - 0.6 (approx.). Culture was kept on ice for 10 min. with constant shaking.Cells were pelleted by centrifugation at 2000 g at 4°C for 8 min. Pellet was resuspended in 40 ml of ice-cold TB buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000 g at 4°C for 8 min. Pellet was resuspended in 8 ml of TB buffer in which final concentration of DMSO was 7% and left on ice for 10 min. 100 μl aliquots were made and snap frozen in liquid nitrogen and stored at -80°C

All the salts (10 mM PIPES, 15 mM CaCl2.2H2O, 250 mM KCl, 55 mM MnCl2.2H2O) except MnCl2 were dissolved in milliQ water and pH was adjusted to 6.7 with 1N KOH. MnCl2 was dissolved separately in mill Q water. MnCl2 was added drop wise while stirring (MnCl2 if added directly will give a brown color to the solution and precipitate out, hence it needs to be dissolved separately). Solution was then filter sterilized and

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

Human lung epithelial type II cells (HPLD) were a kind gift from Dr. T Takahashi, Japan. All the cell lines were maintained in 5% CO2 with the recommended media containing 10% FBS (3% FBS for HPLD) and following the standard guidelines.

A549 (human lung adenocarcinoma epithelial) and murine fibroblast cell line NIH3T3 was purchased from American Type Culture Collection. Murine lung epithelial cell line E-9 and E-10 were a kind gift from Dr. L.M.Anderson, (NCI-FCRDC, Frederick, Maryland)

12% resolving gel (for 25 ml)Water = 8.2 ml 30% Acrylamide = 10.0 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.01 ml 5% stacking gel (for 10 ml)Water = 6.8 ml 30% Acrylamide = 1.7 ml 1.5 mM Tris (pH 6.8) = 1.25 ml 10% SDS = 0.1 ml 10% APS = 0.1 ml TEMED = 0.01 ml

A double cylinder gradient former was used with 12% poly acrylamide gel mix in the inner cylinder and a 3% polyacrylamide gel mix in the outer cylinder that was stirred using a magnetic bead on a magnetic stirrer. A pump was connected to the flow tube and the flow rate adjusted at 5-8 to cast a 12-3% gradient gel. A 5% stacking gel was used. After the protein samples were run on the gradient gel, it was stained in instant blue over night under shaking. 3% resolving gel (for 25 ml)Water = 15.68 ml 30% Acrylamide = 2.5 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.02 ml

The DNA samples were mixed with the appropriate volumes of the 6X loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 30% glycerol in water) and subjected to electrophoresis through 1-1.5% agarose gel in either 1X TBE or 1X TAE buffer. The gel was stained in 1 μg/ml of ethidium bromide solution for 30 min at room temperature and the bands were visualized by fluorescence under UV-light

The lacZ U118 is an amber nonsense mutation(Am) that confers Lac─phenotype and also polarity of the downstream lacYA genes in the operon due to premature Rho-dependent transcription termination within the untranslated region of lacZ. Melibiose is a sugar which can only be utilized in a lacZ (Am) strain at high temperature (39 ̊C, when the native melibiose permease is inactive) if the downstream gene lacY encoded permease is transcribed and translated. Therefore, in lacZ (Am) strains, growth on minimal melibiose plates (0.2%) at 39°C reflects transcriptional polarity relief at the lac locus, and the same was scored after streaking the relevant strains on such medium

Strains were streaked on LBON agar plates and after an overnight incubation at 42°C growth was monitored (compared to that on LBON at 30°C as control). Absence of single colony growth was taken to reflect temperature sensitivity. Whenever needed the phenotype was also quantitatively assessed by plating dilutions of cultures on LBON agar plates and the drop in plating efficiency was scored after overnight incubation at 30°C and 42°C

genome cloned in a ColEI-based replicon, and obtained from Dr. Manjula Reddy. pHYD2556 is spectinomycin resistant and carries the minimal nusA+ open-reading frame with its native ribosome-binding site between genomic nucleotide co-ordinates 3314061and 3315548 cloned downstream of the ara regulatory region in a pSC101-based replicon, and obtained from Dr. Ranjan Sen. pHYD2557 is chloramphenicol resistant and carries a 2.3-kb PCR-amplified region between genomic nucleotide co-ordinates 3314061 and 3316393 (containing yhbC nusA region with its own promoter) cloned in a pSC101-based Ts replicon, and obtained from Dr. Ranjan.Plasmid DNA preparations were routinely prepared from recA strains such as DH5αand were stored in 10mM Tris-Cl (pH 8.0) plus 1mM EDTA at ─20 ̊C

pWSK30 an Ampicillin resistant vector with pSC101 origin of replication and blue-white screening facility (Wang and Kushner, 1991). pHYD272 is a derivative of pMU575, an IncW-based single copy vector with Trimethoprim resistance marker carrying lacZYA reporter genes under proU promoter (Dattananda et al., 1991). pHYD751 a ColE1 replicon plasmid with ampicillin resistance marker and 2.1kb EcoRI-SalI fragment carrying nusG+cloned into EcoRI-SalI sites of pAM34 vector. The plasmid exhibits IPTG dependent replication (Harinarayanan and Gowrishankar, 2003). pHYD763 is a Ts (maintained at 30 ̊C but not at 37 ̊ or 39 ̊C), CmR, pSC101 derivative carrying 3.8 kb BamHI-SacI fragment of nusG+ cloned into BamHI-SacI sites of pMAK705 (Harinarayanan and Gowrishankar, 2003). pHYD1201 a ColE1 replicon plasmid with ampicillin resistance marker and 3.3kb HindIII-SalI fragment carrying rho+cloned into HindIII-SalI sites of pAM34 vector. The plasmid exhibits IPTG dependent replication (Harinarayanan and Gowrishankar, 2003). pHYD1622 is the derivative of pHYD1201 where the Ampicillin resistance marker has been replaced with Chloramphenicol using Wanner method of gene replacement. Cm gene was amplified from pKD3 plasmid (K. Anupama, unpublished). pHYD1623 is the derivative of pHYD751 where the Ampicillin resistance marker has been replaced with Chloramphenicol using Wanner method of gene replacement. Cm gene was amplified from pKD3 plasmid (K. Anupama, unpublished). pHYD2368 is a derivative of pBAD18 (AmpR) with 1.7 kb fragment encompassing RBS and coding region of uvsW from phage T4gt7 into SacI site of pBAD18 (K. Leela, unpublished). pHYD2554 is a derivative of pMBL18 with ampicillin resistance, carrying the 10-kb EcoRI-HindIII fragment between kilobase co-ordinates 3310.06 and 3320.08 of the E. coli

to CCT mutation leading to a Glutamic acid to Glycine change at the 53rd amino acid and a Threonine to Proline change at the 55th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-I119T is a derivative of pLG-H-NS plasmid with ATC to ACC mutation leading to a Isoleucine to Threonine change at the 119th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-P116S is a derivative of pLG-H-NS plasmid with CCA to TCA mutation leading to a Proline to Serine change at the 116th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-Y97C is a derivative of pLG-H-NS plasmid with TAT to TGT mutation leading to a Tyrosine to Cysteine change at the 97th amino acid in the H-NS protein (Willams et al., 1996). pPMrhoCam is a Ts (maintained at 30 ̊C but not at 37 ̊ or 39 ̊C), CmR, pSC101 derivative carrying PuvII-HindIII fragment containing trxArho+ cloned into PuvII-HindIII sites of pPM103 (Martinez et al., 1996). pTrc99A an expression vector with ColE1 origin of replication and ampicillin resistance marker. Provides IPTG dependent induction of the insert (Amann et al., 1988). pUC19 is a high-copy-number ColE1 based E.coli cloning vector (500-700 copies/cell) with an Ampr selectable marker. It is one of a series of related plasmids constructed by Messing and co-workers and contains portions of pBR322 and M13mp19 (Yanisch-Perron et al., 1985). It carries a multiple-cloning site (MCS) region in the lacZα fragment, and therefore allows for blue-white screening of recombinant clones

pAM34 is a pBR322-derived cloning vector with Ampr and Specr selectable markers. The replication of this plasmid is dependent on the presence of IPTG, the gratuitous inducer of the lac operon (Gil and Bouche, 1991). pBAD18 is an expression vector with a pBR322 derived origin of replication and allows for tightly regulated expression of the genes cloned under the PBAD promoter of the araBADoperon (Guzman et al., 1995). The vector also carries the araC gene, encoding the positive and negative regulator of this promoter. pBluescript II KS (pBKS) is also a high-copy-number ColE1 based cloning vector with Ampr selectable marker and blue-white screening facility (obtained from Stratagene). pCL1920 is a low-copy-number vector with pSC101 replicon (~ 5 copies/cell), that carries streptomycin (Str)/spectinomycin (Spec)-resistance marker (encoded by aadA) and also carries a MCS region within the lacZα that allows blue-white screening to detect recombinants (Lerner and Inouye, 1990). pCP20 pSC101-based Ts replicon, CmR AmpR, for in vivo expression of Flp recombinase (Datsenko and Wanner, 2000). pLG339 is a low-copy-number cloning vector with pSC101 replicon that has a Kanrselectable marker (Stoker et al., 1982). pLG-H-NS is a pLG339 derivative where the hns ORF had been cloned into the EcoRI-SalIsites of pLG339 vector (KanR, pSC101) (Willams et al., 1996). pLG-H-NSΔ64 is a derivative of pLG-H-NS plasmid with AT base pair deletion after codon 63 in the hns gene resulting in a frameshift (Willams et al., 1996). pLG-H-NS-L26P is a derivative of pLG-H-NS plasmid with CTG to CCG mutation leading to a Leucine to Proline change at the 26th amino acid in the H-NS protein (Willams et al., 1996). pLG-H-NS-E53G/T55P is a derivative of pLG-H-NS plasmid with GAG to GGG and ACT

pACYC184 is a medium-copy-number cloning vector (~ 20 copies/cell) with Cmr and Tetrselectable markers. It carries the origin of replication from plasmid p15A (Chang and Cohen, 1978), which is related to and yet is compatible with that of ColE1. This property enables pACYC184 to co-exist in cells with ColE1 plasmid vectors, including all the ones mentioned above

Automated DNA sequencing on plasmid templates or on PCR products was carried outwith dye terminator cycle sequencing kits from Perkin-Elmer on an automatedsequencer (model 377, Applied Biosystems), following the manufacturer’s instructions.Manual sequencing was achieved using the SequenaseVersion2.0 DNASequencing Kit from USB Corp. as described in manufacturer’s instructions and thesequencing reaction products were resolved by electrophoresis on a 6% sequencing gel

Native isoelectric focusing was done using Pharmacia Phast Gel Apparatus and precast IEF gel (pH 3-9) from GE healthcare. The samples were prepared in 50 mM sodium buffer (pH 8.0) and applied in the middle portion of the gel. Gels were run as previously described(Olsson et al., 1988) that is at 15°C, pre-focusing at 2000 V (75Vh), sample loading at 200V (15Vh) and run at 2000V (500Vh). Staining was done using Coomassie Blue G-250

Gel-filtration chromatography was performed at room temperature on a BioLogic LP protein purification system (Biorad) with an in-house packed Sephadex G-100 column of size 1.5 X 43 cm; each protein sample was loaded in 0.8-ml volume, and the buffer used for chromatography was 20 mM Tris-Cl (pH 8) with 200 mM NaCl at a flow rate of 0.1 ml per min with 1.5-ml fractions being collected for analysis. Protein elution was detected by measurement of A295.The void volume, V0was determined using blue dextran (2X 106Daltons) and theelution parameter Kavfor each proteinwas calculated from elution volume Veand total bed volumeVtusing the equation:Kav= (Ve–V0)/(Vt–V0)Initially, acalibration curve was derived froma semilogarithmic plotof Kav of protein standardsalbumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and ribonuclease A (13 kDa) on the Y-axis against log10of their molecular masses on theX-axis. The Kavof the ArgPdproteins were calculated based on their elution volume and then the molecular masses were derived from the corresponding point on the calibration curve

directly from lysed cells, log and stationary phase cultures were spun down, samplebuffer (1 X final concentration) was added to the cell pellet and boiled for 10 min,cooled to room temperature, and after a second spin, the clear supernatant was loaded.The gel run was started at constant current of 20 mA. When the dye front crossed thestacking gel the current was increased to 40 mA

The method followed was as described in Sambrook and Russell (2001). Gels of 1.0mmthickness were casted in the commerciallyavailable small gel apparatus. Resolving gelof 12% (15 ml) and stacking gel (4 ml) was made. Gels were polymerised by theaddition of TEMED and APS (1 % v/v of the gel mix). Sample preparation for gelloading was done as follows. Cell lysate or pure protein fractions (around 30 μg) wasmixed with the sample buffer to 1 X and heated at 95ºC for 2-min. To check expression

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

argP+, argPd-S94L, argPd-P108S, argPd-P274Sfragment downstream of the phage T7-promoter, such that the encoded proteins beara C-terminal His6-tag provided by the vector DNA sequence. Theresultant plasmid was transformed into strain BL21(DE3) which has the T7 RNA Polymerase under the isopropyl thio-β-D-galactoside (IPTG) inducible lacUV5promoter.The resultant strains were grownin LB (500-1000 ml) to an A600of around 0.6and were then induced with 1 mM IPTG and harvested after 4-hrs of induction.Bacterial cells were recovered by centrifugation, resuspended in 20 ml of lysis buffer(20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mM DTT and 10 mM imidazole) containing20 μg/ml lysozyme, and lysed by sonication with 30-sec pulses for 10-min. Theprotocol for His6-ArgP(ArgPds)protein purification involved (i) passing the lysate through a 5ml Ni-NTA (Qiagen) chromatographic columnequilibrated with lysis buffer, (ii) washing thecolumn with 100 ml of washing buffer (20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mMDTT; 30 mM imidazole), and (iii) elution of His6-ArgP(ArgPds)from the column with elutionbuffer (20 mM Tris-Cl, pH-8;300 mM NaCl; 10 mM DTT and 250 mM imidazole) andcollection of 1.5 ml eluate fractions (10 fractions). The fractions were tested forprotein by Bradford method and the protein-carrying fractions (generally tubes 2 to 5)were pooled and dialysed in a 1:200 volume ratio against 20 mM Tris-Cl, pH-8 with 10mM DTT, 300 mMNaCl for 5 hrs followedby a change to buffer of composition 20 mM Tris-Cl, pH-8 with 10 mM DTT, 300 mM NaCl and 40% glycerol for 24 hrs. The proteins were concentrated by centrifugation toaround 1 mg/ml by using Amicon filter (pore size 10-KDa) and stored at −20ºC or −70ºC

For preparing ArgP and ArgPd-S94L, -P108S and -P274S proteins, derivatives(designated as pHYD1705, pHYD2678, pHYD2679 and pHYD2680 respectively) of the plasmidvector pET21b (Novagen) was constructed which carries the PCR-amplified

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

CAN is a toxic analog of Arg and is an inhibitor of bacterial growth. Strains were tested for sensitivity/resistance to CAN by streaking them on minimal A-glucose platessupplemented withoutand with40 μg/ml CAN(or other concentrations as indicated) and 40 μg/ml uracil

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

For routine plasmid transformations, following method which is modification of thatdescribed by Cohen et al. (1972) was used. An overnight culture of recipient strain wassub-cultured 1:100 in fresh LB medium and grown till mid-exponential phage. Theculture was chilled on ice for 15-min, and the steps thereafter were performed at 4ºC.20 ml of culture was centrifuged and pellet was re-suspended in 10 ml of 0.1 M CaCl2.After 15-min of incubation on ice, the cells were again centrifuged and re-suspended in2 ml of 0.1 M CaCl2. The suspension was incubated on ice for 30-min. To the 200 μl aliquot of the cell suspensionplasmid DNA (20 to 200 ng in less than 10 μl volume)was added, incubated for half an hron ice and given a heat shock for 90-sec at 41ºC.The cultures was rapidly chilled, mixed with 0.8 ml of LB-broth and incubated at 37ºCfor 1-hr, and plated on an appropriate selective medium at various dilutions. An aliquotof cell suspension to which plasmid DNA was not added served as a negative control

The primers used in this study are listed in Table 2.3.Table 2.3 Oligonucleotide primersa

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

For SEM, C. glabratacells were fixed for 24 h in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.2) at 4 ̊C, post-fixed in 2% aqueous osmium tetroxide for 4 h and dehydrated. After drying to critical point, mounted samples were coated with a thin layer of gold for 3 min using an automated sputter coater and visualized by SEM (JEOL-JSM 5600)

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed at the Electron Microscope Facility, RUSKA LABs, Acharya N. G. Ranga Agricultural University, Hyderabad

Log-phase yeastcells were collected, washed and suspendedin 10 mM Tris-HCl (pH 7.5) containing 50 mg/ml zymolyase-20T. Cell suspension was incubated at room temperature and absorbance was monitored at 600 nm every10mininterval. Initial absorbance of the cultures at 0 minwas normalized to 100%and the graph was plottedas%decrease in the absorbance with respect to time

Resultant precipitate was dissolved in 3 N HCl and reprecipitated in methanol:acetic acid (8:1) solution. Following 16 h incubation at room temperature, the precipitate was washed withmethanol:acetic acid (8:1) solution till green colour of the supernatant disappeared.Finally,pellet was washed thrice with methanol and air dried. Driedpellet was resuspended in 0.5 NHCl and total mannan content was quantified with phenol-sulphuric acid carbohydrate estimation method as described earlier.Commercially available purified glucose was used as the standard

Total mannan from 3% NaOH-extractable supernatant of cell wall was precipitated by Benedict’s solution.Reducing sugars(mostly mannan) from alkali-extractable supernatant reactwith copper(II) sulphate present in Benedict’s solution and forms red copper(I) oxide precipitate.Briefly, equal volume of Benedict’s solution was added to 3% NaOH-extractable cell wall supernatant fraction and heated at 99 ̊C for 10 min

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

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

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

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

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

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

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

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

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

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

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

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

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

ATPconcentrationin yeast cells was measuredby luminometricluciferase-luciferinbased assayusingATPbioluminescent kit(Sigma # FLAA).Briefly, log-phase yeast

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

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

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

Determination of acid phosphatase activity

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

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

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

were maintained in log-phase by continuous passaging in fresh YNB medium every 4 h

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Genomic DNAisolation buffersBuffer A:50 mM Tris-HCl10mM EDTA150 mM NaCl 1% Triton-X 1% SDSBuffer B:50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 M Sorbitol50 mM β-mercaptoethanol(To be added just before use

Buffer C:100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSRNA isolation bufferAE buffer: 3 M Sodium acetate0.5 M EDTA(pH 8.0)Phenol:Chloroform:Isoamyl Alcohol (25:24:1)solution:25 volume of Phenol24 volume of Chloroform1 volume of Isoamyl alcholDNA sampleloading buffer:0.25% Bromophenol blue0.25% Xylene cyanol15% Ficoll

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)

Luria Bertani (LB):0.5% Yeast Extract1% Tryptone 1% NaCl LB-ampicillinand LB-kanamycin plates:LB medium50 μg/ml ampicillin30 μg/ml kanamycinSuper Optimal Broth (SOB): 0.5% Yeast extract2% Peptone 10 mM NaCl2.5 mM KCl10 mM MgCl210 mM MgSO4

Bacterial medium

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

PI-3 kinase reaction set up and phopsholipid extraction

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

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

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

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

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

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)

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

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

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

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

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

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

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

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

Stripping of membranes in buffer containing 0.4 M NaCl yielded slightly better results. Hybond membranes were reused for 5-10 times after stripping

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

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

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

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

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

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

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

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

weretransferred toa sterile 100 mm cell culture dishcontaining 11 mlfresh and prewarmed completemedium andculturedin tissue culture incubatorat37°C and 5% CO2.After 12hincubation, medium was replacedwith fresh prewarmed mediumand cells were allowed to proliferate till they acquire 80% confluence

Freezer stocks of THP-1 and Lec-2 cells were prepared either in commercial cell preservation medium (Gibco) or completemedium supplemented with 10%heat inactivated serum and 10% DMSO. For cryopreservation, 5-6 million cells were resuspended in 0.5 mlfreezing medium in 2 ml cryopreservation vials,stored in an isopropanol bath and were transferred to-70°C freezer. Aftertwo days, freezer stocks were transferred to liquid nitrogen containertill further use. To revive the cells, freezer stocks were taken outfrom liquid nitrogen container and transferred immediately to37°C water bath. After2-3 min, when freezing medium hadthawed completely,cells

SDS-loading buffer was prepared as a 4X stock solutionin H2Oand used at a 1X concentration.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSRunning buffer was preparedas a 10X stock solution and diluted to 1X concentration before use.Buffers for Western blotanalysisTransfer buffer (10X stock solution)0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSTransfer buffer was prepared as a 10X stock solution and diluted to 1X concentration.1X Transfer buffer (1 litre)200 ml of methanol100 ml of 10X transfer buffer700 ml of waterTris-BufferSaline (TBS)25 mM Tris150 mM NaClpH was adjusted to 7.4 with HCl.TBS buffer was prepared asa10X stock solution and diluted to 1X concentration.Blocking and wash buffers (PBS-T and TBS-T)5% Fat-free milk0.1% Tween-20Volume was made to 100 ml with 1X TBS

1 mM sodium orthovanadate1 X protease inhibitor cocktail SDS-PAGE30% Acrylamide solution29 g Acrylamide1 gBis-acrylamideDissolved in 100 ml H2O.10% Sodium Dodecyl Sulfate (SDS)10 g SDS in 100 ml H2OResolving gel mix (12%) (20 ml)6.6 ml H2O8 ml 30% acrylamide:bisacrylamide (29:1) mix5 ml 1.5 M Tris-HCl (pH 8.8)200 μl 10% SDS200 μl 10% Ammonium persulfate(APS)8 μl N,N,N′,N′-Tetramethylethylenediamine(TEMED)Stacking gel mix (5%, 6 ml)4.1 ml H2O1 ml 30% acrylamide:bisacrylamide (29:1) mix750 μl 1 M Tris-HCl (pH 6.8)60 μl 10% SDS60 μl 10% APS6 μl TEMEDSDS loading buffer130 mM Tris-HCl (pH 8.0)20% (v/v) Glycerol4.6% (w/v) SDS0.02% Bromophenol Blue2% DTT

Whole cell lysis buffer (Homogenizing buffer)50 mM Tris-HCl (pH 7.5)2 mM EDTA10 mM sodium fluoride

Oligonucleotides used in this study were designed either by freely available online tool Primer 3 plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/)or Generunner software. Oligonucleotides were commercially synthesised at MWG Biotech Pvt. Ltd., Bangalore, India. Oligonucleotides used in this study are listed in Table 2.3

competent cells pre-inoculum was prepared. A single bacterial colony was picked from LB agar plate that has been incubated for 16-20 hours at 37 °C and inoculated into 3 mlLB medium and incubated overnight at 37 °C temperature with 200 rpm shaking. 1% of this pre-inoculum was sub cultured in 100 ml LB-broth and incubated at 18 °C until OD 600 reached 0.5 -0.6 (approx.). Culture was kept on ice for 10 min. with constant shaking. Cells were pelleted by centrifugation at 2000xg/4°C/8 min. Pellet was resuspended in 40 ml of ice-cold Innoue buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000 xg/4°C/8 min. Pellet was resuspended in 8 ml of TB buffer inwhich final concentration of DMSO was 7% and left on ice for 10 min. 100μl aliquots were made and snap frozenin liquid nitrogen and stored at -80 °C

All the salts (10 mM PIPES, 15 mM CaCl2.2H2O, 250 mM KCl,55 mM MnCl2. 2H2O) except MnCl2were dissolved in water and pH was adjusted to 6.7 with 1N KOH. MnCl2was dissolved separately in water. MnCl2was added drop wise while stirring (MnCl2if added directly will give a brown colour to the solution and precipitates;hence it needs to be dissolved separately). Solution was then sterilized by filteringand stored. To prepare

Of theligation mixture,2μl (of total volume of 10 μl reaction)was added to atube of 100μlultra competent DH5α bacterial cells and incubated in ice for 30 minutes. The tubewas quickly transferred to a water bath maintained at 42°C to give a heat shock for 90 seconds and again quickly transferred to ice. 1ml of LB broth was added to the tube and then incubated at 37°C for 1 hour. The bacterial cells were then pelletdownby centrifugation at 6000 rpm for 5 minutes and plated on LB agarcontaining appropriate antibiotic

A total of 100-200ng of DNA was used in each ligation reaction. Vector to insert ratio of 1:3 to 1:5was maintained. The reaction volume was generally maintained at 10μl containing 1μl of 10X ligation buffer (provided by the manufacturer) and 0.05 Weissunit of T4-DNA ligase. The reaction was carried outat 16ºC for 14-to 16-hrs or at room temperature for 4hours

Digested DNA fragments required for ligation were eluted from the agarose gel after electrophoresis. The gel was visualised over a UV illuminator and section of it containing the desired DNA fragment was carefully sliced out from the gel. The sliced agarose gel was then processed using commercially available gel elution kitsfor this purpose. The elution efficiency was checked by running a small aliquot of DNA sample on agarose gel

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

Overnight grown bacterial culture (3ml)was pellet down by centrifugationat4ºC for10-min at 6000 rpm. The cells were re-suspended in 200μl of Resuspension solution(solutionI). 400μl of freshly prepared Lysissolution(solution II)was then added and mixed by gently inverting the tubesfor 4-6 times and allowed to lyse for 5 min at room temperature.The complete lysis was ascertained by uniformity and clarityof the contents. Subsequently, 400μl of Neutralization solution(solution III)was added and the tubes were inverted 4-6 timesand gently for homogeneous mixing followed byincubation for 5 min on ice. After centrifuging at 12,000 rpm for 15-min, supernatant was decanted into a fresh tube, and0.7 volume of iso-propanol was added.Theprecipitated nucleic acids were then recovered by centrifugation at 12,000 rpm for 30-min. The pellet was washed once with 70% ethanol, air-dried and re-suspended in 100μl of TE-buffer. It was treated with RNase at a concentration of 20μg/ml by incubating at 37ºC for 1hour. It was further extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) mixture. After centrifugation, the clear supernatant was used for recovering the nucleic acids. The nucleic acids were precipitated with 2.5 volumesof ethanolin presence of3 M sodium acetate. In case where high purity plasmid preparations are required (for transfection to cells) the plasmid isolation was carried out with the commercially available midiprep or miniprep kits following the manufacturer’s instruction. Plasmids were observed on 1% agarose gel

2μg of total RNA was reverse-transcribed using SuperScript III Reverse Transcriptase which is a commercially available version of M-MLVRT with reduced RNase H activity and increased thermal stability.According to manufacturer’s protocol1μg of RNA,1μl oligo(dT)(500ng),1μl 10mM dNTPand nuclease freewater was added to afinal volume of 13μlin a PCR tube.Thismixture was then incubated at 65°C for 5 minutesin a thermo cyclerand then quicklytransferredtoicefor 1minute. To this 4μl of 5X first strand buffer 1μl of 0.1MDTTand1μl ofRNaseOUT (40U/μl) were added. Then contents were then mixed and 1μl (200 units/μl) of SuperScript III RT was added. Themixture was then incubated at 50°C for 60 minutesin a thermo cycler.Lastlythe reaction was stopped byincubating the mixture at 70°C for 15 minutes. The cDNA thus prepared was then usedas a template for PCR

drop wiseaddition and kept at 4⁰C for 24h. Cells were then washed with PBS and stained with DNA staining solutionat 370C in darkwith intermittent shaking. The DNA content of cells was measured by flow cytometryusing FACS-Aria (Beckton-Dickenson) at 695 ±20 nm using a 655 long pass filter.The DNA content was then analysed by FACSDivaor FlowJosoftwareto evaluate the various phases of cell cycle. The diploid 2N DNA content was referred as G1/G0 population and the 4N DNA content was referred as G2/M population. Cells with intermediary DNA content (between 2N -4N) content were considered as S phasecells and those below 2N DNA content as sub G0 cells

Thecells were collected at various time points by trypsinization, washed in phosphate buffered saline (PBS, pH 7.2) and fixed in chilled 70% methanol: ethanol (1:1) solution by

Malachite green reagent

DNA loading dye

Neutralization solution(Solution III)

EMSA Buffer

Blocking buffer: 2% BSA

Stripping Buffer

Blocking Buffer

TBST

Transfer Buffer

Running Buffer

Stacking and resolving AcrylamidegelsResolving gel (10 ml)

6X protein loading buffer (Laemmlibuffer)

Cell lysis buffer(RIPA Buffer)

Sodium Chloride (NaCl)ComponentsFinal concentrationFor 100 mlNaCl5M29.22gH2Oq.s

Briefly, cells were cultured on 35 mm culture dishes (Corning) and were transfected with pcDNA3.1 (+) empty vector or pcDNA3.1 (+) profilin-1 clone (encodes for neomycin resistance for selection in mammalian cells) using Lipofectamine 2000 reagent. The following day, cells were trypsinized and plated in four 10 cm culture dishes separately for each cell clones in selective media containing 800μg/ml of Geneticin, G418 (whose concentration was obtained from kill curve based on MTT assay). Over the time,it will select only those cells that have stably incorporated the plasmid into their genomic DNA. Cells were selected in G418 for about two weeks until colonies appeared. Meanwhile, cells were regularly washed with sterile PBSto remove dead cells from culture dishes. Colonies were picked using a pipettewith a sterile tip by loweringitto the surface of the colony of interest, followed by gentle scraping and rapid sucking.About 20 colonies were picked, followed by dilution plating in 96-well plates until single cell per well was obtained to establish a pure colony. Once colonies have reached to fair confluency, they were further expanded by dilution plating and screened for stable expression of profilinby immunoblotting and semi-quantitative RT-PCR. Once the clonal cell line is established, aliquots were freezed for future use and rest were maintained in lower concentration of G418 with proper track of passage number

5X EMSA buffer

6X DNA loading dye

Blocking buffer: 2% BSA

Permeabilisation buffer: 0.2% Triton X100

4% Formaldehyde fixative

(c) 6X Protein loading buffer (Lammeli buffer)

Wild type or H133S mutant of profilin-1 witheither FLAG or un-tagged werecloned in pcDNA3.1 (+).Mdm2 gene upstreampromoter region having p53 binding site was cloned in pLUC vector (designated as p53-Luc). The constructs of NF-κB-SEAP, p65 (RelA), wild type and dominant negativeIKKβ(IKKβ-WT and IKKβ-DN, respectively)were a kind gift fromProf.Bharat B. Aggarwal (M. D. Anderson Cancer Center,Houston, TX). The constitutive active mutant of IKKβ, in which two serine residues are mutated to glutamic acid, at position 177 and 181 (referred as IKKβ-EE or IKKβ-CA) was gifted byProf. GourisankarGhosh (University of California, San Diego, USA).FLAG or Myc tagged Full length andtruncationmutants of PTEN wereprovided by Dr.M.Subba Reddy (CDFD, Hyderabad).For p53 gene knockdownstudies, TP53 mission shRNA were obtained from Sigma Aldrich (St Louis, MO, USA). For PTEN silencing, retroviral vector based PTEN shRNA (shRNA#1-AGGCGCTATGTGTATTATTAT; shRNA#2-CCACAGCTAG-AACTTATCAAA; shRNA#3-CCACAAATGAAGGGATATAAA)wasgifted by Dr. M.Subba Reddy (CDFD, Hyderabad)

To monitor yeast cell death 1 OD600equivalent cellsfrom mid-log and overnight growncultures were pelleted, washed in PBS and the cell pellet was suspended in 100 μL of PBS. 20 μLof 0.4% trypan bluesolution was added to 20 μLof cell suspension and incubated for 10 min. 20 μL of this suspension was placed on a slide, covered with a cover slip, and cell death was measured by scoring dead cells that take up the dye.To monitor cell viability, cells equivalent to 10-5OD600 from mid-log and stationary phase cultures were plated on YPD-agar, incubated at 30°C for 48 h, and colonies were counted to extrapolate viable cell count per OD600

To determine yeast cell mass, cells equivalent to 5 OD600were harvested from mid-log and overnight growncultures, and washed twice with PBS. Cell pellets were dried at 50°C for 20 minand the dry weight of yeast was measured. To assess the cell number, cells in mid-log or stationary phase were counted using a Neubauer chamber and the number of cells present in 1 OD600was calculated

20 mM HEPES500 mM NaCl 2 mM EDTA1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer C)IP7 reaction buffer(10X)250 mM HEPES,pH 7.4500 mM NaCl60 mM MgCl210 mM DTT (1 M stock was made separately, aliquoted into 100 μL and stored at -20oC).10X buffer was made and stored at 4oC. An appropriate amount was added to the reaction mix to get a final concentration of 1X.DTT was added fresh to the reaction buffer just before use

50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC37% sucrose solution

100mM NaCl30mM MgCl250μg/mLcycloheximide 200μg/mL heparin All the components were made in DEPC treated water.Gradient buffer10% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC10% sucrose solutionTo analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.30% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC30% sucrose To analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.50% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC50% sucrose To analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.37% sucrose gradient buffer

Lysis buffer10mM Tris, pH7.4

Oligonucleotides used in this study were designed manually by examining the relevant DNA sequences. Oligonucleotides were commercially synthesised at MWG Biotech Pvt. Ltd., Bangalore, Indiaor Ocimum biosolutions, Hyderabad, India. Oligonucleotides used inthis study are listed in Table 2.4 and 2.5

PCR product was incubated with 1ul of Dpnl restriction enzyme for 2-3 hours at 37ºC, following which Dpn1 treated PCR product was transformed into DH5α competent bacterialcells. Mutant colonies were screened andconfirmed using DNA sequencing

PCR product was incubated with 1ul of Dpnl restriction enzyme for 2-3 hours at 37ºC, following which Dpn1 treated PCR product was transformed into DH5α competent bacterialcells. Mutant colonies were screened andconfirmed using DNA sequencing

The various mutant plasmids were generatedbyusing PCR-based site-directed mutagenesis protocol (Stratagene).Briefly, primers carrying the desired nucleotide changes were used in a PCR reaction to amplify the nascent mutantplasmids from the wild type parent plasmid. The PCR reaction was set up according to manufacturer’s protocol(Table 5)using a high-fidelity Pfu DNApolymerase and donor-plasmids of the desired geneas the template. Following reactionmixture and the cycling conditions were used for site-directed mutagenesis

All the primers (sequences)used for cloning the above-mentioned genes are providedin AppendixI

using gateway cloning method (Invitrogen). P73domain deletions were cloned in SFB destination vector. WWP2, WWP1, HACE1, E6AP, and PPM1G were cloned into SFB (S-protein/Flag/streptavidin binding protein (SBP) triple tag), GFP,and Myc mammalian destination vectors using the Gateway cloning technology (Invitrogen). WWP2 domain deletions were cloned into Myc-destination vector. WWP1 domain deletions were cloned into SFB-destination vector. PPM1G domain deletions were cloned into SFB mammalian destination vector using Gateway cloning. Bacterially expressing GST-p73, GST-∆Np73, GST-PPM1G, MBP-WWP1, MBP-WWP2, GST-WWP2, GST-WWP1 and GST-HACE1 were generated by using gateway technology. Ubiquitin WT and all the mutants were cloned into hemagglutinin (HA) mammalian destination vector. Flag-tagged Dvl2was purchased from Addgene. Dvl2 domain deletions were cloned into SFB-destination vectors. All the plasmid constructs generated in the present study are mentioned in table 2.Table 2: Plasmids used in the study

TAp73α and ∆Np73α were kindly gifted by Alex Zaika, Vanderbilt University. Full-length p73 and ∆Np73 were cloned into Myc and HA mammalian destination vectors

Four week old tomato S-22 cultivar (acts as non-host for Xanthomonas oryzae pv. oryzicola) were syringe-infiltrated with a suspension of Xocstrains and water control. Plants were incubated in green house for 24 h with minimum and maxium temperature of 26 and 28°C, respectively and relative humidity of 65%. Callose deposition assay was performed as a marker for hypersensitvity response in non host plant as described previously (Hauck et al., 2003). Leaf picture was captured at this stage to observe the HR browning of leaf. For assaying callose deposition by aniline blue staining, infilterated leaves were removed from plant,dipped in lactophenol solution and incubated at 65°C in water bath until the cholorohyll is completely removed. Leaves were rehydrated by washing with 50% ethanol, and finally rinsed with water. For aniline blue staining, leaves were incubated in 0.01% aniline blue solution, prepared in 100 mM K2HPO4(pH 9.5), for 15-20 min in dark. Subsequently, leaves were washed with water and observed for callose deposition in epifluorescence microscope (Stereo, Lumar V7, Zeiss) under UV illumination

flash (Thermoscientific). β-Glucuronidase activity for GUS was expressed as nanomoles of MU produced/minute/108 cells

In planta siderophore gene expression was studied by measuring β-glucuronidase activity. GUS marked BXOR1 strain and wild-typeBXOR1 (control) were inoculated in the leaves of 14 day old susceptible Taichung Native 1 (TN-1) variety of rice. After 10 days of inoculation, leaves were crushed and dissolved in 1 ml of MUG extraction buffer without adding MUG substrate (4-methylumbelliferyl β-D-glucuronide). Subsequently, 250 μl extraction buffer containing MUG was added, and incubated at 37°C for appropriate time (Jefferson et al., 1987). Next, 75-μl aliquots were taken from each reaction mixture, and the reaction was terminated by the addition of 675 μl Na2CO3 (0.2 M). Fluorescence was measured against 4-methyl-umbelliferone (MU; Sigma) as standard at excitation/emission wavelength of 365/455 nm, respectively in

grown culture was inoculated in fresh PS medium with or without 50 μM 2, 2’-dipyridyl and grown at 28°C. At regulartime intervals, 1 ml culture was removed to determine OD at 600 nm. Furthermore, for GUS assay, 1 ml culture was centrifuged to obtain the pellet, which was washed once in sterile miliQ water, and resuspended in 250 μl volume of 1 mM MUG (4-methylumbelliferyl β-D-glucuronide) extraction buffer (50 mM sodium dihydrogen phosphate [pH 7.0], 10 mM EDTA, 0.1% Triton X-100, 0.1% sodium lauryl sarcosine, and 10 mM β-mercaptoethanol),and incubated at 37°C (Jefferson et al., 1987). After appropriate time intervals, 75 μl aliquotes were taken from each reaction mixture, and reaction was terminated by adding 675 μl Na2CO3 (0.2 M). Fluorescence was measured against 4-methyl-umbelliferone as the standard at excitation/emission wavelength of 365/455 nm, respectively. Likewise, GFP activity was measured in Varioscan flash (Thermoscientific) at exitation/emission wavelength of 472/512 nm, respectively by taking 200 μl of culture directly

For reporter assay, GUS and GFP marked Xanthomonas oryzaepv. oryzicolastrains and control strains were grown overnight in PS medium. 0.2

2 bed volumes of methanol, and equilibrated with 5 bed volumes of distilled water. In order to reduce the water solubility of siderophore in the supernatant, it was acidified to pH 2 using concentarted HCl. This acidified supernatant was passsed through the column, and finally eluted with 160 ml methanol by collecting approximately 60 fractions (2 ml each) of the flow through. Siderophore assay was done on CAS plate with each collected fraction. Fraction that gave orangish-yellow halo for the siderophore on CAS plate, was combined together, dried in rotary evaporator and finally reconstituted in 1 ml methanol for further quantification using HPLC as described previously (Amin et al., 2009).For HPLC analysis, siderophore samples were filtered through filter membrane (porosity, 0.45 μ). Next, 10 μl sample was injected into Agilent C18 (4.6mm×250mm×5μm) column (gradient:(A=H2O/0.1%TFA), (B= CH3CN/0.1%TFA) 0-30% B in 10 min, 30-45% B in 15 min,45-0%B in 20 min at a flow rate of 1 ml/min). Similarly, standard vibrioferrin (siderophore produced by Xanthomonas) was also estimated through HPLC for comparison. Fe(III) bound vibrioferrin complex was prepared by incubating FeCl3.6H2O and apo-vibrioferrin for overnight. This complex was detected at300 nm (RT 10.998 min), whereas apo-vibrioferrin was detected at 220 nm at RT 10.988 min. The siderophore concentration in the samples were determined by peak area and calculated against the standard curves obtained from standard vibrioferrin. The siderophore from the test samples were detected at 300 nm, which confirms that majority of the vibrioferrin isolated from the culture was present in bound form

Different Xanthomonas oryzaepv. oryzicola strains were grown overnight in PS medium at 28 °C and 200 rpm. 0.2% of the overnight grown culture was inoculated in the the fresh PS medium supplemented with 50 μM 2, 2’-dipyridyl, and grown till OD600 reached to 1. Cultures were centrifuged at 12,000 g for 50 min to get the cell free culture supernantant, which was collected into acid treated bottles. Excess exoplysaccharide was removed by centrifugation for longer time. Siderophore was initially isolated by column chromatography as described previously (Wright, 2010). Briefly, 220 g of XAD-16 resin was soaked overnight and packed into the column (2.4×30 cm), column was wa

concentarion in the samples were determined based on their peak area against standard oxalic acid plot.For GC-MS analysis, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization was performed with the dried HPLC fraction of samples as well as standards as described previously (Šťávová et al., 2011). Briefly, 200 μl BSTFA, and 100 μl hexane were added to the sample, and incubated at 50 °C for 70 min. GC analyses were performed using a Shimazdu GP 2010 plus instrument equipped with an autosampler, and a split injector.Separations were accomplished using a 30-m long DB-5 capillary column, 0.25 mm internal diameter (I.D.) at a constant helium flow rate of 1.5 mL/min. Samples (10 μL) were injected with a split ratio of 10 into the column at 100 °C. The final column temperature program started at 100 °C and attained final temperature 280°C with a gradient increase of 5 °C/min. The MS data (total ion chromatogram,TIC) was acquired in the full scan mode (m/z of 50–500) at a scan rate of 1000 amu using the electron ionization (EI) with an electron energy of 70 eV. The acquired spectrum was searched against standard NIST-05 library

Xanthomonas oryzaepv. oryzicola strains were grown overnight in PS medium supplemented with appropriate antibiotics. 0.2 % of the overnight grown culture was reinoculated in 250 ml of fresh PS medium supplemented with 50 μM 2,2’-dipyridyl, and allowed to grow till OD600reached 1. Cultures were centrifuged to obtain cell free culture supernatant, concentrated on vaccum evaporator, and freeze dried at regular time intervals to remove the water completely. Oxalic acid was estimated from the dried supernatant by using Agilent 1100 series HPLC system as described previously with slight modifications (Ding et al., 2006). In brief, dried supernatant fractions of different cultures were dissolved in mobile phase of pH 2.7, and allowed to stand for 3 h for the precipitation of humic substances. These samples were filtered through membrane filter (porosity, 0.45 μm), and 20 μl volume of the filtrate was injected into the Agilent C18 (4.6 mm× 250 mm× 5 μm) column. The mobile phase used was 10 mM KH2PO4-CH3OH (95:5, pH 2.7), and the samples were separated by isocratic elution at 0.8 mL/min at 26°C temperature. Standard oxalic acid was detected in similar way in mobile phase (pH 2.7 at 210 nm) with retention time (RT) of 6.7 min. Likewise, oxalic acid in the test samples were also detected at 210 nm with RT 6.7

CFUs/ml) onto fully expanded leaf, and pricking with sterile needle to facilitate the entry of bacteria inside the leaves through wound. To detrmine the growth of bacteria inside leaves, 1 cm2 leaf area surrounding the inoculation site was cut at regular time intervals, surface sterilized by dipping in 2% (vol/vol) sodium hypochlorite for 2 min, and washed twice in sterile water. For getting the CFUs, leaves were crushed using mortar and pestle, serially diluted, and plated on PSA medium containing appropriate antibiotics

Exogenous iron supplementation was performed as described previously (Chatterjee and Sonti, 2002). Briefly, leaves of 40-day-old greenhouse-grown rice plants of the susceptible rice cultivar Taichung Native-1 (TN-1) were cut with scissors 2 cm above the junction of the leaf blade and leaf sheath. These cut leaves (25 leaves per flasks) were dipped in 250 ml conical flasks containing 200 ml 1μg/mlof Benzyl amino purine (BAP) in double distilled water. BAP (a cytokine hormone) maintain the detached rice leaves in fresh condition for longer period. For iron supplementation, FeCl3 was added to a final concentration of 50 μM (stock-10 mM). Prior to inoculation with different strains of Xanthomonas oryzaepv. oryzicola, the leaves were maintained overnight on a laboartory bench top. Strains were inoculated into the leaves by needle pricking method by dropping 20μl of bacterial suspension (approx. 1 × 108bacterial

Ferric-iron-reduction activity of Xanthomonas oryzaepv. oryzicolawas measured using ferrozine, a chromogenic ferrous iron chelator, as described previously (Velayudhan etal., 2000; Worst et al., 1998). For estimating the ferric reductase activity, Xanthomonas oryzaepv. oryzicolastrains were grown in 20 ml PS medium carrying appropriate antibiotics for 24 h to OD600 of 1. Cell free PS media was incubated under similarcondition to be used as control. Chromogenic ferrous iron chelator, ferrozine was added to a final concentration of 1 mM, and FeCl3was added as ferric iron source to a final concentration of 50 μM, and incubated at 28ºC. At regular time intervals, 1 ml aliquotes were taken from the test culture and control, centrifuged to remove the cells, and absorbance of the magenta coloured Fe2+-ferrozine complex in the cell free culture supernantant was measured at 535 nm by using control supernatant as reference. The Fe2+reduction activity was quantified as micromoles of Fe2+-Ferrozine complex formed

media containing 50 μM 2’2’-dipyridyland grown for 24 h at 28°C with continuous shaking at 200 rpm. Cells were harvested by centrifugation at 7000 g for 10 min at 4 °C, washed twice with 50 mM phosphate buffer (pH-7.4), and finally resuspended in phosphate buffer. The bacterial suspension was then diluted with chelex-100 treated PS to get a final OD600of 1.0 and incubated at 28°C for 5 min. Iron transport assay was initated by adding 55FeCl3(American radiolabeled chemicals, Inc., St. Louis, USA,specific activity 10.18 mci/mg) to a final concentration of 0.4 μM into the bacterial suspension. The radiolabelled stock solution was diluted with water and 1M sodium ascorbate for 55Fe3+uptake and 55Fe2+uptake studies, respectively. For uptake of FeCl3bound vibrioferrin, both vibrioferrin (7.6 mM stock) and 55FeCl3were incubated in 1:1 ratio by diluting it appropriately with water and uptake was initiated with a final concentartion of 0.4 μM. To stop the uptake, 200 μl of bacterial cell suspension was layered and immediately centifuged (13000 g; 1 min) through 300 μl of di-butylphthalate and di-octyl phthalate (1:1) mixture. The upper aqueous layer and organic solvent was aspirated, and pellet was resuspended in 100 μl Triton-X-100. The suspension was added to 5 ml scintillation cocktail, and radioactivity count was determined in the 3H channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). As control, both Fe2+and Fe3+uptake assays were performed in presence of proton motive force uncoupler carbonylcyanidep-trifluoromethoxyphenylhydrazone (FCCP; 50 μM), to distinguish between non-specific uptake of readiolabelled Fe by the bacterial cells. However, no significant increase in the incorporation of Fe2+and Fe3+ was observed in presence of FCCP, which indicated that iron uptake by these strains is energy-dependent process

In vitro transport assay was performed by using radiolabelled iron to measure the capacity of Xanthomonas oryzaepv. oryzicola strains to transport 55Fe(II) and 55Fe(III) forms of iron as described previously with slight modifications (Ardon et al., 1997; Velayudhan et al., 2000). For iron uptake asssay, Xocwild-type BXOR1 strain, ∆rpfF mutant and the complemented strain harboring full length rpfFgenewere grown overnight in PS medium. 0.2% of the overnight grown culture was inoculated in fresh P