8

8

8

8

muscular hypotonia

motor development

speech and language development

Nobel Peace prize

We deplore all forms of violence

last war

Drugs and chemicals

Statisticalanalysis

TLC study of alkaloids

TLC study of phenols

TLC study of alkaloids

Activation of TLC plate

Preparation of TLC plate

Study of Phytochemicals by Thin Layer Chromatography (Wagner, and Bladt, 1996)

Test for cardiac glycosides

RAPDand SSRscoring and data analysis

Assayof xylanase activity

Dinitrosalicylate reagent (DNS)(per liter)

Citrate phosphate buffer

Reagents

Xylanase activity

Evaluation of bioagents against the pathogen

Laboratory screening of antagonists against the test pa

Production of caseinase

Gel documentation and image analysis

Detection of toxR gene

Detection of tlhgene

Extraction of genomic DNA

Detection of V. parahaemolyticusspecies-specific gene

Isolation of V. parahaemolyticuson HiCrome Vibrio

Isolation and identification of V. parahaemolyticus

Gelatinase production

Gelatinase productio

Distribution of antibiotic resistance genes in Vibriofrom Cochin estuary, shrimp farm and seafood

Reaction resuspension

Post-reaction clean up

Sequencing PCR

Sequencing reaction

Python Molecular Viewer

Quantification of MMP or ΔΨmloss

Assessmentof mitochondrial membrane potential(MMP or ΔΨm)

Glutathione peroxidase (GPx) assay

Superoxide dismutase (SOD) assay

Catalase assay

Catalytic activity

Estimation of bioaccumulation of trace metals inmuscle and gill tissue of fish, Labeo rohita

Grain yield per plot (g)

Post vaccination enhanced expression of activation marker CD69 and Chemokine receptor CCR5 on memory B cells in HBV positive newborns

Freezingor storageof cells

Cytoskeleton extraction buffer (CSE)

Ni-NTA pull down

RNA Interference

pET30a-β-catenin-FL (Antigen for -catenin antibody)

Protein estimation of sonicate

2 Bioassay

Proliferation Assay

Target binding assay

Cytotoxicity assay

ACK lysis Buffer

lgG-isotyping in the sera of the immunized animals

Polymerase chain reaction (PCR)

Genomic DNA extraction

RT-PCR analysis

Ectomorphic endomorph:

Femur

concentrators (Amicon), and subjected to reduction with 0.0.5 M sodium dithionite. For this an appropriate amount of anaerobically prepared dithionite solution was added to the reconstituted Hb and the reaction mixture was quickly passed through a Sephadex G25 gel filtration column (30 em x 1.5 em) equilibrated with 0.05 M Tris HCI (pH 7.4), in order to minimize the duration of contact of dithionite with the protein. The reduced Hb was dialyzed extensively against 0.01 M potassium phosphate buffer (pH 6.5) and loaded onto a CM52 column (1 Ocm x 1.5cm) equilibrated with the same buffer. A linear gradient of 150 ml each of 0.01 M potassium phosphate buffer (pH 6.5) and 0.015 M potassium phosphate buffer (pH 8.5) was employed to elute the protein from the column

Construction of mutant a globins

Reconstitution of a globin and rf chain into HbS tetramers

Paduans

The reaction mixture contained 0.2 mL of enzyme sample, 0.3 mL of buffer and 0.5 mL of p-nitrophenyl-β-D-glucopyranoside (1.0 mM) prepared in 100 mM buffer as the substrate. The reaction was terminated after 30 min of incubation at 70 °C by adding 2 mL of sodium carbonate-bicarbonate buffer (0.1 M, pH 10.0). The liberation of p-nitrophenol was measured at 400 nm and its yield was determined using a standard curve of p-nitrophenol (1-10 μg mL-1) prepared in sodium carbonate-bicarbonate buffer

β-Glucosidase

The activities ofβ-xylosidase, xylan acetylesterase and arbinofuranosidase were measured using 1 mM p-nitrophenylxylopyranoside, p-nitrophenylacetate and p-nitrophenylarabinofuranoside, respectively prepared in sodium citrate buffer (0.1 M, pH 7.0). One mL of reaction mixture containing 0.2 mL of crude enzyme solution, 0.3 mL of sodium citrate buffer (0.1 M, pH 7.0) and 0.5 mL of substrate was incubated at 80 °C for 30 min. The reaction was terminated by adding 2 mL sodium carbonate-bicarbonate buffer (1.0 M, pH 10.0). The activities were determined using p-nitrophenol standard curve (1-10 μg mL-1) drawn using absorbance values measured in spectrophotometer at 400 nm. One unit of the enzyme is defined as the amount of enzyme that liberates 1μmole of p-nitrophenol mL-1min-1 under assay conditions.

Assays for β-Xylosidase, acetylesterase and arbinofuranosidase

Xylanolytic activity was determined according to Archana and Satyanarayana (1997). The reaction mixture containing 0.5 mL of 1% birchwood xylan in glycine NaOH buffer (0.1 M, pH 9.0) and 0.5 mL of cell free sonicated supernatant was incubated at 80 °C in a water bath for 10 min. After incubation, 1 mL DNSA reagent (Miller, 1959) was added to the reaction mixture and the tubes were incubated in a boiling water bath for 10 min, followed by the addition of 400 μL of 33% w/v sodium potassium tartrate. The absorbance values were recorded at 540 nm in a spectrophotometer (Shimadzu, Japan). The liberated reducing sugars were determined by comparing the absorbance values of these with a standard curve drawn with different concentrations of xylose. One unit (IU) of xylanase is defined as the amount of enzyme required for liberating one μmol of reducing sugar as xylose mL-1 min-1under the assay conditions. Composition of Dinitrosalicylic acid (DNSA) reagent NaOH - 10.0 g Phenol - 2.0 g DNSA - 2.0 g Distilled Water - 1000 mL DNSA reagent was stored in an amber bottle at 4 °C till further use. Sodium sulphite (0.05 % v/v) was added just before the use of the reagent.

Enzyme Assays

A stock solution of xylose (1 mg mL-1) was prepared in distilled water. A dilution series ranging from 100-1000 μg mL-1 was prepared from the stock solution. To 1 mL of solution, 1mL of DNSA was added and kept in a boiling water bath for 10 min and then 400 μL of sodium potassium tartrate solution was added and kept it for cooling. The absorbance was recorded in a spectrophotometer (Shimadzu, UV-VIS) at 540 nm

The clear cell-free supernatants were used as the source of crude recombinant xylanase.

Preparation of standard curve of xylose

Tris, pH 7.4, 1 mM dithiothreitol, and 10% glycerol. Protein concentration w~s detennined by densitometry analysis of Commassie stained gels. Protein samples were stored at -70°C until further use

To facilitate the expression of recombinant GST-CDPK4 or its mutants, the desired regions of enzyme were PCR amplified using pGEMT-PfCDPK4 as template and PCR primers which possessed overhangs for XhoI and SmaI restriction enzymes (see List II). Often, the PCR products were cloned in TA cloning vector pGE¥T-I easy. Clones in pGEMT-easy vectors were digested with appropriate restriction enzymes to release the inserts. The released inserts were cloned in expression vector pGEX4T-l to facilitate the expression of recombinant proteins. In some cases, the PCR products were digested directly with restriction enzymes and ligated into expression vectors. The plasmid DNA for expression was used to transform E. coli BL21-RIL (Stratagene) strain for the expression of GST-PfCDPK4 and its mutants. Protein expression was induced by overnight incubation of cells with O.lmM IPTG at 18-20°e. Subsequently, cell pellets were suspended in ice cold lysis buffer, contaiJ;1ing 50 mM Tris, pH 7.4, 2 mM EDTA, 1 mM dithiothreitol, 1% TritonX-100, and protease inhibitors (lmM phenylmethylsulfonyl fluoride, 10~g/ml leupeptin, 1 O~g/ml pepstatin) and sonication was performed for 6 cycles of one minute each. The resulting cell debris was removed by centrifugation at 20,000g for 40 min at 4°C. Fusion proteins from the cell lysates were affinity-purified using glutathione-sepharose resin (Arnersham). Briefly, after the protein binding, the resin was washed with lysis buffer, and bound proteins were eluted with 50 mM Tris, pH 8.0 with 10 mM glutathione. Finally, purified proteins were dialyzed against 50 mM

xpression and Purification of Recombinant GST (Glutath ion e-S-Transferase) fusion PfCDPK4 and its mutant

For synchronization, mostly ring stage parasites (10 to 12 h post-invasion) wen~ used. The parasite culture was centrifuged at 200g for 5min and the supernatant was discarded. To the pellet, 4 ml of 5% sorbitol was added, mixed gently and incubated for 15min at 37°C. The mix was shaken 2 or 3 times and centrifuged at 200g followed by washing 3 times in complete medium (list I). The culture was then maintained at 5% hematocrit in a 37°C incubator

orbitol-synchronization 0/ P./alciparum

THP-1 macrophages and human peripheral blood monocyte derived macrophages were transfected with SMARTpool Bcl-2 siRNA (15 pmol), or ER-a siRNA (100 pmol), or ER-~ siRNA (100 pmol), or with negative control siRNA (15 pmol or 100 pmol) using TranspassR2 transfection reagent. Prior to transfection, the cells were depleted of serum by washing 2x with serum-free media. The transfection complex was prepared by diluting 0.5 J!L of transfection reagent A and 1.0 J!L of transfection reagent B to 400 J!L of serum-free media and siRNA's were added to the mix at an appropriate concentration and incubated for 20 min at room temperature. The formed transfection complexes were transferred gently using a large bore pipette tip to 105 cells/well grown in 24 well plates and incubated for 6 h, following which fresh complete medium was added. Transfection efficiency was estimated by observing Cy3-fluorescence of the negative control siRNA with a Nikon TE2000E fluorescence microscope using a tetramethyl rhodamine filter (530-580 nm). For all transfections, target protein knockdown was assessed 24 h after transfection by probing extracts oftransfected cells on Western blots using appropriate antibodies

siRNA transfection

resuspended in 0.2 ml TE, and extracted successively with phenol, phenol-chloroform, and chloroform. In the aqueous phase, 0.25 volume of J 0 M ammonium acetate and two volumes of chilled ethanol were added and the mixture was incubated at room temperature for 5 min. to precipitate the plasmid DNA. The pure plasmid DNA was recovered by centrifugation at J 2,000 g for J 0 min. at 4 °C, washed with 70 %ethanol, dried and resuspended in TE buffer (pH 8.0). The amount and the purity of the DNA was done spectrophotometrically by recording the absorbance at 260 nm.

Plasmid DNA was prepared by alkaline lysis method of Ish-Horowicz ( 1981 ). 5 ml cultures were grown as described for small scale plasmid preparation. 0.5 ml from the growing culture was inoculated into 250 ml of LB containing ampicillin. The culture was grown for 12 h at 37 °C with vigorous shaking, centrifuged at 3000 g, at 4 °C, for 15 min. and the bacterial pellet was resuspended gently in 1 0 ml TEG buffer. The mixture was incubated at room temperature for 10 min., followed by addition of 20 ml of freshly prepared alkaline-SDS solution. The contents were mixed by inversion and the mixture was kept on ice for 10 min., followed by the addition of 15 ml of chilled potassium acetate solution. The contents were mixed by inverting the tube, and incubated on ice for 10 min. The lysed cell suspension was centrifuged at 5000 g, at 4 °C, for 20 min. The supernatant was taken, and nucleic acids we~,-.. precipitated by adding 0.6 volume of chilled isopropanol. The mixture was incubated on ice for 10 min. followed by centrifugation at 5000 gat 4 °C, for 10 min .. The pellet was washed with 70% ethanol, dried and resuspended in TE buffer. The plasmid DNA was purified further to remove the contaminating proteins and RNA following the PEG purification protocol as described by Sambrook et al ( 1989). Equal volume of chilled 5 M lithium chloride solution was added to DNA . suspension, mixed well and incubated on ice for 10 min. The precipitate was removed by centrifugation at 10,000 g at 4 °C, for 10 min. DNA was precipitated from the supernatant by adding equal volume of isopropanoL The mixture was centrifuged at 10,000 g for 10 min. at 4 °C and the pellet was washed with 70% ethanol. The DNA thus obtained was incubated in TE buffer containing 20 J.tg/ml of DNase free RNase A for 30 min. at room temperature. Afterwards, equal volume of 1.6 M NaCl containing 13% (w/v) PEG 8000 was added to DNA solution. The contents were. thoroughly mixed and centrifuged at 10,000 g, at 4 °C, for 10 min .. The pellet was

Large Scale Plasmid DNA Preparation

administered as and when required. Animals were put on continuous mating with males of proven fertility after administration of the three primary injections and monitored for menstrual cyclicity and conception. Ab titres were determined as described above except that anti-monkey HRPO conjugate was used as the revealing Ab.

Female bonnet monkeys (Macaca radiata) reared at the Primate Facility (Nil, New Delhi) were selected and serum progesterone levels were estimated for atleast three months in samples which were collected biweekly. Animals showing atleast two consecutive normal ovulatory peaks (serum progesterone levels >2 ng/ml) (Bamezai, 1986) were selected for fertility trials. Five animals (MRA 375, 515, 640, 672, 770) immunized with 250 Jlg equivalent of r-bZP3, expressed in SG I3009[pREP4] cells, conjugated to DT, was emulsified with Squalene and Arlacel A, adjuvants permitted for human use, in a ratio of 4: I and administered intramuscularly at two sites. In addition, the primary dose also contained I mg/animal of SPLPS as an additional adjuvant. Animals were boosted at intervals of 4-6 weeks depending on the Ab titers with 250 Jlg of r-bZP3-DT using Squalene and Arlacel A as adjuvants. A second group of 3 monkeys (MRA 384, 502, 661) were immunized using a slightly different protocol. The primary immunization consisted of 125 J.lg of r-bZP3-DT and 125 J.lg of r-bZP3-TT (expressed in BL2I(DE3) cells) using the same adjuvants and immunization protocols mentioned above except that boosters were administered alternately with 250 Jlg of r-bZP3-DT or -TT conjugates using Squalene and Arlacel A as adjuvants. Following completion of the primary immunization and 2 boosters at monthly intervals, bleeds (1-2 ml) were collected biweekly from the antecubital vein for estimation of progesterone levels and Ab titres. Boosters were

Immunization of Female Bonnet Monkeys

Radiolabeling of Recombinant Proteins in Infected S/9 Cells

Peptide antisera were generated in the laboratory against peptides PI, 23-45 aa residues with an extra lysine at the N-terminus (KQPFWLLQGGASRAETSVQPVL VE), P2, 300-322 aa residues (CSFSKSSNSWFPVEGPADICQCC) and P3, 324-347 aa residues (KGDCGTPSHSRRQPHVVSQWSRSA) corresponding to bZP3 precursor protein in rabbits and were used to determine their reactivity with the r-bZP3 protein expressed in E. coli in an enzyme linked immunosorbent assay (ELISA). Microtitration plates were coated with 200 ng of r-bZP3 or I J.tg/well of the peptide. HRPO conjugated goat anti-rabbit Ig at I :5000 dilution was used as revealing Ab.

Reactivity with Anti-peptide Sera

The bZP3 sequence was analyzed using PCgene and Lasergene DNA and protein analysis softwares. The alignment of the bZP3 aa sequence with the homologous sequences from other species was carried out using the Cluster V Multiple Alignment Programme (Higgins and Sharp, 1989).

Analysis of Sequence

Transformation was performed in chilled 1.5 ml eppendorf tubes, using 200 ul of competent cells and about 50 ng of ligated plasmid DNA. Frozen competent cells were thawed in ice and the DNA was added immediately after thawing. The DNA volume was always kept under 30 ul. The DNA was mixed well with the cells by gentle tapping, and the tube incubated in ice for 3 0 minutes with occasional gentle shaking. The tube was then immersed in a 42°C water bath for 2 minutes, to give a heat shock to the cells. The cells were then incubated in ice for 10 minutes. Next, 1 ml LB was to the cells, and the cells incubated in a 37°C water bath without shaking, for one hour. 50 ul aliquots were plated in triplicate from the transformed cell mixture on suitable antibiotic containing agar plates and incubated 0/N at 37°C to select the transformants. In case of JM105 cells, the transformed cells were plated on antibiotic containing agar plates on which 50 ul of 2 % X-gal ( made in dimethyl formamide ) , and 10 ul of 100 mM IPTG had been spread in advance, to select for the lac-phenotype. The lac-colonies appeared colourless while the lac+ colonies were blue. For each batch of transformations, a negative control was included in which no DNA was added to the cells while keeping the rest of the procedure the same as for the test transformations.

Transformation procedure.

were stored at -70°C for at least six months without any significant loss in the competence.

A single ~.coli colony taken from an agar plate was used to inoculate 10 ml of LB and incubated 0/N at 37°C in an incubator-shaker. Next day, 0. 5 ml of this freshly grown culture was used to inoculate 100 ml of LB in a 500 ml flask. The culture was incubated at 37°C in an incubator -shaker and absorbance of the growing culture was monitored at 620 nm. When the A620 reached 0. 4 -0. 5 ( in about 120 -150 minutes), the flask was rapidly chilled by shaking in ice. The cells were harvested in sterile, chilled centrifuge bottles at 4, ooog for 10 minutes at 4 °c. The pellet was gently resuspended in 50 ml sterile, ice cold 100 mM cacl2 and the cells incubated in ice for 30 minutes. The cells were again centrifuged as above and the pellet resuspended in 6.5 ml of sterile, chilled, 100 mM cac12 containing 15 % glycerol. The cells were resuspended very gently, and a 200 ul aliquot was transformed with a standard plasmid DNA to check the competence of the cells. Meanwhile, the rest of the competent cells were incubated in ice for 16 -18 hours, to increase the competence of the cells a further few fold. After ascertaining high transformation efficiency of the competent cells, the cells were dispensed as 200 ul aliquots into prechilled, sterile 1.5 ml eppendorf tubes. These cells

Preparation of competent E.coli cells.

All glassware I plasticware used for transformation procedure was sterile and prechilled.

Transformation of E.coli.

All antisera were obtained from the reagent bank at National Institute of Immunology, New Delhi.

Antisera.

collected by centrifugation at 12,000 X g for 15 min, and washed with 70% ethanol. The pellet was air-dried and resuspended in 20 Jll TE. The clones were checked for the pres~nce of the insert by restriction analysis. The digestion products were checked on 1% agarose gel for the release of the insert. One positive clone was selected from each set of transformations and the plasmid DNA was purified in large amount for the insert preparation.

Transformants picked following blue-white selection were inoculated in 5 ml LB medium containing 100 j...tg/ml ampicillin (LBamp) and grown 0/N. Following day, 1.5 ml aliquots of 0/N culture were harvested by centrifugation at 10,000 X g in a microfuge. The supernatant was discarded and the pellet was resuspended in 100 j...tl of chilled TEG (25 mM Tris-Cl, pH 8.0, 10 mM EDTA and 50 mM glucose) and incubated for 10 min at RT. After incubation, 200 j...tl of freshly prepared alkaline-SDS (0.2 N NaOH, 1% SDS; sodium dodecyl sulfate) was added and the contents were mixed gently by inversion. This was followed by incubation on ice for 10 min. Post-incubation, 150 j...tl of ice-cold sodium acetate solution (3 M, pH 5.2) was added to the mixture and incubated on ice for 15 min. After incubation, the contents were centrifuged at 12,000 X g for 15 min at 4°C and the supernatant was carefully transferred to a fresh tube. DNA was precipitated by adding 0.6 volumes of isopropanol and incubating at RT for 10 min. The DNA pellet was obtained by centrifugation at 12,000 X g at RT for 15 min, air-dried and dissolved in 200 j...tl of TE. To remove RNA contamination, 50 j.lg of DNase free RNase was added and incubated for 1 h at 37°C. Plasmid DNA was then extracted once with an equal volume of phenol equilibrated with TE (I 0 mM Tris, pH 8.0 and 1 mM EDT A) followed by extraction with phenol : chloroform : isoamyl alcohol (25 : 24 : 1) and then with chloroform : isoamyl alcohol (24 : 1 ). DNA was precipitated by addition of 2 volumes of chilled 100% ethanol to the aqueous phase and incubating the contents at -70°C for 30 min. The DNA pellet was

Small scale plasmid DNA isolation and restriction

ITALYusinga96wellmicrolitreplatereadbyELISAmicroplatereader(modelEL3/Sx,BioTeKInstrumentsINC).Thefinalsolutionwasreadatawavelengthof450nm.Theplasmacortisolconcentrationwascalculatedbasedonaseriesofstandards.

PlasmacortisolwasmeasuredbyadirectimmunoenzymaticdeterminationofcortisolkitmanufacturedbyEquiparSriviaG.Ferrari,21/N-21047,SARONNO

Plasmacortisol

phosphoricacidformedisreducedbytheadditionof1-amino2napthol~4-sulphonicacid(ANSA)reagenttoproducethebluecolor.Theactivityofthebluecolorwasreadat680nmagainstreagentblankusingaU.V.Spectrophotometer.Suitablestandardswererunthrougheachbatchofassays.Theenzymeactivitywasexpressedintermsofpgofinorganicphosphorusformedhr'1mg'1protein.

Aftereffluentexposure,thecontrolandeffluentexposedfishtissueswereremovedandplacedinabeakercontainingice-coldSEIbuffer(300mMsucrose,200mMNa2EDTA,50mMimidazole,pH7.23)foranalysisofNa+-K+ATPaseactivity.ThetissueswereimmediatelyfrozeninliquidN2andstoredat-80°Cuntilanalyzed. Thespecificactivitiesofsodium,potassium,magnesiumandcalciumdependentATPaseswereassayedaccordingtothemethodsdescribedbyWatsonandBeamish(1980)and Boeseetal.(1982).AdenosinetriphosphatasecatalysestheconversionofATPandADP.Duringthisconversion,onemolecule ofphosphorusisliberated.ATPaseAdenosinetriphosphate^...^Adenosinediphosphate+PTheinorganicphosphorusliberatedwasassayedaccordingtothemethodofFiskeandSubbarow(1925).Inthismethodtheproteinisprecipitatedwithtrichloroaceticacid.Theproteinfreefiltrateistreatedwithaceticacidmolybdatesolutionandthe

Na+K+ATPase,Mg2+ATPaseandCa2+ATPase

ThecircadianrhythmofbimodalO2uptakeofcontrolandeffluenttreatedfisheswerestudiedseparatelyat28°±1°C.TheamountsofO2extractedfromwaterandairwereseparatelydeterminedforadayatregularintervalsof3hreach.TotalO2uptakeateachtimewasobtainedbysummingupthevaluesforaquaticandaerialrespirationobtainedatthecorrespondingtime.Throughoutthepresentstudy,theinitialO2contentofthewaterwaskeptconstant(6±0.5mgF1)

CircadianrhythmofbimodalO2uptake

Forstudyingtheaerialrespirationoffishesinair,respirometersweredesignedinvolvingtheprinciplesofmonometrictechniques.Thesetup(Figure10and11)consistsofarespiratorychamberconnectedtoagraduated‘U’tubecontainingBrodie’sfluid.KOHisusedasCO2absorbent.Thedifferenceinthelevelofthefluidinthemanometerforagiventimeisusedinthefollowingequationandthegasutilizediscalculated.VixhV=-...........-10,000Where,‘V’isthevolumeofthegasutilized‘Vi’isthevolume ofgasintherespiratorychamber‘h’isthedifferenceintheleveloftheBrodie’sfluidinthemanometerand10,000isthepressureofmanometricfluid(Brodie’sfluid)inmm

AerialRespiration

Theexperimentalsetup(Figure8and9)forthedeterminationofO2uptakesimultaneouslyfromairandwaterwassimilartothatusedearlierbyNatarajan(1972),Rani(1994)andVijayalakshmi(1996).Aclosedglassrespirometerof5litrecapacitywasfilledwith3.5litrefreshtapwater.Athermocolfloatwithasemicircularholeatitsperipherywasplacedoverthewater,whichseparatedtheair-waterinterphaseoftherespirometer.Theair-phaseoftherespirometerwasattachedtoafluidmanometer.Asthefishcomestothewatersurfaceandtakesair-gulp,thereisapressurechangeintheair-phasecausinganimbalanceinthemanometricfluid.AgraduatedsyringefilledwithpureO2(takenfrommedicalO2cylinder)isusedtorestoretheimbalanceofthemanometricfluid.TheamountthusneededshowstheaerialO2uptakeofthefish.TheexpiredCO2wasabsorbedbythepelletsofKOHinthepetridishoverthemanometricfluid.Theconcentrationofdissolvedoxygenoftheambientwaterwasestimatedbefore andaftertheexperimenttomeasuretheaquaticO2uptakebythefish.ThedifferenceintheDOandtheamountofwaterindicatestheactualaquaticO2uptake.Winkler’svolumetricmethod(Welch,1948)wasusedtoestimatetoDOofthewatersamples.Darkenedrespiratorychamberswereusedwithdimensionsthatwereclosetothoseofthefishinorderthatthefishshouldremaininmoreorlessthesamepositionbut havesufficientroomtomoveitsopercula.Theflowofwaterthroughtherespirometer wasregulatedandmeasuredbymeansofaflowmeter.APhilipsO2electrode(PI1056)waskeptinawaterjacketmaintainedatthesametemperatureastheclosedcirculation.SamplesoftheinflowandoverflowwatercouldalsobeledovertheO2electrode

Bimodalrespiration

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

Aquaticrespiration

Theeffectof2%,5%and7%effluentexposureontheoxygenuptakewasmeasuredatexperimentalconditions,viz.,(a)whenaccesstoairwasprevented(aquaticconsumption),(b)whenitwasallowed(bimodalrespiration)and(c)underaerialconditions(aerialrespiration)

Effectofeffluentexposureontheoxygenconsumption

Respirationstudies

All the cell lines were grown and maintained in Dulbecco' s modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% antibiotic-antimycotic (penicillin, streptromycin and amphotericin B). The cells were maintained at 37<>C with 5% C02 in a humidified CD2 incubator (Nuaire-IR Autoflow CD2 Water-Jacketed incubator)

ell culture media and cell lines

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

f. 5 μl of water was then spotted on each spot for 30 sec and removed using Whatman filter paper strips. This step was repeated once. g. 1-2 μl of SAP matrix was then applied to each spot and allowed to dry. h. The chip was then placed in the SELDI machine

a. 5 μl of 10 mM HCl was added to each spot on the chip and removed after 5 min. using Whatman filter paper strips. b. Washing was given by spotting 3 μl of water for 30 sec on each spot followed by removal using Whatman filter paper strips. This step was repeated two times. c. 10 μl of low stringency/ high stringency buffer was then added to the spot and kept in humid chamber for 5 min. followed by removal using Whatman filter paper strips. d. 3 μl of sample prepared in low stringency/ high stringency buffer was then added to the spot and incubated in humid chamber for 30 min. e. Washed the spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH 7.0 for 30 sec and removed using Whatman filter paper strips. This step was repeated five times.

Activation of CM10 (weak cation exchange ) array

Fresh overnight culture of the E. coli strain (DH5α) was subcultured 1:100 in 250 ml LB/SOB media at 18 ̊C and 2500g and allowed to grow to an A600=0.55. Culture was chilled on ice and centrifuged at 2500g at 4 ̊C for 10 min. The cell pellet was redissolved in 80 ml ice-cold Inoue transformation buffer (55 mM MnCl2, 15 mM CaCl2, 250 mM KCl, 10 mM PIPES pH6.7). This cell suspension was centrifuged at 2500g at 4 ̊C for 10 min. and the cell pellet was resuspend in 20ml ice-cold Inoue buffer with 1.5 ml DMSO. This mixture was then placed on ice for 10 min. Aliquots of this suspension were dispensed into chilled, sterile microfuge tubes that were snap-frozen in a bath of liquid nitrogen. Tubes were stored at ─70 ̊C until required. For transformation the cells were thawed on ice and plasmid DNA was added followed by the standard transformation protocol

Preparation of ultracompetent cells

PonceauS stain Instant Blue (Biorad)

Protein loading dye (6X) Tris-Cl (pH 6.8) 300 mM SDS 12% (w/v) Bromophenol blue 0.6% (w/v) Glycerol 60% (v/v) 600 mM β-mercaptoethanol

Stains and Dyes

C-1 (5,5' ,6,6' -tetrachlorol,1' ,3,3' tetraethyl benzimidazoly 1 carbocyanine iodide) is an anionic mitochondrial vital dye (10mm stock prepared in DMSO) that is lipophilic and becomes concentrated in the mitochondria in proportion to the membrane potential; more dye accumulates in mitochondria with greater potential and ATP generating capacity. The dye exists as a monomer at low concentrations that emit a green fluorescence (530nm) but at high concentrations forms J aggregates that emit red fluorescence (590nm). The ratio of the two fluorescences gives a ratiometric comparison of mitochondrial membrane potential. Following appropriate treatment, 107 (in 1mL medium) cells were transferred to an MCT containing 10pL of the working stock (0.4mM ) of the dye (final concentration of 4pM), and incubated at 37°C for exactly 10 min in the dark. This was followed by centrifugation at 1811 x g for 3 min at RT. The pellet obtained was resuspended in M199 medium containing 10% FBS and centrifuged at 1811 x g for 3 min at RT. Two more such washes were given, after which the pellet was resuspended in 2mL M199 + 10% FBS and fluorescence measured at 485nm/ 530nm and 535nm/ 590nm

Assay for measuring Mitochondrial Membrane Potential (JC-1 Staining)

Germany) as per manufacturer's protocol. Briefly, the gel was solubilised by incubating it with buffer QG (composition proprietary) at S0°C for 10 min. The solubilized gel was loaded onto a binding column and centrifuged at 12000 x g for 1 min. The flow through was discarded and the column was washed once with buffer PE containing ethanol. The DNA bound to the column was eluted using the elution buffer provided with the kit, or alternatively with nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration= A26o X SOX dilution factor.

To elute DNA from agarose gel, samples were loaded on a low-melting agarose gel. The samples were resolved and visualized under UV transilluminator, and the band of interest was excised quickly using a scalpel blade. The volume of gel slice was quantified by weighing and the DNA eluted using MinElute Gel Extraction kit from Qiagen (Hilden

Elution of DNA from agarose gel

Band intensities in gel autoradiograms were determined by densitometry with the aid of the Fujifilm Multi Gauge V3.0 imaging system.Equal areas of radioactive bands (preferably the unbound probe) were boxed and the PSL (Photostimulated luminescence) valueswere further considered. For Kd(dissociation constant)calculations, the values thus obtained for each lane were expressed as a percentage with respect to the PSL for the lane without any protein taken as 100%

Densitometry

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

Plasma membrane H+-ATPase activity assay

dithiothreitol and1X protease inhibitor cocktail. Cell suspension was rapidly frozen at -80 ̊C,thawed and lysed with 0.5mm acid-washed glass beadsin a homogenizer (FastPrep®-24,MP Biomedicals)at maximum speed of 60 secfive times. Homogenate wasdiluted with 5mlTris-HCl (0.1M; pH 8.0)solutioncontaining 0.33M sucrose, 5mM EDTAand 2mM dithiothreitoland centrifuged at 1,000g for 3 minat 4 ̊C. Supernatant was collected and centrifuged again at 3,000g for 5 minat 4 ̊C to remove unbrokencells. The resulting supernatant was centrifuged at 19,000g for 45 minat 4 ̊C to obtain total membrane fraction. Total membrane pellet was resuspendedin 100μl membrane suspension buffer and stored at -80 ̊Ctill further use. Total protein concentration in the membrane fraction was estimated using BCAprotein assay kit (Thermo Scientific, US) with bovine serum albumin (BSA) used as astandard

Isolation of total membrane fractions from C. glabratastrains were carried out as described previously (Fernandes et al., 1998). Cells grown to log-phase under different environmental conditionswere harvested, washed and suspended to afinal density of 20 OD600cells in 1 ml solution containing100mM Tris (pH 10.7),5mM EDTA,2mM

Total membrane preparation

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

Whole cell acidification assay

Measurement of plasma membrane H+-ATPase activity

A single colony of E.coliBW23473strainfrom a freshly-streaked LBplate was inoculated in50ml LB medium. Culture was incubated overnight at 37°C with shaking at 200rpm. 25ml of the overnight-grown BW23473 culturewas transferred to500ml pre-warmed LB medium andincubated at 37°C till the OD600reached to 0.4. After incubation, cultureswere transferred to an ice-water bathandcentrifugeat 1,000g for 15 minat 4°C. Cells were washed twice with 500ml ice-coldwater, thrice with250ml ice-cold 10% glycerol solution and resuspendedin 1ml 10% glycerol solution. Cell suspension wasnormalized to final cell densityof 3x 1010cells/ml and dispensed in 50μl volume into sterile ice-cold microcentrifuge tubes. Aliquots weresnap frozen inliquid nitrogen and stored at -70 ̊C for further use

Electro-competentcell preparation

5-10 ml saturated bacterial culture harboring the desired plasmid was harvested at 5,000 g for 3 min. Plasmid DNAwas isolated using QIAprep Spin Miniprep Kit (Qiagen, USA) or GenElute™ HP Plasmid Miniprep kit (Sigma-Aldrich, USA) as per manufacturer’s instructions

Bacterial plasmid isolation

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

Genomic DNA isolationby glass bead lysis method

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

Opsonizationof C. glabratacells

undertissueculture conditionsfor 45-60min andfixed in 3.7% formaldehydeas described earlier.For DAPI staining, Vectashield mounting medium containing DAPI was used and slides were visualized under confocal microscope.For heat killing, yeast cells were harvested from 1 ml culture, washed, resuspended inPBS andwere incubated at 95°C for 5 min

PMA-treated THP-1 macrophages were infected with C. glabratacells to a MOIof 1:1 in four-chambered slides and incubated at 37°C and 5%CO2. After 1 hcoincubation, each chamber was washed thrice with PBS to eliminate extracellular yeast cellsand medium was replaced with fresh prewarmed RPMI medium containing100 nM Lysotracker Red DND-99.Infected THP-1 macrophageswere incubated

Lysotracker staining

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

DNA loading dye

Agarose gel

TAE

For DNA electrophoresis

Stripping Buffer

Leupeptin

in 5% fat free milk solution in TBST (1:7000) for 45 min at room temperature and then washed thrice.The detection of signal was performed with ECL detection reagent (Amersham Biosciences) followed by detection of signal either on X-ray film (Hyperfilm-ECL, Amersham Biosciences)or in a chemidoc system (Proteinsimple, California, USA).The blot was reprobed with anti-tubulinor anti-GAPDHantibody to ensure equal loading of extracted protein

Materials and Methods472.2.7 Estimationof protein concentration in cellular lysatesBradford method(Bradford, 1976)was used to determine the quantity of protein in various samplesin a 96-well plate. Bradford’s reagent was prepared by diluting Bradford dye with water in the ratio of 1:5.For estimating the concentration of protein in a particular sample, 50μl volume reactionwas set and200μl of freshly prepared Bradford’s reagent was added. The complex givesa purplish colorwhose intensity is proportional to the amount of protein present in the sample. A standard curve was also generated using increasing concentrations of BSA (50 μg/ml, 100 μg/mland 200μg/ml).Cell lysatesof test samples werediluted to1:50 in the same volume.Each sample (including blank and standards) was taken in duplicates.The concentration of protein was measuredusing the ELISA reader at 570 nm. The unknown protein concentration (X) was calculated as follows:where,OD1& OD2: Optical densities of Standard (Std) 1 & Standard (Std) 2, respectively.BSA: Bovine serum albuminX×50 (dilution factor)/1000 = YConcentration of unknown protein (μg/μl) = Y × OD2.2.8 Immunoblotting(Western Blotting)Immunoblotting was performed as essentailly described by Lee (Lee, 2007). Equal amounts of protein were resolved on a denaturating SDS-polyacrylamide gel (8-12%). After completion of the run, the gel was transferred onto PVDF membrane and placed in the blotting cassette. The cassette wasthenput intothe mini transblot apparatus and transfer was done for 2-3 hours at a constant voltage of 80 V, depending on the size of the protein. Post transfer, membrane was rinsed in TBS containing 0.1% Tween-20 (TBST) and blocked with 5% non-fat milk in TBST for 1 h at 37ºC,on a gentle shaking rotator. After blocking, membrane wasrinsed thrice in TBST and incubated with primary antibodydiluted in TBST (ranging from 1:1000 to 1:10000, depending upon antibody used) for either3h at room temperature or overnight in the cold room.The membrane was then washed thrice with TBST and incubated withhorseradish peroxidase(HRP)-conjugated secondary antibody diluted

Immunoblotting(Western Blotting)

Extraction buffer

MTT reagent

For Cytotoxicity assays

Blocking buffer

Yeast were grown till mid-log phase 0.6-0.8 OD600in an appropriate medium and 10 mL of culture was pelleted at 2500 x g. Cells were suspended in 350μLof AEbuffer,mixed with 50μLof 10% sodium dodecyl sulphate and 400μLacid phenol(pH4.3) and immediately shaken vigorously on a dry bath (Eppendorf)at 65°C for 15 min.The tube was then quickly chilled on iceand centrifuged at 12000 x gfor 15 min to separate the aqueous phase from the phenol. After centrifugation, the aqueous phase was transferred to a new tube and extracted with an equal volume of chloroform.RNA was precipitated by adding 50 μLof 3 M sodium acetate (pH 5.3) and equal volume of 100% ethanol followed by incubation at -20°C for 2 h, andcentrifugation at maximum speed for 30 min at 4°C. The pellet obtained was washed in 70% ethanol, dried at room temperature anddissolved in an appropriate volume of DEPC-treated water. The concentration of RNA was estimated by measuring A260using a Nano Drop Spectrophotometer (ND1000). To monitor different classes of rRNA levels, 10 μg of total RNA from each strain was resolved on a 1.2% formaldehyde-agarose gel

RNA extraction by hot-phenol method

0.83mL1.5 M Tris-HCl,pH 6.8 50μL10% SDS 50μL10% Ammonium persulfate (APS)8 μLN,N,N′,N′-Tetramethylethylenediamine (TEMED)Resolving gel mix (12%) (20 ml)6.6 mLH2O 8 mL 30% acrylamide:bisacrylamide (29:1) mix 5 mL1.5 M Tris-HCl,pH 8.8 200 μL10% SDS 200 μL10% Ammonium persulfate (APS)8 μLN,N,N′,N′-Tetramethylethylenediamine (TEMED)

Whole cell lysis buffer for yeast (Homogenizing buffer) 50 mM Tris-HCl,pH 7.52 mM EDTA yeastprotease inhibitor cocktail SDS-PAGE 30% Acrylamide solution 29 g acrylamide 1 g bis-acrylamide dissolved in 100 mLH2O. 10% sodium dodecyl sulfate (SDS) 10 g SDS in 100 mLH2O Stacking gel mix (6%)(5 mL)3.4mLH2O 0.63mL 30% acrylamide:bisacrylamide (29:1) mix

Buffers for SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

final supernatant, beads were boiled in the equal volume of 2X SDS loading buffer (Lamelli Buffer) for 5 min. at 95°C. Bound protein complexes were collected by brief centrifugation (1 min.) at 14000 RPM and the supernatant containing the eluted protein fraction was resolved in SDS gel and analyzed by western blotting technique. For denaturing immunoprecipitation, cells were harvested in 1X PBS andpelleted. Cell pellet was resuspended in denaturing lysis buffer (50mM Tris-HCL, 100mM β-mercaptoethanol, 1% SDS, 5mM EDTA,0.5mM PMSF, 1 mg/ml aprotinin and 1 mg/ml pepstatin) (200μl for 100mm culture dish) and boiled for 10 min at 99°C. Cells suspensionwas briefly sonicated. Ice-cold 1X NETN (800μl) was added to the suspension after sonication and incubated on ice for 20min.,cells were centrifuged at 14000 rpm for 10 min. The supernatant was collected and used for immunoprecipiation by following the standard protocol

Protein-A or protein G beads (50 μl of 50% agarose beads) were washed twice with NETN lysis buffer. 2-5 μg of specific antibody was added to beads (in 1ml NETN) and were incubated with beads for 1 h at 4°C on a rotary shaker. Beads were collected by spinning at 500 g for 2 min.and supernatant was removed. 200-600 μg of totalprotein (mammalian cell lysate or bacterial cell lysate) was added to the beads and incubated for 2 hrs at 4°C on a rotary shaker. Beads were washed four times with the lysis buffer, each time by centrifuging at 500 g for 2 min at 4°C. After discarding the

Immunoprecipitation

ForEPS isolation,X. oryzaepv. oryzicolastrains were plated on PS agar plateand incubated at 28°C. Bacterial lawn was dissolved in 15 ml 1X PBS and 100 μl formamide, and centrifuged at 12,000 g for 6-8 min at RT. Before centrifugation, 1 ml cell suspension was diluted, and plated to get the CFUs. For EPS precipitation, 250 ml chilled acetone was added to the supernatant, and kept at 4°C for overnight (Dharmapuri and Sonti, 1999). EPS was pelleted down at 7000 g for 10 min at 4°C, washed with 10 ml acetone, and kept for drying. After drying, it was dissolved in appropriate volume of water, and quantitated by colorimetric method for estimation of pentoses and hexoses by phenol-sulphuric acid method (Dharmapuri and Sonti, 1999)

EPS isolation and estimation

For DNA precipitation after digestion, 500 μl nuclease free water was added to the digested DNA fragment. Equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the mixture and centrifuged at 13,000 g for 10 minat RT. Upper aqueous phase containing DNA fragment was transferred to fresh microcentrifuge tube and DNA was precipitated by adding 0.7 volume of iso-propanol and 1/10thvolume of sodium acetate. Precipitated DNA was washed with 70% ethanol, pellet was air dried for 20-30 min at RT and dissolved in nuclease-free water