Sequencing of ADHT gene

Inhibition of haemocytes spreading behavior

Oral toxicity bioassay

Organic carbon

pH

Calculation

Physical Characterizatio

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

Procedure:

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

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

DNA sequencing

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

Test for canavanine (CAN) sensitivity

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

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

Zymolyasedigestion assay

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

Measurementof intracellular pH (pHi)

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

Protein extraction and immunoblotting

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

Post-hybridization washes

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

Ligation of DNA

biosensor strain 8523/KLN55was inoculated in fresh medium, and grown with the ethyl acetate extract isolated from the test strain as described earlier. After 30 h of growth, cells were pelleted by centrifugation, washed once with sterile water and resuspended in sterile miliQ waterfor measuring the GFP fluorescence intensity at excitation and emission wavelength of 472 and 512 nm, respectively. 1 DSF unit is equivalent to increase in fluorescence by 1 arbitary unit in DSF biosensor strain

For DSF extraction, X. oryzaepv. oryzicolastrains were grown in PS media to an OD600 of 1.2 as described earlier. Supernatant was collected by pelleting down the cells at 7000 g for 10 min. Next, water-saturated ethyl acetate was added to the cell-free culture supernatant in a ratio of 2:1, and mixed properly for 5-10 min. The mixture was centrifuged at 5000 g to separate the DSF containing organic phase. The ethyl acetate layer (organic phase) was evaporated at 37°C, remaining residue was dissolved in methanol, and assayed for DSF by using Xccbiosensor strain 8523/KLN55 (Newman et al., 2004). Biosensor strain is a DSF minus strain comprised of DSF responsive endoglucanase promoter fused to promoterless gfpand expressed through plasmid (Peng::gfp). To check the DSF production by a particular strain, 0.2% inoc

Isolation and detection of DSF

Radioactive counts measured in2x106labelled C. glabratacells and lysates were considered as ‘input’ and ‘output’ values, respectively. Percentage adherence was calculated by following equation.%Adherence=Output radioactive countsInput radioactive countsX 100

Adherence of C. glabratacells to Lec2, Chinese hamster ovarian (CHO) cells, wasdetermined as described previously (Cormack et al., 1999). Briefly, Lec2 cells were seeded at a cell density of 5x105cells per wellin a 24-well tissue culture plate.Cells were incubated in a cell culture incubator (Thermo Scientific) set at 37°C and 5%CO2for 12 h. Post incubation, the medium was discarded in a reservoir and Lec2 monolayer was washed thrice with sterile 1X PBS without disturbing the monolayer. Lec2 cells were fixed with 3.7% para-formaldehyde for15 min followed by twoPBS washes. 1 mlof 1X PBS containing antibiotics, penicillin (100 units/ml) and streptomycin (100 μg/ml), was added to each well, plates were sealed with PARAFILM, Cole-Parmer(PM-996) and stored at 4°C until use.C. glabrata cells,to be tested for their adherence potential, were grown in CAAmedium for 24 h.100 μl of 24 h-grownculture was re-inoculated in fresh 5 ml CAAmedium containing 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN label mix (JONAKI, India)in a 15 ml polypropylene tube.Cultures were allowed to grow for 16-20 h at 30°C with shakingat200 rpm to radiolabel the cells. Radiolabelled C. glabratacells were harvested by spinning down1 ml of labelled yeast cultures,andthe cell pellet was washed thrice with sterile 1X PBS to remove any residual S35(Met:Cys-65:25) labelling mix from the medium. Post washes, the pellet was resuspended in 1 ml PBS, OD600was measured andcell suspension of 0.4 OD600wasprepared.Next, 24well plates containing fixed Lec2 cells were taken out from 4°C and PBS from the wells wasdiscarded by inverting the plates. Wells were washed once with PBS and 2x106labelled yeast cells were added to eachwell, andincubatedfor 30 min at room temperature.Post incubation, plates were centrifuged at 1,000 rpm and the wells were washed thrice with 1X PBS to remove non-adherent C. glabratacells. Lec2 cells were lysed with 5% SDS in PBS by scraping the wells, lysates were collected and transferred to a vial containing scintillation fluid

Adherence assay

Reverse transcriptase “Superscript III” (Invitrogen, 18080-051) was used to perform cDNA synthesis. Briefly, 500 ng of DNase I-digested RNA was incubated with 1 μl of 10 mM dNTP and 50 μM oligo(dT) at 65°C for 5 min in a 10 μl reaction mixture followed by cooling on ice for 5 min. Post incubation, 10 μl of cDNA synthesis mixture was added which contained 2 μl of 10XRT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNase out (40 units) and 1μl of Superscript III (200 units). Tubes were incubated at 50°C for 1 h and thereaction was terminated at 85°C for 5 min. The quality of synthesized cDNA was checked by using it as a template in a PCR reaction to amplify the housekeeping gene CgACT1. Amplification of CgACT1was indicative of proper cDNA synthesis

Complementary DNA (cDNA) synthesis

Reverse transcriptase “Superscript III” (Invitrogen, 18080-051) was used to perform cDNA synthesis. Briefly, 500 ng of DNase I-digested RNA was incubated with 1 μl of 10 mM dNTP and 50 μM oligo(dT) at 65°C for 5 min in a 10 μl reaction mixture followed by cooling on ice for 5 min. Post incubation, 10 μl of cDNA synthesis mixture was added which contained 2 μl of 10XRT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNase out (40 units) and 1μl of Superscript III (200 units). Tubes were incubated at 50°C for 1 h and thereaction was terminated at 85°C for 5 min. The quality of synthesized cDNA was checked by using it as a template in a PCR reaction to amplify the housekeeping gene CgACT1. Amplification of CgACT1was indicative of proper cDNA synthesis

Complementary DNA (cDNA) synthesis