Isolation of plasmid DNA

Total RNA was isolated by TRIzol method using the manufacturer’s protocol. Briefly, medium was removed from culture dish and recommended amount of TRIzol wasadded directly on to the dish and kept at room temperature for 5 minutes for lysis of cells. The cellular homogenate was then transferred to a 1.5ml microcentrifuge tube. For each mlof TRIzol, 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 of 12,000 rpm for 10 minutes. The upper aqueous phase was transferred into a fresh micro centrifuge tube and RNA was precipitated by adding 500μl of iso-propanol. The RNA pellet was obtainedby centrifugation at 12,000 rpm for 30 minutes at 4°C. The pellet was washed with 1ml of chilled 70% ethanol followed by centrifugation at 12,000 rpmfor 5minutes. The supernatant was removed and the pellet air-dried for about 5 minutes. The pellet was resuspendedin 30-50μl RNase free deionisedwater and dissolved at 55ºC followed by quantificationusingnanodrop spectrophotometerfor further use.The RNA integrity was checked by evaluating the 18S and 28S rRNA signals by running 1μl of total RNA on denaturing agarose gel stained with ethidium bromide

Total RNA isolation from cultured cells

nvolved use of GFP based vector system, the expression of the transgene was visualized under fluorescent microscope with excitation filter of 485+20 nm

Transient transfection of plasmid DNA in cellswas performed usingLipofectamine 2000transfection reagentaccording to manufacturer’s protocol. Briefly, 0.5 to 1million cellswere seeded in a 35mm tissue culture dish one day prior totransfection. For each 35mm dish, 4μg DNA was mixed in 250μl of Opti-MEMin one polypropylene tube. In another tube 10μl of Lipofectamine 2000 was diluted in250μl Opti-MEM and incubated at room temperature for 5 minutes. DNA and Lipofectamine 2000 were mixed together and allowed to form complexes for 30minutes at room temperature. Meanwhile, the adherent cells were washed twicewithPBS and 1ml of Opti-MEM was added. 500μl of complexes were then added to each dishcontaining cells and medium. After 6-8 hrs, the medium containing complexes wasremoved and complete medium was added and transgene expression was evaluated 24-48 hrs after transfection. Since most of the experiments

Transient transfection in adherent cells

Themixture is incubated in a water bath at 37⁰C for 15 min and afterwards transferred on ice and 4μl of DNA loading buffer is added. The samples were then run on a polyacrylamide gel electrophoresis which had been pre-run for 30 min. Electrophoresis was carried out at 4⁰C for 3h till the bromophenol blue migrated to 2cm above the bottom of gel. The gel was taken out and kept on Whatman filter paper sheet and covered by saran wrap followed by drying in a gel dryer at 80⁰C for 1h under suction. The dried gel was exposed to phosphoimager screen by keeping in phosphoimager cassette overnight

A binding reaction mixture was prepared by adding the following components to a microcentrifuge tube on ic

Binding reaction

The reaction was carried out by incubating at 37⁰C for 30 min. The reaction was stopped by adding 2μl of 0.5M EDTA, pH 8.0 and keeping on ice. A spin column was prepared using 1ml syringe and packed with sterile Sephadex G50 slurry and reaction mixture is applied on the top. The eluate is collected in different microcentrifuge tubes and radioactivity was counted using Geiger counter. The tube showing 7 to 9X106was used for experiment. The column containing the unincorporated [γ-32P] ATP was discarded in radioactive waste bin. The radiolabelled oligonucleotides were annealed with their corresponding complementary unlabelled oligonucleotides. A 50 fold molar excess of the latter was used for annealing for conversion of labelled single strand to double strand. Thetubes were kept in boiling waterbath for 3 min followed by room temperature for 30 min. The tubes were transferred to ice and the oligonucleotides were diluted to 4fmoles/μl using sterile H2O

The oligonucleotides were labelled at their 5'end with 32P using T4 polynucleotide kinase (T4 PNK) enzyme in a reaction given belo

end labelling of the oligonucleotides

Electrophoretic mobility shift assay

Adherent cells growing either on cover slips or chamber slides were fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed with PBS thrice for 5 min each and blocking was done in 2% BSA(preparedin PBScontaining 0.3% Triton-X 100) for 1h.The cells were incubatedwith primary antibody(dilutedin PBScontaining 0.3% Triton-X 100)for 2h at room temperature or overnight at 4⁰C.The cells were washed with PBS thrice for 5 min each followed by incubation withAlexa Fluor 488-or 594-conjugatedsecondary (anti-mouse/rabbit) antibodiesfor 1h. Then the cells were mounted on microscopicslides using Vectashieldmountingmediumcontaining nuclear dye DAPI. Imaging was done byeither the laser scanning confocal LSM510 or LSM 750 (Carl Zeiss, Oberkochen, Germany) or fluorescence inverted (Olympus 1X51, Tokyo, Japan) microscope

Immunofluorescence Microscopy

Equal amount of proteins were loadedon an appropriate percentageof denaturing SDS-PAGE gel. After completion ofthe run, the gel was over laid on a PVDF membranecut to the size of gel and sandwiched between filter paper sheets and kept inthe blotting cassette in the presence of transferbuffer. Finally the cassette was put in themini transblotapparatus and blotting was done for 2-3hours at a constantvoltage of 80Vat 4⁰C. For blocking the nonspecific sitesmembrane was incubated with blocking solution(5% non-fat milk solution in TBST)with gentle shaking for 1 hourat room temperature. Excess milk from the membrane was washedoff with TBST and themembrane was incubatedwith primary antibody diluted in 1XTBST for 3 hours atroom temperature or overnight at 4°C withshaking. After incubation the membrane was washedwith TBST and incubatedwithappropriate secondary antibody (conjugated with horse-radish peroxidase)diluted in5% fat free milk solution (in TBST) for 1hat room temperature.The blotwas later washed thricefor 10min eachwith TBST and processed for the detection of proteinsignal using ECL-prime chemiluminescencedetection reagent followed by detectionof signal either on X-ray filmor in a chemiluminescence detectionsystem(Proteinsimple, California, USA)

Immunoblotting

BCA (Bicinchoninic acid) method was used to determine the proteinconcentrationin various samples. The Cu2+ions from cupric sulphate (present inBCA reagent B) reagent arereduced to Cu+by the protein in an alkaline medium. The cuprous ion (Cu+) then combines with BCA (present in BCA reagent A) to give a purple colour whose intensity is proportional to the amount of protein present in the samples. This intensity is measuredby colorimetry at 562 nm. BCA reagent was prepared by mixing reagent A with reagent B in avolumeratio of 50:1. A standard curve was generated using increasing concentrations of BSA (2-10μg) in a 25μl reaction, in a 96 well plate. Cell lysates were also dilutedto same volume in parallel wells. 200μl of BCA reagent was then added to each well and incubated at 370C for 30 minutes. The absorbance readings were then takenin a spectrophotometer at 562 nm. Total protein was quantified by calculation of the slopes of regression lines ofabsorbanceand BSA standards

Protein estimation

For preparation ofcellular homogenate from adherent cell culture, the medium was first removed and cells were washed with ice cold 1X PBS. The cells were then scraped in 1X PBS and pellet down by gentle centrifugation (4000 rpm for 2 minutes) at 40C. Cell lysis buffer was then added to the cell pellets and lysis was allowed for 30 minutes on a rotor at 4⁰C. Post lysis, cellswere centrifuged at 13000 rpm for 10min at 4°C. The pellet was discarded and supernatantwascollectedas cell homogenate

Extraction of total cellular protein

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

Cell cycle analysis

Cells were seeded in replicates of five @ 3X103cells per wellinfive different 96well cell culture platesand grown in complete media. The method described earlier was slightly modified and followed (Gillies et al., 1986). After every 24h of seeding, one plate was stained with 0.2% crystal violet in 2% ethanolfor 15 minutestill 4thday i.e. 96h.One plate was stained just after the cells get attached to use as 0h time point. Excess dye was removed from the plates by washing with ample amount of water. Crystal violet dye incorporated in the cells was extracted using 0.1% SDS solution by shaking for 10 minutes on a shaker. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. The experiment was repeated at least three times and the average absorbance was plotted for each time point to generate a growth curve

Cell growth Assay

Table2.2: Cell types used in the present study

In the present thesis, various cell types were used for which the details are provided in the Table 2.2. SiHa, HeLa, HaCaT, U2OS, SaOs , A549,HPLD andHEK-293cells were grown in Dulbecco’s modified Eagle’s medium (HyClone, Thermo Scientific, Logan, Utah, USA) supplemented with 2 mM glutamine (Gibco BRL), 100 U/ml penicillin and streptomycin (Gibco BRL, Carlsbad, CA, USA), and 10% fetal bovineserum (Gibco BRL, Carlsbad, CA, USA) under humified conditions at 37°C and 5% CO2.Cells were grown in cell culture dishes till they attained 70% confluency. For sub culturing, these were then trypsinised using 0.05% Trypsin EDTA solution and incubated for 5 minutes at 370C for cells to be detached from surface. The detached cells were then collected by gentle tapping the dish and pipetting. Trypsin was then inactivated by addition of FBS containing culture medium, transferred to a 15 ml tube and centrifuged at 1500 rpm for 2 minutes in a hanging bucket centrifuge. The cell pellet was then resuspended in complete medium and counted in Neubauercell counting chamber. Viability of the cells was checked by trypan blue exclusion method.Appropriate number of cells wasthen sub cultured in fresh cell culture dishes with culture medium as per the experimental requirements

Maintenance of cell lines

Cell biology methods

Cell lysis Buffer

Fixative

TAE

Resuspension solution(Solution I)

Polydeoxy (Inosinate-cytidylate) (Poly dI-dC)

Cytoplasmic extractionbuffer (without protease inhibitors)

Fixative : 4% Formaldehyde

Cell lysis buffer(RIPA Buffer)

Phosphate Buffered Saline (PBS)

Ammonium persulfate(APS)

Acrylamide (29:1)

Phenylmethylsulfonyl fluoride (PMSF)

Benzamidine

Aprotinin

Leupeptin

NP-40ComponentsFinal concentrationFor 10 mlNP-4010%1mlH2O9ml

Dithiothreitol (DTT)ComponentsFinal concentrationFor 5 mlDTT1.0M0.7725gH2Oq.s

Ethylenediamine tetraacetic acid (EDTA), pH 8.0ComponentsFinal concentrationFor 500 mlEDTA0.5M93.05gH2Oq.sThe pH is adjusted to 8.0 using 10M NaOH

Ethylene Glycol Tetraacetic acid (EGTA), pH 7.0ComponentsFinal concentrationFor 50 mlEGTA0.1M1.902gH2Oq.sThe pH is adjusted to 7.0 using 10M NaOH

Potassium Chloride (KCl)ComponentsFinal concentrationFor 100 mlKCl2M14.91gH2Oq.s

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

Potassium Chloride (KCl)

HEPES pH 7.9ComponentsFinal concentrationFor 100 mlHEPES1M23.83gH2Oq.sThe pH wasadjusted to 7.9 using 10M NaOH

Stock solution

forpreparation ofregular buffers and solutions viz. Tris, Glycine, SDS, Sodium Chloride, Potassium Chloride, Disodium Phosphate,NP-40, Tween 20, TritonX100, Formaldehyde, Glycerol, Agarose, Acrylamide,Bis-Acrylamide,Ammonium per sulphate (APS), TEMED,BSA, Propidium Iodide, RNase Aetc. were obtained from Sigma(St Louis, MO, USA). PVDF membrane, X –ray films and western blotting detection reagent (ECL prime) were obtained from GE Healthcare (Little Chalfont, UK). Proteaseinhibitor tablets were obtained from Roche (Penzberg,Germany). Anti mouse and anti-rabbit secondary antibodies tagged to HRP (Horse radish peroxidise) were obtained from Bangalore Genei(Peenya, India). Secondary antibodies for Immunofluorescence (anti mouseIgGand anti rabbitIgG) conjugated to Alexa Fluor (488 and 594) from Molecular Probes, Invitrogen and Vectashield mounting medium with DAPI wasobtained from vector laboratories(Burlingame, CA, U.S.A).Antibodies from different sources were used in the present study. The list of different antibodies used in the present thesis is provided in Table 2.1.Table 2.1: List of antibodies used

Media for cell culture (DMEM and Ham’s F12) and foetal bovine serum (FBS) were obtained from Gibco, Invitrogen (Carlsbad, CA, USA). Cell culturereagents such asTrypsin, Phosphate Bufferedsaline (PBS), Antibiotics, Glutamine, etc. were also obtained from Gibco, Invitrogen (Carlsbad, CA, USA). Chemicals for cell culture experiments Aphidicholin, Nocadazole, Polybrene, and Puromycinwere obtained from Sigma (St Louis, MO, USA). Cyclosporine A, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), wortmannin, UO126, SP 600125, cycloheximide, camptothecin, Tacrolimus/FK506 , Tween 20 and Malachite green were obtained from Sigma-Aldrich (St. Louis, MO, USA). Specific calcineurin substrate RII peptide, calmodulin, eIF-2α inhibitor salubrinal, MG-132 and caspase inhibitor z-VAD FMK were obtained from Calbiochem (San Diego, CA, USA). Cytotoxicity detection kit (LDH) was obtained from Roche Diagnostics, (Mannheim, Germany).Live /Dead cytotoxicity assay kit was obtained from Molecular probes, Life technologies, USA.Lipofectamine-2000 and Opti-MEM for transient transfections were also obtained from Invitrogen(Carlsbad, CA, USA).Growth media for bacteria (LB) was obtained from HiMedia laboratories (Mumbai,India). Enzymes used for recombinant DNA experiments (Restriction endonucleases, DNA ligase) were obtained from New England Biolabs (Ipswich, MA, USA). Markers for DNA and protein gels were from Fermentas (Vilnius, Lithuania). Various kits used for macromolecular isolation (Plasmid isolation kit-Mini and midi, Gel extraction kit, PCR purification kit, RNA isolation kit) were procured from Qiagen(Hilden, Germany) or HiMedia (India).Trizol reagent for RNA isolation was obtained from Invitrogen (Carlsbad, CA, USA). BCA protein estimation kit was from Pierce (Rockford Illinois, USA). Cell fractionation kit was obtained from Fermentas (USA). Kitfor TUNEL assay kit wasobtained from Invitrogen(Carlsbad, CA, USA). PCR reagents (PCR buffer, dNTPs, MgCl2, Taq DNA polymerase) were obtained from Fermentas. Polymerasefor long PCRs (AccuTaq) was obtained from Sigma. Reverse transcriptase (SuperScript III) was obtained from Invitrogen. Various chemicals required

Media, reagents, chemicals and antibodies

Maintenance of cell lines

Inpresent thesis, various cell lines have been used as mentionedearlier. Cells were either cultured in DMEM or RPMI medium containing 10% fetal bovine serum (FBS)along with antibiotics such as penicillin (100 U/ml), and streptomycin (100 μg/ml).In general, cells were grownin tissue culture T-75 flaskupto 85-90% confluency. Cells are washedwith PBS, followed by trypsinization with 0.05% Trypsin EDTA solution. Cells were detachedfrom the surfaceeither by gentle tapping or gentlepipettingor incubated for 5 minutes at 37°C. Culture medium containing serum was then added to inactivate trypsin. After careful mixing, cells were transferred to a 15 ml tube and centrifuged at 800 rpm for 5minutes. The cell pellet wasre-suspended in a fresh culture media containing FBS. The cell viability was checked by trypan blue staining, followed bycounting in Neubauer cell-counting chamber. Appropriate number of cells wasthen either sub-culturedin the ratio of 1:4 to 1:6or seeded in culture dishes as per the experimental requirements.Cells were maintained in humidified incubator at 37ºC in 5% CO2-95% air, throughout the experiment

Extraction buffer

MTT reagent

For Cytotoxicity assays

6XEMSA sample loading dye

5X EMSA buffer

Native EMSA PAGE

10XBinding buffer

For Electrophoretic Mobility Shift Assay (EMSA)

For preparation of Ultra competent cells

Inoue buffer

6X DNA loading dye

Agarose gel

TAE

For DNA electrophoresis

Nuclear lysis buffer (without protease inhibitors

Cytoplasmic extraction buffer (without protease inhibitors)

For Cell fractionation

Blocking buffer: 2% BSA

Permeabilisation buffer: 0.2% Triton X100

4% Formaldehyde fixative

For Immunofluorescence(IF)

Stripping buffer

Blocking buffer

TBS-T

Transfer buffer

(f) Running buffer

(e) Stacking polyacrylamide gel

(d) Resolvingpolyacrylamide gel

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

(b) Celllysis buffer B(For IB)

Cell lysis bufferA(For IP)

II. For Immunoprecipitation(IP)and Immunoblotting(IB)

Table 2.1: Commonly used buffers and solutionsI. General buffers(a)Phosphate Buffered Saline (PBS)

The following antibodies were used in the present study:Primary antibodies against GAPDH (anti-rabbit), FLAG (anti-mouse), Immunoglobulin (IgG, anti-rabbit or anti-mouse),profilin-1 (anti-rabbit), tubulin (anti-mouse) and ubiquitin (anti-rabbit) were obtained from Sigma Aldrich Chemicals(St Louis, MO, USA). Antibodies againstAKT (anti-rabbit), cleaved caspases-3, 8 and 9 (anti-rabbit),HA-tag(anti-rabbit), Myc-tag (anti-rabbit), p21 (anti-rabbit), phospho-p53 (anti-mouse), PTEN (anti-mouse), phospho-AKT (Ser473; anti-rabbit), phospho-GSK-3β (Ser9; anti-rabbit), phospho-IKKα/β (Ser177/181; anti-rabbit), phospho-IκBα (Ser32; anti-rabbit), and phospho-p65 (Ser276; anti-rabbit) were obtained from Cell Signaling Technologies(Danvers, MA, USA), whereas antibodies for cox-2 (anti rabbit), c-Rel (anti-rabbit), ICAM-1 (anti-rabbit), IKKα/β (anti rabbit), IκBα (anti-rabbit), Mdm2 (anti-rabbit), PARP-1/2 (anti-rabbit), Rel-B (anti-rabbit), p50 (anti-rabbit), p53 (anti-mouse), p65 (anti-rabbit) were obtained from Santa Cruz Biotechnology(Santa Cruz, CA, USA).HRP (Horse radish peroxidase)-conjugated secondary antibodies (anti mouse and anti-rabbit) were obtained from Bangalore Genie(Peenya, India). For immuno-fluorescencestudies, secondary antibodiesconjugated toAlexa Fluor (488 and 594, anti-mouse and anti-rabbit) were obtained from Molecular Probes, Invitrogen(Eugene, OR, USA)

Antibodies

S. cerevisiae strains were routinely grown either in rich YPD mediumorsyntheticcomplete medium (SC)(Section 2.1.5.1) at 30°C with continuous shaking at 200 rpm unless otherwise stated. In general, S. cerevisiae frozen glycerol stocks were revived on 2% YPD medium by streaking and allowed to grow for 1-2 days. S. cerevisiae strains harbouringaplasmid containingthe URA3geneas the auxotrophy selectionmarker were revived on synthetic complete medium lacking uracil (SC-Ura).To prepare liquid cell culture, a single colony of each S. cerevisiae strain was inoculated either in YPD or SC-Ura medium and grown for 14-16 h. S. cerevisiae strains streaked on plates were sealed with paraffin film (parafilm M) and stored at 4°C for a maximum period of 2 weeks.Protein over expression in yeast was carried in presence of galactose instead of glucose as the carbon source, as the plasmid pYesGex 6p2 carries the GALpromoter under which yeast proteins were expressed

Strains and culture conditions

20 mM HEPES pH 6.8100 mM NaCl2 mM EDTA5 mM DTTYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer A)

Buffer A

EDTA (pH 8.0)186.1 g of EDTA.2H2O was dissolved into 800 mL of water stirredvigorously and the pH was adjusted with NaOH pellets. When the pH of the solution reached8.0 EDTA dissolvedcompletely and was made upto 1000 mL with water.Tris-HCl buffer (1M)121.1 g of Tris base was dissolved in 800 mLof water and pH was adjusted to 7.2 using concentrated HCl Tris-EDTA (TE) buffer 10 mM Tris-HCl, pH 8.01 mM EDTA Tris-Acetic acid EDTA (TAE) buffer 40 mM Tris base 1mMEDTApH was adjusted to 8.4with glacial acetic acid. TAE buffer was prepared as a 50X stock solution and used at 1Xconcentration.Tris-Saline20 mM Tris-HCl, pH 7.20.9% NaCl

PhosphateBuffered Saline (PBS) 137 mM NaCl 2.7 mM KCl 10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 using HCl and NaOH beforeautoclaving. PBS was prepared as a 10X stock solution and diluted to 1X concentration before autoclaving

Common buffers

Yeast synthetic complete medium without leucine(SC-Leu)0.67% Yeast Nitrogen Base without amino acids 76mg/L His76mg/L Ura76 mg/mL Trp76 mg/mL Met2% DextroseYeast sporulating medium1% Potassium acetate0.05% Dextrose

Yeast extract Peptone Dextrose (YPD)1% Yeast extract2% Peptone 2% Dextrose Yeast synthetic complete medium(SC)0.67% Yeast Nitrogen Base with amino acids 2% Dextrose1.92 g/LYeast Synthetic Drop-Out media supplement without Uracil76 mg/L uracilYeast synthetic complete medium without histidine(SC-His)0.67% Yeast Nitrogen Base without amino acids 1.92 g/L Yeast Synthetic Drop-Out media supplement without histidine2% DextroseYeast synthetic complete medium without uracil(SC-Ura)0.67% Yeast Nitrogen Base without amino acids 1.92 g/LYeast Synthetic Drop-Out media supplement without Uracil2% DextroseYeast synthetic complete medium without methionine(SC-Met)0.67% Yeast Nitrogen Base without amino acids 380mg/L Leu76 mg/L His76mg/L Ura2% DextroseYeast synthetic complete medium without tryptophan(SC-Trp)0.67% Yeast Nitrogen Base without amino acids 380mg/L Leu76mg/L His76mg/L Ura76 mg/L Met2% Dextrose

Yeast media(Media composition was followed as described by Sigma product data sheet)

All S. cereviseae and bacterial strains and plasmids used in this study are listed in Table 2.1and 2.2

Strains and plasmids

Cells were plated in a manner that they were 30-50% confluent on the day of transfection.Cells were washed with serum-free medium,and the serum-free medium was added to the cells as per plate size. SiRNA was diluted in the serum-free medium, and oligofectamine was diluted in serum-free media, separately (Table 10). Both the complexes were incubated at room temperature for 5 min. Diluted siRNA wasmixed gently with diluted oligofectamine and incubated at room temperature for 15 min. The final transfection mixture was added dropwise to the cells and mixed properly by gentle rocking. Cells were incubated for 4 hrs.,and the growth medium containing 10% FBS was added to the plates without removing the previous medium. Cells were incubated overnight at 37°C in a CO2 incubator. After overnight incubation, the siRNA transfection was repeated using the same protocol. Cells were harvested after 24-48 hours of second round siRNA transfection. The knockdown was detected bychecking the protein levels throughwestern blotting. (Note: SiRNA transfection is carried out in antibiotic free medium)Table 10: SiRNA transfection methodology

SiRNA

Table 8: Lipofectamine plasmid-transfection methodology

For transfection with Lipofectamine, cells were plated in antibiotic-free medium 24 h before transfection and were transfected at a confluency of 70-80% as per the manufacturer’s protocol. The plasmid of interest was incubated in serum free media,and Lipofectaminewas incubated in serum free media forseparately5minutes. The plasmid and the Lipofectamine mixtures(Table 8)were mixedgentlyand incubated at room temperature for 20 min.;thetransfection mixture was added dropwise to the cells. Transfection media was replaced with the fresh complete medium after 6 hrs.of transfection and cell are harvested after 24 hours

Plasmid transfection using Lipofectamine 2000

Amplified PCR products were run on Agarose gel to check for the amplification ofgene of interests

PCR amplification of the gene of interests was carried out by following the method mentioned in table 4.Table 4: PCR methodology

Polymerase chain reaction (PCR)

The following chemicalswere used in the present study: Ampicillin, EDTA (USB), dNTPs, Taq DNA polymerase(Fermentas), Pfu DNA polymerase (Stratagene), DpnI (New England Biolabs), Plasmid miniprep, midiprep, and maxiprepkits(Qiagen, and Invitrogen), glycine, EGTA, NaCl, Tris (Fisher Scientific), NH4Cl, acrylamide(SRL), Cisplatin, Doxorubicin, MG132,Cadmium chloride,Nonident P-40, propidium iodide (PI),bis-acrylamide, SDS, TEMED, Ammonium persulphate (APS), CoomassieBrilliant Blue, DAPI, IPTG, kanamycin, Aprotinin, pepstatin, PMSF, -Glycerophosphate, Sodium Fluoride (NaF),Biotin, and DMSO(Sigma), Luciferase assay kit (Promega#1500), Gateway cloning kit, DMEM, FBS, RPMI, Opti-MEM medium,Met-/Cys-DMEM, dialyzed FBS,trypsin-EDTA, L-glutamine, PBS, Lipofectamine 2000, Oligofectamine, (Invitrogen), PEI(Polysciences), milkpowder (Warana), protein G agarose beads, Streptavidin sepharose beads, Glutathionesepharose beads, MBP beads (GE Healthcare), S-protein beads (Novagen/Calbiochem), HA beads(Covance), LB media(Himedia)

Chemicals and reagents

development. Absorbance was measured at 490 nm, and concentration of glucose production was calculated against glucose standard. Cellulase activity is expressed as micromoles of reducing sugar (glucose) released per minute per 109cells. For plate assay, cell-free culture supernatant of X. oryzaepv. oryzaestrains were inoculated in wells of 0.2% CMC agarose plates. In addition, cellulase assay was also performed by spotting the colony on 0.2% CMC PSA plates. Plates were incubated for 8 to 24 h and stained with congo red to observe the halo formation as described previously (Wood and Bhat, 1988). Extracellular xylanase activity in different X. oryzaepv. oryzae strains was measured using 0.2% 4-O-methyl-D-glucurono-D-xylanremazol Brilliant Blue R (RBB-Xylan) (Sigma-Aldrich) as substrate (Biely et al., 1988)on 1% agarose plates. Xylanase activity is indicated by production of halo around the bacterial colony (Ray et al., 2000). Similarly, for lipase activity p-nitrophenyl butyrate was used as substrate. Lipase activity was calculated by measuring the level of p-nitrophenol released upon hydrolysis of p-nitrophenyl butyrate at 410 nm (Acharya and Rao, 2002). Lipase activity was expressed as micromoles of p-nitrophenol released permin per109cells. For plate assay, colonies were spotted on 1% PSA plates containing 0.5% Tributyrin in 100 mM Tris (pH 8) and 25 mM CaCl2 and halo formation was observed for lipase activity

For extracellular enzyme assays, X. oryzaepv. oryzae strains were grown in PS, MM9 and XOM2 media to an OD of 0.6, and centrifuged at 12,000 g for 10 min to collect the supernatant. The supernatant was taken as an extracellular fraction and cell pellet was plated by dilutionplating to get the CFUs per milliliter of culture. Extracellular cellulase activity was measured using phenol-sulphuric acid (H2SO4) method, which measures pentoses and hexoses (concentration of glucose released) upon cellulase activity (DuBois et al., 1956). Briefly, a specific amount of supernatant was taken and volume was adjusted to 300 μl by adding 50 mM acetate buffer (pH-5.4). To this, 1% carboxy methyl cellulose (CMC) substrate solution was added and mixed well. This mixture was incubated at 28°C for 30 min, and the reaction was stopped by adding 1 ml ice-cold ethanol. Solution was mixed well, kept on ice for 5 min and centrifuged at 12,000 g for 5 min. Supernatant was recovered and 5% phenol was added to it, mixed well followed by adding 1 ml H2SO4. The tube was incubated at RT for 20 min for co

Extracellular enzyme assays

200 rpm in LBbroth supplemented with appropriate antibiotics (plasmid antibiotic marker). Cells were harvested by centrifugation at 12,000 g for 5 min. Plasmids were extracted using Qiagen plasmid miniprep ormidiprep kit following the manufacturer’s instructions. Concentration of the extracted plasmid DNAs was measured using spectrophotometer at 280 nm and stored at -20°C

E.colistrains carrying plasmids were inoculated and grown overnight at 37°C and

Plasmid DNA purification

A microtipful cells of bacterial strain from appropriate medium was resuspended in 20 μl sterile water and incubated at 98°C for 10 min for cell lysis. 2 μl of heat-lysed cell suspension was used as template in 25 μl PCR reaction

Xanthomonasand E.colicolony PCR

and finally resuspended in 100 μl sterile water. Bacterial cell suspension was aliquoted in 20 μl volume. The above procedure was followed for all the three strains and cell suspension of three different strains were mixed together in 1:1:1 ratio. For conjugation to occur, 20 μl of the above mixture was spottedon the LB agar plate and incubated at 37°C for 12-16 h. Next, the conjugation drops were streaked on LB agar plate containing appropriate antibiotics to select the S17-1 recipient containing recombinant plasmid.S17-1 was directly conjugated with Xanthomonasstrain. S17-1 strain containing recombinant plasmid (3 ml) and recipient Xanthomonasstrain (100 ml) was grown overnight with appropriate antibiotics. Cells were harvested and washed thrice as mentioned earlier. Xanthomonasstrain was finally dissolvedin 600-700 μl sterile water and S17-1 strain was dissolved in 3 ml sterile water. 50 μl Xanthomonascell suspension and 10 μl S17-1 cell suspension were mixed together and 20 μl was spotted on PS agar plate. After 40 h of incubation at 28°C, each conjugation drop was dissolved in 400 μl water separately and plated on PS agar medium with rifampicin (counter-selectable marker) and plasmid specific antibiotics for specific selection of Xanthomonascolony with recombinant plasmid

Since compatible conjugation does not exist between Xanthomonasand E.coliDH5α strain.Therefore, upon getting the appropriate clones in DH5α, conjugation was performed with S17-1 (recipient strain) and PRK600 (helper strain). All the three strains (DH5α with clone, S17-1 and PRK600 strain of E.coli) were grown overnight at 37°C with constant shaking at 200 rpm in 3 ml LB broth. Cells from 1 ml overnight grown cultures were harvested by centrifugation followed by three washes with s

Xanthomonas conjugation

shaking at 200 rpm. 1% of overnight grown culture was inoculated in 100 ml fresh PS medium and grown to obtain log-phase culture. Log phase Xanthomonas culture was kept on ice for 10-15 min, aliquoted in 50 ml pre-chilled centrifuge tubes and centrifuged at 4000-5000 g at 4°C for 10 min. Supernatant was discarded and pellet from each tube was gently resuspended in 10-20 ml sterile chilled water. Next, cells were harvested by centrifugation at 4000 g at 4°C for 10 min and supernatant was discarded. Harvested cells were washed twice and finally resuspended in adequate amount of prechilled sterile water. 100 μl of cell suspension was aliquoted in sterile 1.5 ml microcentrifuge tubes and kept on ice. For transformation, Xanthomonaselectrocompetent cells and appropriate amount of plasmid DNA was mixed, and kept on ice in laminar hood. This mixture was added to 1 mm electroporation cuvettes (Biorad) and tapped gently to allow the cells to settle properly in order to avoid air bubbles. Competent cells were electroporated (1800 V, 25 μF, 200 Ω, 1mm cuvette) followed by immediate addition of fresh PS broth in the cuvette, mixed properly and taken in the microcentrifuge tubes. Microcentrifuge tubes containing transformed cells were incubated at 28°C for 2 hours with continuous shaking for recovery. After recovery, cells were plated on specific medium with appropriate antibiotics and incubated in 28°C plate incubator

For electrocompetent cell preparation, single colony of desired Xanthomonasstrain was inoculated in 5 ml PS medium and grown overnight at 28°C

Xanthomonastransformation

E.coliDH5α strain was transformed with plasmids carrying appropriate inserts to generate clones, and Xanthomonas deletion strains. Ultracompetent cells stored at -80°C were thawed on ice for 5-10 min. 5 μl ligated plasmid was added to 100 μl ultracompetent cells and incubated on ice for 30 min. Next, competent cells were subjected to heat shock at 42°C for 90 seconds. Cells were immediately transferred on ice for 2-3 min. Next, 1 ml LB medium was added and cells were allowed to recover for 1 h on a shaker incubator set at 37°C. After the recovery, cells were centrifuged at 3000 g for 3 min. Medium supernatant was discarded and cells were resuspended in 100 μl fresh sterile medium. Cells were plated on LB agar containing appropriate antibiotics. Plates were incubated at 37°C for 12-16 h

E.colitransformation

A single colony of E.coliDH5α strain was inoculated in 5 ml LB medium and incubated at 37°C for overnight. 1% of overnight grown culture was inoculated in 500 mlfresh LB medium and incubated at 37°C for 2-3 h till the OD600 reached to 0.4-0.5. Culture was chilled on ice for 5 min followed by centrifugation at 3000 g for 15 min at 4°C. Harvested cells were washed gently with 200 ml ice-cold TFb-I buffer. Cells were collected by centrifugation at 3000 g for 5 min at 4°C and gently resuspended in 20 ml ice-cold TFb-II buffer. Bacterial cell suspension was kept on ice for 15 min and was aliquoted in 100 μl volumes in chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80°C

Preparation of E.coliultracompetent cells

Xanthomonas strains were grown in PS medium for 14-16 h at 28°C with continuous shaking at 200 rpm. 1 ml of bacterial cultures were ten-fold serially diluted in water and 100 μl volume of each dilution was plated on PS agar plates to get the colony forming units (CFUs). Similarly, 5 μl volume of each dilution was spotted on PS agar plates containing different concentration of streptonigrin and different detergents for intracellular iron and membrane sensitivity assay, respectively. Plates were incubated at 28°C and images were captured after 2-8 days of incubation depending upon m

Serial dilution plating and spotting assay

For growth analysis of Xanthomonasstrains, a loopful of bacterial colony was inoculated in appropriate broth medium and grown for 14-16 h. 0.2% of overnight grown culture was used to inoculate the test medium (for iron limitation, PS with 50 or 100 μM of 2,2’-dipyridyl, and for iron supplementation, different concentrations of either FeCl3or FeSO4was added). Cultures were transferred to a shaker incubator set at 28°C and 200 rpm. Absorbance of cultures was measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences)at 600 nm at regular time-intervals till 48 h. Absorbance values were plotted with respect to time and generation time was determined from the logarithmic (log) phase of bacterial growth using the following formula.G = Generation time (h)T1= Initial time point taken for analysisT2= Final time point taken for analysisNf = Absorbance at time T2(Final OD)Ni= Absorbance at time T1(Initial OD)

Growth analysis and determination of generation time

Xanthomonasstrains were routinely grown in rich PS medium, at 28°C with continuous shaking at 200 rpm unless otherwise stated (New Brunswick Scientific, Innova 43, U.S.A.). In general, Xanthomonas frozen glycerol stocks were revived on PSA medium by streaking,and allowed to grow for 3-4 days. To prepare liquid culture, a loopful of each Xanthomonasstrain was inoculated in PS medium and grown for 24-30 h. Xanthomonasstrains on plates were stored at 4°C for a maximum period of 1 week. For growth of X. oryzaepv. oryzaein Minimal (MM9); (Kelemu and Leach, 1990)and XOM2 media (minimal media which induces hrp genes in X. oryzaepv. oryzae),(Tsuge et al., 2002), first the strains were grown in PS medium to a cell density of 109cells/ml and then centrifuged at 5000 g to concentrate the cells and washed twice with sterile water to remove media components sticking to the cells. Washed cells were inoculated in MM9 and XOM2 medium and grown for overnight.Escherichia coliDH5α,used for cloning purposes, was revived on LB medium containing nalidixic acid and grown at 37°C with continuous shaking at 200 rpm. LB medium was supplemented with appropriate antibiotics to grow the bacterial strains carrying plasmids. For plasmid purification, bacterial strains were grown overnight in LB broth medium containing suitable antibiotics.Antibiotics were used at a final concentration of 50 μg/ml rifampicin, kanamycin, streptomycin and trimethoprim; 100 μg/ml ampicillin; 25 μg/ml nalidixic acid; 10 μg/ml cephalexin, chloramphenicol and gentamicin; 20 μg/ml cyclohexamide and 5 μg/ml tetracyclin

Strains and culture conditions

Microbiological methods

PBS was prepared as a 10X stock solution and used as a 1X concentration.Tris-HCl buffer0.5 M Trizma BasepH was adjusted to 7.6 using concentrated HCl.Tris-Cl buffer was prepared as a 10X stock solution and used as 1X concentartion.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1mM EDTATris Acetic acid-EDTA (TAE) buffer40 mM Tris base0.5 M EDTApH was adjusted to 8.5 with glacial acetic acidTAE buffer was prepared as a 50 X stock solution and used at 1 X concentartion.Potassium Phosphate buffer (0.1 M)1 M Potassium phosphate dibasic (K2HPO4)1 M Potassium phosphate monobasic (KH2PO4)61.5 ml of 1 M K2HPO4was mixed with 38.5 ml of 1 M KH2PO4, pH was adjusted to 7.0 and volume was adjusted to 1 L with H2O

Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 before autoclaving

Common buffers

10 mM Ferric ethylenediaminetetraacetic acid (Fe(III)EDTA)100 mM Magnesium chloride (MgCl2)Working solution 0.18% Xylose 670 μM L-Methionine10 mM Sodium glutamate14.7 mM Potassium dihydrogen phosphate (KH2PO4)40 μM Mangenese sulphate (MnSO4)240 μM Ferric ethylenediaminetetraacetic acid (Fe(III)EDTA)5 mM Magnesium chloride (MgCl2)1.2% AgarLuria Bertani (LB)0.5% Yeast extract1% Tryptone1% Sodium cholride (NaCl)Media and solutions were sterilized either by routine autoclaving at 121°C and 15 psi for 20 min or by filtration through membrane of 0.22 μM porosity

Peptone Sucrose (PS)1 % Peptone1 % SucroseFor preparing plates, 1.2 % agar was added to the medium before autoclaving.Minimal Medium (MM9)Stock SolutionMinimal Salt (2X) for 250 mL 5.25 g di-Potassium hydrogen phosphate (K2HPO4) 2.25 g Potassium dihydrogen phosphate (KH2PO4)0.5 g Ammonium sulphate (NH4)2SO40.25 g Tri-Sodium citrate (Na3citrate)1 M Magnesium sulphate heptahydrate (MgSO4.7H2O) -250 μl 25 mg/mL L-Methionine-1 ml25 mg/mL Nicotinic acid-1 ml10 mg/mL Glutamic acid-25 ml20% Glucose-12.5 ml3% Agar-100 mlPlant mimicking medium (XOM2)Stock preparation100 mM L-Methionine1 M Sodium glutamate1 M Potassium dihydrogen phosphate (KH2PO4)10 mM Manganese sulphate (MnSO4)

Bacterial media

Allbacterial strains and plasmids used in this study are listed in Table 2.1

Strains and plasmids

Identification of selected micro-organism

3.0–5.0, phosphate buffer for pH 6.0–8.0 and Tris-HCl buffer for pH 9.0) were used. •pH stability: The pH stability of the selected tannases was examined in the range of 3.0–9.0 by incubating the enzyme samples for 6 h in different buffers. Tannase activity was estimated under standard assay conditions. •Temperature tolerance: Temperature tolerance of the tannases was examined by assaying their activity at different temperatures in the range of 20 to 80ºC. •Temperature stability: Temperature stability of the tannases was determined by incubating them in the temperature range of 20 to 70 ºC for 6 h. After the incubation tannase activity (%) was determined under standard assay conditions. •Organic solvent stability: In order to determine the suitability of the selected tannases for organic synthesis, their stability was determined in different organic solvents. Experimentally, 10 mg of each of the crude lyophilized tannase from the selected cultures were mixed with 1.0 ml of the following organic solvent: a) Hexane b) Methanol c) Propanol d) Isoamyl alcohol e) Petroleum ether f ) Chloroform The mixture was incubated for 6 h at optimal temperature and the organic solvents were then decanted and the residues were dried in a vacuum desiccator. These dried samples were dissolved in 1.0 ml of citrate phosphate buffer (50 mM, pH 5.0) and the tannase activity was determined under standard assay conditions. The tannase activity thus obtained from each culture were compared with initial tannase activity. Finally, on the basis of tannase titres produced per ml and desirable biochemical properties, the best tannase producer was selected for further investigations

The tannases obtained (at high titres) from selected cultures were evaluated for the following important biochemical properties. 1. pH tolerance and stability 2. Temperature tolerance and stability 3. Organic solvent stability •pH tolerance: pH-tolerance of the selected tannases was examined in the range of 3.0–9.0. Buffers (0.05 M) of different pH (citrate phosphate for pH

Preliminary biochemical characterization of tannases from the potent tannase producers

The reaction mixture contained 10 μl of culture filtrate, 490 μl of double distilled water (DDW) and 300 μl of methanolic rhodanine solution. This mixture was incubated for 5 min at 30°C in a water bath. The reaction was stopped by adding 0.3 ml of methanolic rhodanine solution (0.667 %), which resulted in the formation of complex between gallate and rhodanine. This was followed by the addition of 0.2 ml of KOH solution (0.5N) and the tubes were further incubated at 30°C for 5 min. The total reaction mixture in each tube was diluted with 4.0 ml of distilled water. Tubes were further incubated at 30°C for 10 min. The absorbance was measured at 520 nm against a control having distilled water in place of culture filtrate. The absorbance thus obtained was used to calculate the amount of gallic acid present in the culture filtrate, from the standard gallic acid curve prepared in the range of 100-1000 μg/ml.

The procedure of Sharma et al. (2000) was used to estimate the gallic acid in the culture filtrate. Reagents: Methanolic rhodanine solution (0.667% w/v): Prepared by dissolving 0.667 g of rhodanine in 100 ml of methanol.Potassium hydroxide (0.5 N): 2.8 gpotassium hydroxide dissolved in100 ml of distilled water.

Gallic acid estimation (Sharma et al., 2000)

The tannin sample (1.0 ml) was added to 2.0 ml BSA solution in a 15 ml glass centrifuge tube. The solution was mixed and allowed to stand at room temperature for 15 min and then centrifuged at 10000 rpm for 15 min to separate the precipitated tannin-protein complex as pellet. The supernatant was discarded and the pellet and the walls of the tube were washed with acetate buffer without disturbing the pellet. Now, the pellet was dissolved in 4.0 ml of SDS-triethanolamine solution and to this, 1.0 ml of ferric chloride reagent was added and was mixed immediately. After 30 min of addition of ferric chloride, the absorbance was noted at 510 nm on spectrophotometer. All observations were carried out in triplicates. The concentration of the tannin was determined with the help of tannic acid (Sigma) standard curve prepared in the range of 0.2 to 1.0 mg/ml

The procedure of Hagerman and Butler (1978) was used to estimate the tannin content in different tannin sources. Reagents: Bovine serum albumin (BSA) 1.0 mg/ml: 10.0 mg of bovine serum albumin was dissolved in 10.0 ml of 0.2 M acetate buffer, pH 5.0, containing 0.17 M sodium chloride. Sodium dodecyl sulfate (SDS)-triethanolamine solution: The solution contained 1.0% SDS and 5.0% (v/v) triethanolamine in distilled water. Ferric chloride reagent (0.01 M): 1.62 g of ferric chloride was dissolved in 1.0 L of 0.01 N hydrochloric acid.

Tannin estimation (Hagerman and Butler, 1978)

To 1.0 ml of suitably diluted culture filtrate, 5.0 ml of solution C was added. It was incubated for 10 min at room temperature. To this, 0.5 ml of Folin Ciocalteau’s reagent (diluted 1:1 with distilled water) was added. The solution was vortexed and kept in dark for 30 min. After incubation, absorbance was read at 660 nm against a reagent blank. Protein content was calculated (in mg/ml) using standard curve of bovine serum albumin (BSA) prepared in the range 100-1000 μg/ml

The total protein content in the culture filtrate was estimated by Lowry’s method as described below: Reagents: Solution A: 2.0% Na2CO3 in 0.1 N NaOHSolution B: 0.5 % CuSO4 in 1.0 % Sodium potassium tartarate Solution C: 50.0 ml of solution A was mixed with 1.0 ml of solution B Folin Ciocalteau’s reagent

Protein estimation (Lowry et al., 1951)

For estimation of tannase activity the reaction mixture (4 ml) contained 1.0 ml of 1.0% tannic acid (prepared in citrate-phosphate buffer, pH 5.0), 2.0 ml of citrate-phosphate buffer (pH 5.0) and 1.0 ml of appropriately diluted culture supernatant. The reaction mixture was incubated at 40°C for 30 min in a water bath. The reaction was stopped by adding 4.0 ml of 2.0% BSA solution. In the control reaction, BSA was added prior to incubation. Now the tubes were left for 20 min,at room temperature, for precipitating the residual tannins and subsequently centrifuged at 10,000 rpm for 20 min. The end product, gallic acid thus formed was estimated by diluting 20 μl of the supernatant to 10 ml with DDW. Now, the absorbance at 260 nm was read against a blank (DDW) in a UV spectrophotometer (1601, Shimadzu Corporation, Japan). One unit of tannase: One tannase unit is defined as the amount of enzyme that releases 1 μmol of gallic acid from the substrate (tannic acid) per ml per min under standard assay conditions

In this method, tannase activity was estimated through spectrophotometric method by determining the concentration of the end product i.e., gallic acid, by estimating the absorbance at 260 nm. Reagents: •Tannic acid (1.0%): The solution was prepared by dissolving 1.0 g of tannic acid in 100 ml of citrate-phosphate buffer of the desired pH.•Bovine serum albumin (BSA): BSA (2.0%) was prepared in citrate phosphate buffer (pH 5.0)

Estimation of tannase activity (Deschamp et. al., 1983)

For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity

For fungal cultures, spores were harvested from 72 hour old cultures grown on PDA/Tannic acid agar slants by adding 10 ml of sterilized normal saline and a few drops of sterilized Tween-80 followed by vortexing. The spore suspension was filtered through sterile cotton filter to ensure that mycelial filaments are removed. The spores were counted using a haemocytometer (Neubaeur). Approximately, 5X106 spores were inoculated in 50 ml of tannase production medium in 250 ml Erlenmeyer flasks. These flasks were then incubated at 30±1 and 37±1°C in an incubator shaker (model G25KC, New Brunswick Scientific, NJ, USA) at 200 rpm

Quantitative assay

was observed by the formation of a clear zone of hydrolysis around the bacterial/fungal colony. Tannase production, in terms of the diameter of the zone of hydrolysis around the colony, was measured (in mm) after 24 (bacteria) and 48 hours (fungi) of incubation. The diameter of the hydrolytic zone was measured at three points and the average was calculated. The microorganisms showing a zone of tannic acid hydrolysis were considered as tannase producers. The potent tannase producers were further tested quantitatively for the amount of enzyme produced in broth.

The procedure of Bradoo et al. (1996), involving point inoculation of the microorganisms on tannic acid agar plates was followed. The plates were incubated at 37 and 30±1°C for bacterial and fungal isolates. The presence of tannase activity

Qualitative screening for tannase producer/s

A total of 150 fungal and 150 bacterial isolates were screened qualitatively and quantitatively for their ability to produce the enzyme, tannase.

Screening and selection of potential tannase producers

Microorganisms were isolated from the above mentioned sources using direct plating method. Serial dilution of the different soil samples with normal saline was carried out and the different dilutions were spread plated on to potato dextrose agar (PDA) for isolation of fungi and on to nutrient agar (NA) for the isolation of bacteria. The plates were incubated at either 30 or 37±1°C in a bacteriological incubator so that the different organisms could grow and form visible colonies. The different fungal and bacterial colonies isolated by the procedure mentioned above were purified by subculturing on respective media, and subsequently screened for tannase production. The new isolates, alongwith different cultures obtained from laboratory stock culture collection, were revived on potato dextrose agar (PDA) slants. These cultures were regularly subcultured and stored at 8±1°C in a BOD incubator. Their purity was periodically checked by microscopic examination.

In the present investigation, microorganisms including both bacteria and fungi were isolated from soil samples collected from different geographical locations in India. Microorganisms were also isolated from the bark of trees as well as from the soil near the roots of those trees. Some cultures were also procured from the laboratory stock culture collection.

Collection of samples

Isolation of tannase producing microorganism/s

Pharmacia (Uppsala, Sweden).Column chromatography matrices (Sephadex G series), DEAE-Cellulose E-Merck Germany Silica Thin Layer Chromatography Plates 60 F254, All solvents used in the present investigation were purchased from E-Merck Sisco Research Laboratories (SRL), SD fine chemicals Ltd, Qualigens, Central Drug House (CDH), Thomas BakerChemicals/components used in the preparation of various media were obtained from these companies Local commercial sourcewheat bran, rice bran, wheat straw, corn cob, acacia arabicajambula leaves, aamla, Indian plum, jowari, black tea, kangra orthodox black tea Double distilled water (DDW) was used for preparation of reagents, stock solutions, buffers and different media. All glass and plastic wares used were from Borosil, Schott Duran and Qualigens.

Tannic acid, DEAE-cellulose, phenyl sepharose, acrylamide, bisacrylamide, TEMED, ampholine PAG plates, bromophenol blue, chitosan, chitin, silica, celite, DEAE-sephadex, amberlite XAD-7, glutaraldehyde, cholic acid, saponin, sodium taurocholeate, SDS, tauro cholic acid, sodium choleate, triton X-100, Tween-80, EDTA, phenyl methyl sulfonyl fluoride, p-Chloromercuric benzoic acid, N bromosuccinimide, Phenyl boronic acid, O-phenanthrolin, sodium deoxycholate, phenanthrolin, N-ethylmaleimide, dithiothreitol, β- Mercaptoethanol, bromoacetic acid. gallic acid and its esters (methyl, propyl, ethyl, butyl gallate), epigallocatechin gallate , epigallocatechin, caffeine, epicatechin, epicatechin gallate, 2,2-diphenyl-1-picrylhydrazyl (DPPH) (E)-2-hexenal (Z-3-hexenol), 1-penten-3-ol, 3,7-dimethyl-1,5,7-octatrien-3-ol, linalool, linalool oxides (furanoid), geraniol, methylsalicylate, epoxylinalol, α-irone (2,5-dimethyl-α-ionone),2,7-epoxymegastigma-4,8-diene and 1,3-dioxolane

Chemicals and Reagents

Experiments involving mice were performed at the CDFD animal facility, VIMTA Labs Ltd., Hyderabad, India(www.vimta.com) in strict accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The protocol was approved by Institutional Animal Ethics Committee (IAEC) of the Vimta Labs Ltd. (IAEC protocol approval number: PCD/CDFD/05). Procedures used in this protocol were designed to minimize animal suffering

Ethics statement

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

Glass bead lysis method

Estimation of cytokine production by THP-1 macrophages upon infection with C. glabratacells

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

Glass bead lysis method

Yeast genomic DNA isolation

C. glabratastrains were routinely grown in rich YPD medium or synthetically defined YNB medium, or YNB medium supplemented with CAA, unlessstatedotherwise.To obtain overnight grown liquid cultures, C. glabratacells were inoculated in appropriate medium and incubated at 30°C under constant agitation (200 rpm) to maintain proper aeration.To revive the frozenstocks,about one tipfull of frozen culture was streaked either on YPD-agar or on CAA-agar medium. In general, frozen stocks of C. glabratastrains were revived on YPD-agar medium.However,C. glabratastrains harbouring plasmidscontainingURA3as a selectable marker were revived on CAA-agar medium. After streaking, plates were allowed to grow for 24-48 h at 30°C and were stored at 4°C for a maximum period of two weeks. For long term storage, freezer stocks of C. glabratastrainswere prepared in 15% glycerol and stored at -80° C.Escherichia colistrain DH5αwas revived on LB-agar medium from frozenstock and incubated at 37°C for 14-16 h. DH5α strainwas used for transformation purpose and maintaining plasmids. Bacterial strains harbouring plasmids containing selection markerswere revived on LB-agar medium supplemented with appropriate antibiotics.Bacterial liquid cultures were either grown in LB broth or LB broth containing suitable antibioticsand incubatedin a shakerincubator set at 37°C, 200 rpm for 14-16 h. For preparation of bacterial frozenstocks, 1 ml overnight grown bacterial culture was added to500 μl of 50% glycerolto obtain final concentration of ~16 % glyceroland stored at -80°Cuntil use

Strains and culture conditions

PMA-treated THP-1 cells were seeded toa24-well tissue culture plate to a cell density of 1 million cells per well and allowed to grow for 12 h. After12 hincubation,spent medium was replaced with fresh pre-warmed RPMI-1640 medium andcells were allowed to recover for 12 h before use.C. glabratacells were grown in YNB medium for 14-16 h at 30°C and 200 rpm. 1 ml of theseC. glabratacells were harvested in 1.5 ml centrifuge tubes, washed twice with 1X sterile PBS and the cell density was adjusted to 2x107cells/ml. 50 μl of this cell suspension was used for infection to a MOI of 1:1. Two hours post infection, wells were washed thricewith 1X sterile PBS to remove the non-phagocytosed yeast cells and 1 ml of fresh pre-warmed RPMI-1640 medium was added. Plates were incubated under tissue culture conditions at 37°C and 5% CO2for 24 h. Supernatants were collected in 1.5 ml microfuge tubes, centrifuged at 3,000 rpmto remove the particulate matter, if any, and stored at -20°C until use. Estimation of different cytokines were performed using BD OptEIA ELISA kits as per the supplier’s instructions

Estimation of cytokine production by THP-1 macrophages upon infection with C. glabratacells

To perform survival analysis of C. glabratacells in macrophages, PMA-treated THP-1 cells were seeded to 24-well tissue culture platesto afinal celldensity of 1 millionper well. C. glabratacells were grown in YNB medium for 14-16 h at 30°C and 200 rpm. 1 mloftheseC. glabratacells were harvested in 1.5 ml centrifuge tubes, washed twice with 1X sterile PBS and the cell density was adjusted to 2x106cells/ml. 50 μl of this cell suspension was used to infect PMA-activated macrophages to a MOI (multiplicity of infection) of 0.1. Two hours post infection, THP-1 cells were washed thrice with 1X sterile PBS to remove the non-phagocytosed yeast cells and 1 ml of fresh pre-warmed complete RPMI-1640 medium was added.At different time points, infected THP-1 macrophages were osmolysed with1 ml sterile water. Post lysis,lysates were collected by scraping the wells using 1 ml microtip. Lysates were diluted in 1X sterile PBSand appropriate dilutions were plated onYPD-agar plates. Plates were incubated at 30°C for 24-48 h and colony forming units (CFUs) were counted. Final CFUs/ml were determined by multiplying CFUs withappropriate dilution factor and percentage phagocytosis was calculated by dividing CFUs obtained at 2 h post infection by total numberofyeast cells used for infection. Fold replication was calculated by dividing the CFUs obtained at 24 h post infection by CFUs obtained at 2 h post infection

THP-1 macrophageinfection assay to monitor the intracellular survival and replication of C. glabrata

phorbol myristateacetate (PMA) (Tsuchiya et al., 1982). For PMA treatment, THP-1 cells were allowed to grow till 70-80% confluence and were collectedin a centrifuge tube by centrifugationat 1,000 rpm for 3 min. THP-1 cell pelletswere resuspended in 4-5 ml of pre-warmed complete RPMI-1640 medium, 100 μl of this cell suspension was appropriately diluted in PBS (1X) and viability was determined by counting trypan blue stained cellsusing hemocytometer. THP-1 cell suspension was diluted appropriately to obtainafinal cell density of 106cells/ml with pre-warmed complete RPMI-1640 medium. PMA was added totheTHP-1 cell suspension at a final concentration of 16 nM and mixed well by gently inverting the tubes. PMA-treated cells were seeded either in 24-well cell culture plates or in cell culture dishes and allowed to grow for 12 h under tissue culture conditions i.e. at 37°C and 5% CO2.After 12 h incubation, spent medium was replaced withfresh pre-warmed complete RPMI-1640 medium and cells were allowed to recover for another 12 h