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  1. May 2019
    1. Bacterial strains
    2. All the bacterial strains that were used in this study are derivatives of Escherichia coliand their genotypes have been listed in Table 2.1 Bacterial strains were routinely stored on solid agar plates at 4°C and also as thick suspensions in 40% glycerol either at −20°C or at −70°C. Plasmid harboring strains, were reconstructed whenever necessary by fresh transformations

      Center For DNA Fingerprinting and Diagnostics, Hyderabad

    2. " %  &

      Shivalika Saxena


      Studies on Transcription Translation coupling and the consequences of its failure in Escherichia coli Modulation of Rho dependent transcription termination by H NS family of proteins

    1. 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%
    2. Densitometry
    3. 5% glycerol)containing (i) 5′-end-labeled DNAfragmentof 1200 cpm radioactive count(ii) 1 μg each of bovine serum albumin andpoly(dIdC)(iii) the protein at the indicated monomer concentrations and (iv) when required,co-effectorsat specified concentrations. The reaction mixture was incubated at room temperature for 30-minsand the complexes were resolved by electrophoresis on a non-denaturing 5%polyacrylamide gel (39:1 acrylamide:bisacrylamide)in 0.5X TBE buffer pH8.3, at 12.5V/cm for 3 hrs at 18°C.The gels were then dried on a gel drier at 80°C for 45 minsand the radioactive bands were visualised with a Fujifilm FLA-9000 scanner.For DNA bending EMSA, co-effectors were not added in the binding reaction but at aconcentration of 0.1 mM in both the gel and running buffer
    4. The DNA templates were obtained by PCR from E. coligenomic DNA. After 5-end labeling, the PCR fragments were purified by electroelution following electrophoresis on 6% native polyacrylamide gels (Sambrook and Russell,2001). EMSA reactions were performed in 20 μl reaction volume inEMSA binding buffer(10 mM Tris-Cl at pH 7.5, 1 mM EDTA, 50 mM NaCl, 5 mM dithiothreitol, and
    5. Electrophoretic mobility shift assay (EMSA)
    6. Primer extension analysis to map thetranscription start site was carried out as describedby Conway et al. (1987) and Rajkumari et al. (1997). 20 pmolof primer was labelled at its 5′-end with 32P-γ-ATP as described above. 106cpm equivalent of labelled primer was mixed with 10μg of total cellular RNA. Sodium acetate pH-5.5 was added to a final concentration of0.3 M and the nucleic acids were precipitated with ethanol, washed with 70% alcohol,air-dried and dissolved in hybridization buffer (9 mM Tris-Cl, pH-8 and 0.35 mMEDTA) and incubated overnight at 43ºC for annealing. Reverse transcriptase reactionwas performed by the addition of 5 mM MgCl2, 1 mMdNTP’s, 1 X RT buffer, highconcentration (10 units) of Superscript III Reverse Transcriptase (Invitrogen) to the mixture of annealedlabelled primer and RNA. The reaction was incubated at 43ºC for 1-hr following whichthe nucleic acids were precipitated with absolute alcohol and 0.3 M CH3COONa, pH-5.5. The precipitate was air dried and dissolved in water and gel-loading dye (95%formamide, 20 mM EDTA, 0.05% each of xylene cyanol and bromophenol blue) wasadded. The samples were heated at 90ºC for 2-min before loading on a 6% denaturingpolyacrylamide gel for electrophoreticresolution alongside a sequencingladder
    7. Primer extension analysis
    8. Oligonucleotides and PCR products were end labeled using phage T4-polynucleotidekinase (PNK, New England Biolabs) with 32P-γ-ATP. The radiolabelling reactionmixture (50 μl) contained 1 X of buffer provided by the company, 10 units of T4-PNKand 50 μCi of32P-γ-ATP. The reaction mix was incubated for 1-hr at 37ºC and thereaction was stopped by adding 10 μl of 0.5 M EDTA. The labeled oligonucleotides andDNA fragments were purifiedeither by the Qiagen PCR purification or nucleotide removal kit.Labelling efficiency was checked by scintillation counting
    9. Radiolabelling of oligonucleotides and PCR products
    10. Site directed mutagenesis of plasmid DNA was carried out by using QuikChange kit(Stratagene) with a pair of complementary oligonucleotide primers carrying thenecessary sequence modifications. In this process, the plasmid (around 20-100 ng)containing the fragment of DNA where nucleotidehas to be altered, was used astemplate and “linear PCR” of 20 cycles was set up using Pfu Turbo DNA polymerase toamplify the whole plasmid with extension time calculated according to a rate of 500-bp/min. The reaction mix was digested with DpnIfor 1-hr(to destroy the original inputplasmid DNA) following which it was transformed directly to a highly competent DH5cells. The mutated plasmid was confirmed by sequencing
    11. Site directed mutagenesis
    12. Automated DNA sequencing on plasmid templates or on PCR products was carried outwith dye terminator cycle sequencing kits from Perkin-Elmer on an automatedsequencer (model 377, Applied Biosystems), following the manufacturer’s instructions.Manual sequencing was achieved using the SequenaseVersion2.0 DNASequencing Kit from USB Corp. as described in manufacturer’s instructions and thesequencing reaction products were resolved by electrophoresis on a 6% sequencing gel
    13. DNA sequencing
    14. and a colourless upper aqueous phase. The upper aqueous phase in which RNA existsexclusively, was transferred to a fresh microfuge tube and RNA was precipitated byadding 0.5 ml of isopropyl alcohol for each ml of Trizol used. Samples were incubatedat 15 to 30ºC for 10-min and centrifuged at 12000 rpm for 10-min at 4ºC. RNA formeda gel like precipitate at the bottom of the tube. Supernatant was removed and RNA waswashed with 75% ethanol (by adding 1 ml of ethanol per ml of Trizolemployed). RNAcould be stored after this step in –20 or –70ºC for more than a year. RNA pellet was airdried for 15-to 30-min following which it was dissolved in nuclease free water. Theconcentrations and purity of RNA samples were determined spectroscopically as wellas by visual inspection on formaldehyde-agarose gel in MOPS buffer (Goodet al., 1996). Before loading onto the gel, RNA was mixed with loading buffer and heated at90ºC for 3-min
    15. For isolation of RNA, cells were grown in minimal A medium supplemented with 0.2%glucose upto A600of 0.6. Cells were harvested by centrifugation and total RNA wasisolated by using Trizol (Invitrogen) according to manufacturer’s instructions. 1 ml ofTrizol was used to lyse cells equivalent of approximately 4 ml of overnight culture.Homogeneous lysis was achieved by gentle pipetting repeatedly. The homogenized samples were incubated at room temperature for 5-min to permit complete dissociationof nucleoprotein particles. Following homogenization, 0.2 ml of chloroform for each 1ml Trizol reagent was added and vigorously shaken with hand for 15-sec and incubatedfurther for 3-min at RT. It was then centrifuged at 12000 rpm for 10-min at 4ºC, whichseparates out the homogenate into lower phenol chloroform phase (red), an interphase
    16. Isolation of total cellular RNA
    17. require high fidelity,Taq DNA Polymerase from MBI Fermentas was used. However,for precise amplifications either Herculase Fusion or PfuDNA polymerasefrom Stratagene was used. Approximately, 10-20ng of plasmid or 100 to 200 ng ofchromosomal DNA was used as a template in a 50 μl reaction volume containing 200μM of each dNTP, 20 picomoleeach of forward and reverse primer and 1.5 units of DNA polymerase.In the case of colony PCR performed to examine multiple colonies for presence of the plasmid clones, E. coli cells from afreshly grown plate wereresuspended in 50 μl of sterile Milli-Q water to get a cell suspension (~109cells/ml)and 4 μl from this was usedas the source of DNA template. To verify various pMU575 clonesdescribed in this study, by colony PCR,the vector specific primer pairs JGJpMUF and JGJgalK were used. The expected amplicon for pMU575 alone is ~300-bp, while that carrying the cloned fragment would be >300-bp.For each PCR reaction, the samples were subjected to 30-cycles of amplification and the typical conditions were as follows (although there were slight alterations from one set of template/primerto another):The initial denaturation was carried out at 95°C for 4-min and the cycle conditionswere as given below:Annealing 45ºC to 50°C 1-minExtension 68°C (1-min/kb of DNA template to be amplified)Denaturation 95°C 1-minAfter 30 cycles of PCR, the final extension step was carried out again for 10-min at68°C
    18. For amplification of short length (100-200-bp)DNA fragmentsor that do not
    19. Polymerase chain reaction (PCR)
    20. Molecular techniques
    21. Native isoelectric focusing was done using Pharmacia Phast Gel Apparatus and precast IEF gel (pH 3-9) from GE healthcare. The samples were prepared in 50 mM sodium buffer (pH 8.0) and applied in the middle portion of the gel. Gels were run as previously described(Olsson et al., 1988) that is at 15°C, pre-focusing at 2000 V (75Vh), sample loading at 200V (15Vh) and run at 2000V (500Vh). Staining was done using Coomassie Blue G-250
    22. Native Isoelectric Focusing
    23. Gel-filtration chromatography was performed at room temperature on a BioLogic LP protein purification system (Biorad) with an in-house packed Sephadex G-100 column of size 1.5 X 43 cm; each protein sample was loaded in 0.8-ml volume, and the buffer used for chromatography was 20 mM Tris-Cl (pH 8) with 200 mM NaCl at a flow rate of 0.1 ml per min with 1.5-ml fractions being collected for analysis. Protein elution was detected by measurement of A295.The void volume, V0was determined using blue dextran (2X 106Daltons) and theelution parameter Kavfor each proteinwas calculated from elution volume Veand total bed volumeVtusing the equation:Kav= (Ve–V0)/(Vt–V0)Initially, acalibration curve was derived froma semilogarithmic plotof Kav of protein standardsalbumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and ribonuclease A (13 kDa) on the Y-axis against log10of their molecular masses on theX-axis. The Kavof the ArgPdproteins were calculated based on their elution volume and then the molecular masses were derived from the corresponding point on the calibration curve
    24. Gel-filtration chromatography
    25. directly from lysed cells, log and stationary phase cultures were spun down, samplebuffer (1 X final concentration) was added to the cell pellet and boiled for 10 min,cooled to room temperature, and after a second spin, the clear supernatant was loaded.The gel run was started at constant current of 20 mA. When the dye front crossed thestacking gel the current was increased to 40 mA
    26. The method followed was as described in Sambrook and Russell (2001). Gels of 1.0mmthickness were casted in the commerciallyavailable small gel apparatus. Resolving gelof 12% (15 ml) and stacking gel (4 ml) was made. Gels were polymerised by theaddition of TEMED and APS (1 % v/v of the gel mix). Sample preparation for gelloading was done as follows. Cell lysate or pure protein fractions (around 30 μg) wasmixed with the sample buffer to 1 X and heated at 95ºC for 2-min. To check expression
    27. Sodium dodecyl sulphate-polyacrlyamidegel electrophoresis (SDS-PAGE)
    28. Protein concentrations were estimated by the method of Bradford (1976). The A595wasmeasured after complexation with Bradford reagent. Bovine serum albumin was usedas standard against whichthe unknown protein concentrations were estimated
    29. Protein estimation
    30. Overexpression and purification of ArgPand ArgPdproteins
    31. argP+, argPd-S94L, argPd-P108S, argPd-P274Sfragment downstream of the phage T7-promoter, such that the encoded proteins beara C-terminal His6-tag provided by the vector DNA sequence. Theresultant plasmid was transformed into strain BL21(DE3) which has the T7 RNA Polymerase under the isopropyl thio-β-D-galactoside (IPTG) inducible lacUV5promoter.The resultant strains were grownin LB (500-1000 ml) to an A600of around 0.6and were then induced with 1 mM IPTG and harvested after 4-hrs of induction.Bacterial cells were recovered by centrifugation, resuspended in 20 ml of lysis buffer(20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mM DTT and 10 mM imidazole) containing20 μg/ml lysozyme, and lysed by sonication with 30-sec pulses for 10-min. Theprotocol for His6-ArgP(ArgPds)protein purification involved (i) passing the lysate through a 5ml Ni-NTA (Qiagen) chromatographic columnequilibrated with lysis buffer, (ii) washing thecolumn with 100 ml of washing buffer (20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mMDTT; 30 mM imidazole), and (iii) elution of His6-ArgP(ArgPds)from the column with elutionbuffer (20 mM Tris-Cl, pH-8;300 mM NaCl; 10 mM DTT and 250 mM imidazole) andcollection of 1.5 ml eluate fractions (10 fractions). The fractions were tested forprotein by Bradford method and the protein-carrying fractions (generally tubes 2 to 5)were pooled and dialysed in a 1:200 volume ratio against 20 mM Tris-Cl, pH-8 with 10mM DTT, 300 mMNaCl for 5 hrs followedby a change to buffer of composition 20 mM Tris-Cl, pH-8 with 10 mM DTT, 300 mM NaCl and 40% glycerol for 24 hrs. The proteins were concentrated by centrifugation toaround 1 mg/ml by using Amicon filter (pore size 10-KDa) and stored at −20ºC or −70ºC
    32. For preparing ArgP and ArgPd-S94L, -P108S and -P274S proteins, derivatives(designated as pHYD1705, pHYD2678, pHYD2679 and pHYD2680 respectively) of the plasmidvector pET21b (Novagen) was constructed which carries the PCR-amplified
    33. Biochemical techniques
    34. Typically 200-300 ng of DNA was used in each ligation reaction. The ratio of vector toinsert was maintained between 1:3 to 1:5 for cohesive end ligation and 1:1 for blunt endligation. The reaction was generally performed in 10 μl volume containing ligationbuffer (provided by the manufacturer) and 0.05 Weiss unit of T4-DNA ligase, at 16ºCfor 14-to 16-hrs. On using the rapid ligation kitfrom Fermentas, incubation was at 22ºC for 1-2 hrs
    35. Ligation of DNA
    36. PCR products were purified using the PCR Purification Kit (Qiagen) as per the manufacturer's instructions
    37. DNA fragments to be used for specific purposes like ligation or radioactive labeling were eluted from the agarose gel after electrophoresis. The gel piece containing thedesired band was sliced out from the gel and the DNA was purified using commerciallyavailable purification kits for this purpose. The efficiency of elution was determined bychecking a small aliquot of DNA sample on the gel
    38. Purification of PCR products
    39. Purification of DNA by gel elution
    40. Around 0.5 to 1 μg of DNA was regularly used for each restriction digestion. 2to 5units of restriction enzyme were used in the total reaction volume of 20 μl containing 2μl of the corresponding buffer supplied at 10 X concentration by the manufacturer. Thereaction was incubated for 2 hrs at the temperature recommended by the manufacturer.The DNA fragments were visualised by ethidium bromide staining after electrophoresison a 0.8 to 1% agarose gels. Commercially available DNA size markers were run alongwith the digestion samples to compare with and to estimate the sizes of the restrictionfragments
    41. Restriction enzyme digestion and analysis
    42. TheDNA samples were mixed with appropriate volumes of 6 X loading dye (0.25%bromophenol blue and 0.25% xylene cyanol and 30% glycerol in water) and subjectedto electrophoresis through 0.8 to 1 % agarose gel in TAE buffer. The gel was stained in1 μg/ml ethidium bromide solution for 15-min at room temperature and visualised byfluorescence under UV-light in a UV-transilluminator
    43. Agarose gel electrophoresis
    44. werethen recovered by centrifugation at 12,000 rpm for 30-min. The pellet was washed oncewith 70% ethanol, air-dried and re-suspended in 100 μl of TE-buffer. It was treatedwith RNase at a concentration of 20 μg/ml by incubating at 37ºC for 1-hr. It was furtherextracted with an equal volume of phenol:chloroform mixture followed bychloroform:isoamyl alcohol (24:1) mixture. After centrifugation, the clear supernatantwas used for recovering the nucleic acids. The nucleic acids were precipitated with 200μl of alcohol in presence of 0.3 M sodium acetate (Sambrook and Russell, 2001). In casewhere high purity plasmid preparations are required (DNA sequencing) the plasmidisolation was carried out with the commercially available kits following themanufacturer’s instruction. Plasmids were observed on 1% agarose gel
    45. 1.5 ml of stationary phase culture wascentrifuged and cell pellet was re-suspended in 567 μl of TE buffer. To this 30 μl of10% SDS, and 3 μl of proteinase K (20 mg/ml) were added in that order and the cellsuspension was mixed and incubated at 37ºC for 1-hr. When the suspension was clear, 100 μl of 5 M NaCl was added and thoroughly mixed followed by the addition of 80 μlCTAB/NaCl (10% cetyl trimethyl ammonium bromide in 7 M NaCl). The suspensionwas incubated at 65ºC for 10-min, brought to room temperature and extracted with anequal volume (780 μl) of chloroform isoamyl alcohol (24:1), and aqueous phasetransferred to fresh tube. The aqueous phase was further extracted successively, firstwith phenol:chloroform:isoamyl alcohol (25:24:1) and then with chloroform isoamylalcohol (24:1). DNA was precipitated fromthe clear supernatant by the addition of 0.6volumes of iso-propanol. The chromosomal DNA was either spooled out or pelleted atthis stage and washed with 70% ethanol air dried and dissolved in 100 μl of TE-buffer
    46. Isolation of chromosomal DNA
    47. 1.5 ml of cells from an overnight culture waspelleted by centrifuging in cold (4ºC) for10-min at 6000 rpm. The cells were re-suspended in 200 μl solution I (50 mM glucose; 25 mM Tris-Cl, pH-8; 10 mM EDTA, pH-8) with vortexing. 400 μl of freshly preparedsolution II (0.2% NaOH, 1% SDS) was added and mixed by gently inverting the tubes.Subsequently, 300 μl of solution III (prepared by mixing 60 ml of 5 M CH3COOK,11.5 ml glacial acetic acid, 28 ml water) was added and the tubes were invertedrepeatedly and gently for homogeneous mixing followed by incubation for 5-min onice. After centrifuging at 12,000 rpm for 15-min, supernatant wasdecanted into a freshtube, an equal volume of iso-propanol was added, the precipitated nucleic acids
    48. Isolation of plasmid DNA
    49. Recombinant DNA techniques
    50. A differential gene expression microarray with respect to argP was performed by Genotypic Technology Pvt.Ltd., Bengaluru. The experiment was performed on an oligonucleotide microarray having 10828 probes for coding region(on average three probes were designed for each 4294 coding regions) and 4380 probes for non-coding region (on average two probes were designed for 2240 non-coding regions). The RNA was labelled using Cy3 and single channel detection was used. Data was analysed using GeneSpring GX Version 7.3
    51. Microarray details
    52. supplemented with amino acids and appropriate antibiotic and grown at 37ºC to an A600of 0.5-0.6. Around 0.1-0.5 ml of culture was made up to 1 ml with Z-buffer and lysedwith addition of one drop of chloroform and 1-2 drops of 1% SDS solution. 0.2 ml offreshly prepared 4 mg/ml ONPG was added to start the reaction and incubated at roomtemperature till the color of the reaction mixture turned yellow. 0.5 ml of 1 M Na2CO3was added to stop the reaction and the time duration from initial addition of ONPG tothe stopping of reaction was noted.The absorbance of reaction mix was taken at 420nm and 550 nm. The A600of the culture used was also noted. The enzyme specificactivity (in Miller units) was calculated using following equation:β-galactosidase specific activity = [1000 X A420-(1.75 X A550)] / t X v X A600Where t isthe time period in minsand v the volume of culture used in ml.Each value reported is the average of at least three independent experiments, and the standard error was <10% ofthe mean in all cases
    53. β-galactosidase assay was performed according to Miller (1992). An overnight grownculture of the bacterial strain was sub-cultured in glucose Minimal A medium
    54. β-galactosidase assay
    55. Thialysine or thiosine (S-Aminoethyl-L-cysteine)is a toxic analog of Lys. Strains were testedfor sensitivity/resistance to thialysine by streaking them on minimal A-glucose platessupplemented without and with100-200 μg/ml thialysine(Steffes et al., 1992)
    56. Test for thialysine resistance
    57. For testing ArgR+/–phenotype, the colonies werestreaked on minimal A-glucose plates containing uracil (40 μg/ml) and CAN(65 μg/ml). Uracil wasadded to the medium to sensitize an argR+strain to CAN. An argR+strain is inhibited at65 μg/ml CANon a uracil-containing plate, whereas on a plate without uracil, argR+would grow even at 700-800 μg/ml CAN. Uracil represses the carAB transcription, whichencodes the carbamoyl phosphate synthase enzyme (CarAB). This results in reducedamounts of carbamoyl phosphate, which is the common intermediate between pyrimidineand Arg biosynthetic pathways. Reduced carbamoyl phosphate levels would result indecreased flux through the Arg biosynthetic pathways. This in turn would result indecrease in Arg pools inside the cell. An argR mutant would be derepressed for the Argbiosynthetic pathway and is resistant even to 300 μg/ml CANin a uracil-containing plate
    58. Test for ArgR+/–phenotype
    59. Test for canavanine (CAN) sensitivity
    60. CAN is a toxic analog of Arg and is an inhibitor of bacterial growth. Strains were tested for sensitivity/resistance to CAN by streaking them on minimal A-glucose platessupplemented withoutand with40 μg/ml CAN(or other concentrations as indicated) and 40 μg/ml uracil
    61. The colonies to be tested were streaked on the surface of minimal A-glucose plates containing either 0.4-0.7 M NaCl with 1 mM glycine betaine, and incubated at 37oC. NaCl-tolerant strains grew toform single colonies in 36-60 hrs whereas NaCl-sensitive ones did not. As controls, MC4100 (WT) and other previously identified NaCl sensitive mutants were streakedfor comparison
    62. NaCl-sensitivity testing
    63. agar platesLac+colonies will appear dark pink colonies whereas Lac–will remain colourless
    64. A. lacphenotype
    65. Scoring for phenotypes
    66. Competent cells for high efficiency transformations were prepared by a method ofInoue et al. (1990) with few modifications. An overnight culture of the strain (routinelyDH5α) was sub-cultured into fresh sterile LB-brothin 1:100 dilutions and grown at 18ºC to an A600of 0.55. The cells were harvested by centrifugation at 2500 rpm for 10-min at 4ºC. This was re-suspended in 0.4 volumes of INOUE buffer and incubated inice for 10 min. The cells were recovered by centrifugation at 2500 rpm at 4ºC for 10-min and finally re-suspended in 0.01 volume of the same buffer. Sterile DMSO wasadded to a final concentration of 7%. After incubating for 10-min in ice, the cells werealiquoted in 100 μl volumes, snap frozen in liquid nitrogen and stored at –70ºC
    67. Preparation of high efficiency competent cells
    68. For routine plasmid transformations, following method which is modification of thatdescribed by Cohen et al. (1972) was used. An overnight culture of recipient strain wassub-cultured 1:100 in fresh LB medium and grown till mid-exponential phage. Theculture was chilled on ice for 15-min, and the steps thereafter were performed at 4ºC.20 ml of culture was centrifuged and pellet was re-suspended in 10 ml of 0.1 M CaCl2.After 15-min of incubation on ice, the cells were again centrifuged and re-suspended in2 ml of 0.1 M CaCl2. The suspension was incubated on ice for 30-min. To the 200 μl aliquot of the cell suspensionplasmid DNA (20 to 200 ng in less than 10 μl volume)was added, incubated for half an hron ice and given a heat shock for 90-sec at 41ºC.The cultures was rapidly chilled, mixed with 0.8 ml of LB-broth and incubated at 37ºCfor 1-hr, and plated on an appropriate selective medium at various dilutions. An aliquotof cell suspension to which plasmid DNA was not added served as a negative control
    69. A. Calcium chloride method
    70. Transformation
    71. the infection mixture was centrifuged, washed in 5 ml of citratebuffer and plated without phenotypic expression
    72. To 2 ml of fresh overnight culture of recipient strain, 108pfu equivalent of phage lysatewas added and incubated at 37ºC without shaking for 15-min to facilitate phageadsorption. The un-adsorbed phage particles were removed by centrifugation at 4000rpm for 5-min and pellet of bacterial cells was re-suspended in 5 ml of LB-brothcontaining 20 mM sodium citrate to prevent further phage adsorption. This wasincubated for 30-min at 37ºC without shaking to allow the phenotypic expression of theantibiotic resistance gene. The mixture was then centrifuged, and the pellet was resuspendedin 0.3 ml citrate buffer. 100 μl aliquots were plated on appropriate antibioticcontaining plates supplemented with 2.5 mM sodium citrate. A control tube withoutaddition of P1 lysate was also processed in the same way. In the case of selection ofnutritional requirement,
    73. Phage P1 transduction
    74. 0.3 ml of overnight culture of the donor strain in Z-broth was mixed with 107plaqueforming units (pfu) of a stock P1 lysate prepared on strain MG1655. Adsorption wasallowed to occur at 37ºC for 20-mins. To 0.3 ml of infectionmixture, 10 ml of Z-broth was added and incubated at 37ºC withslow shaking until the visible lysis of the culture occurred (in 4-6 hrs). The lysate wastreated with 0.3 ml of chloroform, centrifuged and the clear lysate was stored at 4ºCwith chloroform.Preparation of P1 lysates on recA mutant strains were also donesimilarly, but with a higher multiplicity of infection (i.e. 108starter P1 phage).To quantitate the P1 phage lysate preparation, titration was done using P1 phagesensitive indicator strainsuch as MG1655. 100 μl each of dilution of phage (typically10–5, 10–6) were mixed with 0.1 ml of fresh culture grown in Z-broth. After 15-min ofadsorption at 37ºC without shaking, each mixture was added on a soft agar overlay ofZ-agar plates and incubated overnight at 37ºC. The phage titer was calculated from thenumber of plaques obtained on the plates
    75. Phage P1 lysate preparation by broth method
    76. Genetic techniques
    77. purification of DNA fragments werefrom Qiagen or HiMedia. The oligonucleotide primers used in this study were mainly synthesised by Ocimum Biosolutions or MWG Biotech. The radioactive chemicals were procured from BRIT Mumbai
    78. Chemicals were obtained from commercial sources. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were mostly from HiMedia laboratories. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4-DNA ligase, DNA-polymerases and DNA size markers were obtained from companies including New England Biolabs, MBI Fermentas and Stratagene.RNA isolation chemicals like Reverse transcriptase, trizol, RNA loading buffers and dyes and RNA size markers were obtained from Invitrogen and Sigma. Protein markers were obtained from MBI Fermentas. Kits for plasmid isolation,
    79. Chemicals
    80. Antibiotics were used at the following final concentrations in various media as given inTable 2.4.Table 2.4Concentrations of antibiotics (μg/ml)
    81. Antibiotics
    82. Waterto 3 mlTEMED 10 μlDenaturing (urea) sequencing gel (6%) composition10 X TBE 50 ml40% acrylamide 75 mlUrea 210 gm (7 M)Waterto 500 mlThis was filtered through a 0.45/0.22 μ milipore filter.For casting the gel 35 ml of the sequencing gel mixure was mixed with 150 μl10% APS and 25 μlTEMED
    83. Formaldehyde agarose gel(For 50 ml)DEPC treated water 43 mlMOPS buffer 5.3 mlAgarose0.63 gmFormaldehyde2.6 mlThe above mix was boiled without formaldehyde to dissolve agarose and then at around 50ºC formaldehyde was added just before casting the gel.40% Acrylamide solutionAcrylamide39 gmBis-acrylamide 1 gmWater to 100 mlNon denaturing gel composition (50 ml)40% acrylamide solution 5 ml10 X TBE 5 mlH2O 40 ml10% APS 250 μlTEMED 30 μlSDS PAGE gel (12%)For resolving Gel (15 ml):30% Acrylamide solution 6 ml1.5 M Tris-Cl (pH 8.8)3.8 ml10% SDS150 μl10% APS 150μlWaterto 15 mlTEMED 10 μlFor stacking gel (3 ml):30% Acrylamide solution 500 μl1 M Tris Cl (pH 6.8) 380 μl10% SDS 30 μl10% APS 30 μl
    84. Storage buffer for proteinTris-Cl (pH 8.0) 20 mMNaCl 300 mMDTT10 mMGlycerol 40 % Hybridization bufferTris-Cl (pH 8.0) 9 mMEDTA 0.35 mMSample buffer (for SDS-PAGE)Tris-Cl (pH 6.8) 150 mMSDS (20%) 6% v/vGlycerol 30% v/vβ-mercaptoethanol (5%) 15%Bromophenol blue 0.6% (w/v)EMSA binding bufferTris-Cl (pH 7.5) 10 mMNaCl 50 mMEDTA1 mMGlycerol 5 %DTT 5 mMDenaturing gel loading buffer with dyeFormamide 95%EDTA 20 mMXylene Cyanol 0.05 gmBromophenol blue0.05 gmNon denaturing gel loading buffer with dyeTris-Cl (pH 7.5) 250 mMBromophenol blue 0.02%Glycerol 20%
    85. MOPS bufferMOPS 4.16 gm0.5 M EDTA 1.0 mlSodium acetate 0.68 gmWater (nuclease free) to 500 mlIt was filter sterilized and stored in an amber colored bottle. This was prepared as 10 Xstock solution and used at 1 X concentration.INOUE (PIPES) bufferPIPES (free acid) 10 mMCaCl2.2H2O15 mMKCl 250 mMMnCl2.4H2O 55 mMpH was adjusted to 6.7 with 1 N KOH.PIPES gets into solution when the pH is greater than 6.7. MnCl2was dissolvedseparately and added drop by drop with stirring. The pH was adjusted to 6.7 and filtersterilized and stored at –20ºC.Z buffer (for β-Galactosidase assay)Na2HPO416.1 gmNaH2PO45.5 gmKCl0.75 gmMgSO4.7H2O 0.246 gmβ-mercaptoethanol 2.7 mlWaterto 1000 mlpH was adjusted to 7.0 and stored at 4ºC.SDS running bufferTris-base 30.3 gmGlycine144 gmSDS 10 gmWaterto 1000 mlIt was prepared in 10 X concentration and diluted to 1 X for running
    86. Citrate bufferCitric acid (0.1 M)4.7 volumeSodium citrate (0.1 M) 15.4 volumeTE bufferTris-Cl (pH 8.0) 10 mMEDTA 1 mMTBE bufferTris-Borate 90 mMTris-Borate 90 mMEDTA (pH 8.0) 2 mMThis was prepared as 10 X stock solution and used at 1 X concentration.TAE bufferTris-acetate 40 mMEDTA (pH 8.0) 2 mMThis was prepared at 50 X concentrated stock solution. Both TBE and TAE were usedas standard electrophoresis buffers
    87. Buffers and solutions
    88. LB agarLB medium 1000 mlBacto-agar 15 gmZ broth (for P1 transduction)LB medium100 mlCaCl2(0.5 M) 0.5 mlZ agar (for P1 transduction)Z broth 100 mlBacto-agar0.75 gm
    89. Amino acids when required, were added to a final concentration of 40 μg/ml. Whengrowth on other carbon sources was to be tested, glucose was substituted withappropriate sugar at 0.2%.Glucose-minimal A medium, pH 7.4This medium was same as Glucose-minimal A medium described above except for the difference in K2HPO4and KH2PO4which were as mentioned below:K2HPO414.0 gmKH2PO42.7 gmGlucose-minimal A medium, pH 5.8This medium was same as Glucose-minimal A medium described above except for the difference in K2HPO4andKH2PO4which wereas mentioned below:K2HPO41.5 gmKH2PO412.4 gmGlucose /Glycerol-minimal A 19 (18 or 17) amino acidmediumThis medium is essentially the same as glucose/glycerol-minimal A medium described above except that all 19 or 18 or 17 otherthan either Lys or Lys and Arg or Lys and Arg and His amino acids were added after autoclaving at a final concentration of 40μg/ml from autoclaved 4 mg/ml stock solutions.Minimal A agarIt contains 1.5% bacto-agar (Difco) in minimal A medium. The plates were pouredafter mixing double strength minimal A with 3% agarthat had been autoclaved separately.LB mediumTryptone 10.0 gmYeast Extract5.0 gmNaCl 10.0 gmWaterto1000 mlpH adjusted to 7.0 to 7.2 with 1 N NaOH
    90. All media and buffers were sterilised by autoclaving at 121ºC for 15 mins. Mediaand buffers used in this study are given below:Glucose /Glycerol-minimal A mediumK2HPO410.5 gmKH2PO44.5 gm(NH4)2SO41.0 gmSodium citrate, 2H200.5 gmWater to 1000mlAfter autoclaving the following solutions were addedMgSO4(1M) 1 mlGlucose (20%) 10 mlOr Glycerol (80%)5 mlVitamin B1 (1%) 0.1 ml
    91. Media
    92. The primers used in this study are listed in Table 2.3.Table 2.3 Oligonucleotide primersa
    93. Primers
    94. bindingsite lie upstream of the MCS to ensure the high level expression of any genecloned in MCS. A stretch of hexa-histidine (His6)-encoding codons followed by stopcodon is incorporated downstream of MCS to give a C-terminally His6-taggedrecombinant protein (EMD Biosciences).6. pBAD18:It is an expression vector with a pMB9derived origin of replication and allows for tightly regulated expression of genes cloned under the PBADpromoter of the araBADoperon (Guzman et al.,1995). The vector also carries thearaCgene, encoding the positive and negative regulator of this promoter.7. pCP20: pSC101-based Ts replicon, chloramphenicol resistant, ampicillin resistant, for in vivoexpression of Flp recombinase (Datsenko and Wanner, 2000)Plasmid DNA preparations were routinely made from recAstrainDH5αandwerestored in 10 mM Tris-Cl (pH-8.0) with 1 mM EDTA at –20ºC. The plasmid constructsused in this study are given in Table 2.2.Table 2.2Plasmid constructs
    95. The plasmid vectors used in this study were as follows:1.pCU22: It is a derivative of pUC19 used to prepare supercoiled DNA for in vitrotranscription where two strong phage fdtranscription terminators flank MCS. This ensures that the transcripts originated from vector based promoters will not interferewith the transcription from the cloned promoter and that the transcript originated fromthe cloned promoter will be terminated after the MCS (Ueguchi and Mizuno,1993).2.pMU575: It is an IncW-based, single-copy, trimethoprim resistance bearingpromoter probe vector. It carries its MCS upstream of a promoterless galK’-lacZfusion. This fusion has the first 58 codons of galKfused to the 8th codon oflacZ, andthe resultant hybrid polypeptide possesses functional β-Galactosidase activity(afterassembly as a tetramer). Translation of the hybrid gene is controlled by the ribosomebinding site of galK. There are stop codons in all the three reading frames between MCS and initiation codon of galKso that there is no interference caused bytranslational read-through from inserts cloned into MCS region. A strong pheRterminator located upstream of the MCS prevents read through from any vector-basedpromoter into the lacZgene (Andrews et al.,1991).3. pTrc99A:It is an expression vector with ColE1 origin of replication and ampicillin resistance marker. It provides IPTG dependent induction of the cloned gene (Amann et al., 1988)4. pCL1920: It is a pSC101-based, low-copy-number vector with spectinomycin and streptomycin resistance marker carrying the MCS in lacZαregion and henceprovides the advantage of screening the insertions using α-complementation (Lernerand Inouye,1990).5. pET21b: It is a ColE1-based, high-copy-number expression vector bearing ampicillinresistance marker. A strong T7 RNAP-recognised promoter and an efficient ribosome
    96. Plasmids
    97. TheE. coli strains used in this study with their genotypes are shown in Table 2.1. All strains other than BL21 (DE3) employed in protein overexpression experiments are derivatives of E. coli K12. Bacterial strains were routinely stored on solid agar plates at 4ºC and also as thick suspensions in 40% glycerol at –70ºC. Plasmid harboring strains were freshly prepared by transformation of the required plasmid. The bacteriophage P1kc from the laboratory collectionwas used for routine transduction tomove a locus from one strain to anotherand is referred to as P1 throughout this thesis.Table 2.1 E. coli strains used in this study
    98. Strains and bacteriophages
  2. shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
    1. Centre for DNAFingerprinting and DiagnosticsHyderabad, India
    2. Carmelita NoraMarbaniang
    3. ArgP protein of Escherichia coli: roles in osmoregulation,gene regulation and inter-relationship with LysG of Corynebacteriumglutamicum
    1. For TEM, C. glabrata cells were digested with zymolyase 20T for 3 h at 30◦C, centrifuged at 1,000 g and washed with YPD medium. Cell fixation was performed as described for SEM and dehydrated samples were embedded in araldite 6005 resin. After complete polymerization at 80 ̊C for 72 h, ultra-thin (50-70 nm) sections were preparedwith a glass knife on Leica Ultra cut (UCT-GA-D/E-1/00)microtomeand mounted on copper grids. Aqueous uranyl acetate-stained and Reynolds lead citrate-counterstained samples were viewed under Hitachi H-7500 transmission electron microscope
    2. Transmission electron microscopy
    3. For SEM, C. glabratacells were fixed for 24 h in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.2) at 4 ̊C, post-fixed in 2% aqueous osmium tetroxide for 4 h and dehydrated. After drying to critical point, mounted samples were coated with a thin layer of gold for 3 min using an automated sputter coater and visualized by SEM (JEOL-JSM 5600)
    4. Scanning electron microscopy
    5. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed at the Electron Microscope Facility, RUSKA LABs, Acharya N. G. Ranga Agricultural University, Hyderabad
    6. Electron Microscopy
    7. Log-phase yeastcells were collected, washed and suspendedin 10 mM Tris-HCl (pH 7.5) containing 50 mg/ml zymolyase-20T. Cell suspension was incubated at room temperature and absorbance was monitored at 600 nm every10mininterval. Initial absorbance of the cultures at 0 minwas normalized to 100%and the graph was plottedas%decrease in the absorbance with respect to time
    8. Zymolyasedigestion assay
    9. Resultant precipitate was dissolved in 3 N HCl and reprecipitated in methanol:acetic acid (8:1) solution. Following 16 h incubation at room temperature, the precipitate was washed withmethanol:acetic acid (8:1) solution till green colour of the supernatant disappeared.Finally,pellet was washed thrice with methanol and air dried. Driedpellet was resuspended in 0.5 NHCl and total mannan content was quantified with phenol-sulphuric acid carbohydrate estimation method as described earlier.Commercially available purified glucose was used as the standard
    10. Total mannan from 3% NaOH-extractable supernatant of cell wall was precipitated by Benedict’s solution.Reducing sugars(mostly mannan) from alkali-extractable supernatant reactwith copper(II) sulphate present in Benedict’s solution and forms red copper(I) oxide precipitate.Briefly, equal volume of Benedict’s solution was added to 3% NaOH-extractable cell wall supernatant fraction and heated at 99 ̊C for 10 min
    11. Total mannan estimation
    12. Cell wall β-glucan measurement was carried out as describedpreviously with some modifications(Kapteynet.al.,2001). Briefly, cell wall fractions were washed multiple times with 1 N NaCl. Washed cell walls were boiled twice in 50 mM Tris-HCl(pH 7.8) containing 2% SDS, 100 mM Na-EDTA and 40 mM β-mercaptoethanol for 5 min to remove non-covalently linked proteins and other contaminants. SDS-treated cell wall fraction was collected and rinsed thrice with water. For β-glucan isolation, cell wallswere extracted three times, each for 1 h, in 0.5 ml 3% NaOH at 75 ̊C and centrifuged at 1,200 g.All 3% NaOH supernatant fractions were saved for isolation of mannan as described below. 3% NaOH-extractable cell wall pelletwasneutralized twice in 100 mM Tris-HCl (pH 7.5) and once in 10 mM Tris-HCl (pH 7.5) and digested with 5 mg/ml zymolyase-20T in 10 mM Tris-HCl (pH 7.5) for 14-16 h at 37 ̊C. This treatment liberates approximately 90-95% glucose into the supernatant. Total glucan content in the cell wall was measured by estimating glucose from both the solubilised supernatant and zymolyase-20T insoluble pellet fractions with phenol-sulphuric acid carbohydrate estimation method using purified glucose as the standard
    13. Total β-glucan estimation
    14. min. Cells were normalized to equal OD600, resuspendedin 1 ml 50 mM Tris-HCl (pH 7.5) and transferred to 2 ml microcentrifuge tubes. Cells were lysed with glass beadsin a homogenizer (FastPrep®-24,MP Biomedicals)asdescribed earlier.Brokencells were washed from glass beadswith 500 μl Tris-HCl (50 mM, pH 7.5) and pelleteddown at 15,000 g for 10 minto obtainall cell wall and membrane content. Pellet was then boiled for 10 minin 1mlTris-HCl(50mM; pH 7.5)solutioncontaining 2%SDS. SDS-extractable material(mannoproteins)was savedand remaining pellet wasboiled again in 500 μl Tris-HCl(50 mM; pH 7.5)buffer containing 2%SDS. Cell wallwas collectedby centrifugation at 15,000 g for 10 min, washed twice with1 ml waterandresuspendedin 100 μl 67 mM potassium phosphatebuffer. This washed cell wall materialwas used for β-glucan estimation as described below
    15. Yeast cell wall was isolatedas describedpreviously(De Groot et al., 2004). Briefly, cells grown underdifferent environmental conditions were harvested at 5,000 g for 5
    16. Crude cell wall isolation
    17. Cell wall isolation, zymolyasedigestion assay and β-glucan estimation
    18. Cells grown to log-phase in YPD medium were spotted on CAAmedium and overlaid with a nitrocellulose filter. Cells were allowed to grow at 30 ̊C for 18-20 h. After incubation, the filter was washed with water to remove cells and membrane-bound CPY was detected by immunoblotting withpolyclonal anti-CPY antibody (Thermo Scientific) at a dilution of 1:15,000
    19. Carboxypeptidase Ysecretion assay
    20. CPY activity was measured as described previously (Jones,2002). A 2.5 mg/ml stock solution of CPY-specific substrate N-benzoyl-L-tyrosine p-nitroanilide(BTPNA, prepared in dimethyl formamide) was diluted 5 times with 0.1 M Tris-HCl (pH 7.5). 100 μl diluted substrate solution was added to a 96-well plate containing 25 μl cell suspension (5 x 107cells). After 18 h of incubation at 37 ̊C, plate contents were clarified by centrifugation and colour formation was quantified by absorbance at 405 nm. Background absorbance measured using BTPNA-free cell cultures was subtracted from BTPNA-loaded cell cultures and absorbancevalues were normalized to total number of viable cells to enumerate total cellular CPY activity
    21. Carboxypeptidase Y(CPY) activity assay
    22. ammonium molybdate, respectively, to the assay buffer.For specific inhibition of vacuolar membrane H+-ATPaseactivity, vacuolar membrane fractions were incubatedwith 1-2.5 μM bafilomycin for 5 minprior to the activity assay.ATPase activity was initiatedby adding ATP to the assay buffer to afinal concentration of 5 mM and incubating the reactionat 30 ̊C for 30-60 min.Reaction was stopped by adding an equal volumeof a stop-developing solution (1% (w/v)SDS, 0.6 M H2SO4, 1.2%(w/v)ammonium molybdate and 1.6%(w/v)ascorbic acid). Amount of inorganic phosphate (Pi) liberated was measured at A750nmafter 10 minincubation at room temperature. Standard curve prepared with 0-50 micromoles of KH2PO4 was used for the determination of total Pi. The ATPase activity of the vacuolarmembrane H+-ATPase was expressed in micromoles of Pireleased per milligram protein per min
    23. Vacuolar membrane H+-ATPase activitywas measured inbothcrude membrane fraction and purifiedvacuolar membrane fraction asdescribed previously(Woolfordet al.,1990).Activity inthe crude membrane fractions was carried out with 2.5-10 μgprotein in 50 μl assay buffer (5 mM MgCl2, 25 mM MES/Tris-HCl(pH 6.9)and 25 mM KCl). For activity inthe purified vacuolar membrane fraction, a totalof300 μl reactionmix was setup with of 2.5-10 μgprotein samples.Residual activities from other ATPases such as mitochondrial ATPases, plasma membrane H+-ATPase and phosphataseswere inhibited by adding 2 mM NaN3, 200 μM NaVO4and 0.2 mM
    24. Vacuolar H+-ATPase activity measurement
    25. Vacuole membraneswere isolatedwith slight modifications of Cabrera’s method(Cabrera et.al.,2008). Log-phase, YPD medium-grown cells wereinoculated in 1 lt YPDmedium to an initialOD600of 0.1. Cells were incubated at 30 ̊C with shaking at 200 rpm till the cell density reached to OD600of 0.8-1.0.Cells were harvested by centrifugation at 5,000 g and washed once with 30 ml 2% ice-cold glucose solution. Cells were incubated in 15 ml solution containingglycine-NaOH(50 mM; pH10)andDTT(2 mM) at 30 ̊C for 10 min. After incubation, cells were normalized to adensity of1000OD600and resuspendedin 15 ml spheroplasting buffer containing 10-15mg of zymolyase20T.Cells were incubated at 30 ̊C for 45-60 minor till the spheroplasting was completed.Spheroplasts werecollected by centrifugation at 4,500 rpmfor 5 minat 4 ̊C, washed gently with15 ml 1.2 M sorbitol solutionandresuspendedin 3.5 ml 15%ficoll solution made in PS buffercontaining 1X protease inhibitor cocktail. This suspension was homogenized on ice with 20-25 strokes in a loose-fitting Dounce homogenizer. Homogenate was transferred to an ice-cold,ultra-clear Beckman ultracentrifuge tube, overlaid witha gradient of3 ml 8%ficoll solution, 2.5 ml 4%ficoll solutionand 2.5 ml PS buffer lacking ficoll and centrifuged at 1,10,000g(30,000 rpm)for 90 minat 4 ̊Cin a pre-cooled Beckman ultracentrifuge with SW41-Ti swinging bucket rotor.Centrifugation was carried out with slow acceleration and deceleration settings.White creamy vacuole membrane layer wascollected from the interfaceof 0and4% ficoll gradientwithout mixing the layers.Total protein concentration in thevacuole fraction was estimated using BCAprotein assay kit as described earlier
    26. Purified vacuole membrane isolation
    27. Crude fractionation of total membraneswas carried outviadifferential centrifugation asdescribed previously (Moranoand Klionsky,1994)with slight modifications. Cells grown tolog-phase in YPDmedium werecollected, washed,normalizedto 10 OD600and resuspendedin 1 ml spheroplast buffer containing 1-2mg of zymolyase20T (MP Biomedicals).Following incubation at 30 ̊Cfor 30-45 min,spherolplastswerecollected by centrifugation at 800 g for 3 minat 4 ̊C and resuspendedin 1 mlice-cold Tris-EDTA (pH 7.5). Spheroplastswere lysed with 100 μl 0.5mm glass beads on a vortex mixer with 10 secpulsegiven thricewith intermittent ice-breaks.Cellsuspension was centrifuged at 800 g for 5 minat 4 ̊C to pellet unbrokenspheroplastsdown andthesupernatant was centrifuged at 15,000 g for 5 minat 4 ̊C to obtainthemembrane fraction pellet.Pellet was washed once with ice-cold Tris-EDTA (pH 7.5), resuspendedin 50 μl of the samebuffer and stored at -20 ̊Ctill further use. Protein concentration of pellet fraction was estimated using BCAprotein assay kit with BSA as thestandard
    28. Crude vacuolar membrane extraction
    29. Vacuolar H+-ATPase activity measurement
    30. A calibration curve of fluorescence intensity values versuspH was prepared for BCECF-AM-loaded wt cells by incubatingcellsin YPD medium containing 50 mM MES, 50 mM HEPES, 50 mM KCl, 50 mM NaCl, 0.2 M ammonium acetate, 10 mM NaN3, 10 mM 2-deoxyglucoseand5 μM carbonyl cyanide m-chlorophenylhydrazone, titrated to five different pH values in the range of 4.0-8.0. Fluorescence intensity values were measured by excitation at 440and 490 nm with emission at 535 nm and a graph was plotted between the ratio of intensity at 490 to 440 nm versuspH. Similar to pHi calibration curve, a polynomial distribution of fluorescent intensity signal and pH was observedfor BCECF-AMprobe
    31. In vivovacuolar pH calibration curve
    32. fluorescence by excitation at 440 (pH-independent) and 490 nm (pH-dependent) with emission at 535 nm. Ratio offluorescence intensity at 490 to440 nm was used tocalculatethe vacuolar pH. Background fluorescence was removed by subtracting the fluorescence intensity values of cells without BCECF-AM from the fluorescence intensity values of the probe-loaded cells
    33. Vacuole pH inyeast cells was determined asdescribed previously (Padilla-López and Pearce, 2006). Briefly, log-phase,YPD medium-grown yeast cells were harvested and suspended in 200 μl YPD medium containing 50 μM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Invitrogen # B1150) to the final cell density of 4 x 107 cells. Cells were incubated at 30 ̊C for 30 min at room temperaturefollowed by three washeswith YPD medium. Washed cells were resuspended in 1 ml YPD medium and 200 μl cell suspension was used for recording
    34. Measurement of vacuole pH
    35. Vacuolar morphology of C. glabratacells was examinedby staining vacuoleswith FM4-64 (Molecular Probes, Invitrogen). FM4-64 is a lipophilic dye that exhibits long wavelength red fluorescence when boundto lipids. FM4-64 binds to the plasma membrane and follows the endocytic pathway to reach the vacuole(Vida and Emr, 1995).Log-phase,YPDmedium-grown cells were harvested and washed with 1X PBS. 1 ODcells were resuspendedin 50 μl YPDmedium containing 30 μM FM4-64 andincubated at 30 ̊C for 30-45 min. After incubation, cells were washed thricewith YPD mediumand resuspendedin 100 μl of the samemedium. Cells were observed under confocal laser scanning microscope(Zeiss LSM 510 Meta)with 63X objective lens,2.5X final zoom, pinhole set at 108 μm and emission filterset to LP 565nmto capture fluorescence image.Along with the fluorescenceimage, aphase contrastimage was alsocaptured for each sample
    36. Staining of yeast vacuoleswith FM4-64
    37. cellswere collected and washed with chilled sterile water.1 OD600cells were resuspendedin 20 μl chilled10%TCA solution containing 8 mM EDTA (pH 8.0) and incubated at room temperature for 15-20 min.Followingincubation, cellsuspension was centrifuged at 12,000 rpm for 5 minat 4 ̊Cand supernatant was transferred to a fresh 1.5 ml microcentrifuge tube. 10 μl of this supernatant fraction was diluted 75-foldwith ATPassay mix dilution buffer provided with the kit. 50 μl of diluted suspension was added to anequal volume of ATPassay mix (Sigma # FLAAM) which containedfirefly luciferase and luciferin with MgSO4, EDTA, DTT and BSA inTricine buffer.Luminescence was measured inluminometer (Varioskan flash-3001,Thermo Scientific). Total ATP was quantified usingpurified ATP as the standardand expressed in moles/OD cells
    38. ATPconcentrationin yeast cells was measuredby luminometricluciferase-luciferinbased assayusingATPbioluminescent kit(Sigma # FLAA).Briefly, log-phase yeast
    39. Determination of intracellular ATPlevels
    40. Estimation of total glycogen in cells was performed asdescribed previously (Parrou et al., 1997) with slightmodifications.Briefly, YPD medium-grown C. glabratacells were harvested, washed once with 1 ml ice-cold waterandresuspendedin 250 μl sodium carbonate(0.25 M)solution. After incubation at95 ̊C for 4 hin water bath with occasional stirring, cell suspension was cooled and pH of the suspension was adjusted to 5.2 by adding 150 μl 1 M acetic acid. Tothis suspension,600 μl 0.2M sodium acetatewas added and cell suspension was incubated with 1-2 U/ml of α-amyloglucosidase from A.niger(Sigma #A7420)at 57 ̊C for overnight with constant agitation.Resultant glucose liberated by α-amyloglucosidase digestion was collected in the supernatant fraction and quantifiedby phenol-sulphuric acid methodof carbohydratedetermination.For quantification, commercially available purified glucose was used as a standard and total glycogen incells was expressed as μg/2 x 107cells tonormalizeagainstcell density
    41. Estimation of glycogenlevels
    42. Trehalose from C. glabratacells was extracted by trichloro acetic acid (TCA)solutionas described previously (Lillie et al.,1980). Cells grown in YPDmediumwere collected at different time pointsof growth and washed thrice with ice-cold sterile water. Cells were immediatelystored at-20 ̊Ctill further use.For trehalose isolation, 10-20 OD600cells were thawed in 500 μl TCA (0.5 M) solutionon ice and incubated at room temperaturefor 1 h.Supernatant fraction was collected by sedimenting cells at 14,000 rpm for 5 minat 4 ̊C.TCA extractionwas repeated withcells once more and the resultingsupernatant was mixed with the earlier fraction.Extractedtrehalose was measuredby phenol-sulphuric acid methodof carbohydratedeterminationwithcommercially available purified trehalose(Becton, Dickinson and Co.) as a standard.Total trehalosecontent was normalized to the cell densityand expressed as μg/2 x 107cells
    43. Estimation of trehalosecontent
    44. Estimation oftrehalose, glycogen and ATPlevels
    45. Quantitative measurement of periplasmic acid phosphatase activity in phosphate-starved C. glabratacells was performedas mentioned previously (Orkwis et al., 2010). A total of 0.5 OD600YNB-grown and phosphate-starved cells were collected, washed thrice with cold water and oncewith cold 0.1 M sodium acetatebuffer (pH 4.2). Washed cells were resuspendedin 500 μl sodium acetate (0.1 M)and incubated at 30 ̊C with constant stirring. After 10 min incubation, 500 μl freshly-prepared solution of 20 mM p-nitrophenyl phosphate in 0.1 M sodium acetate(pH 4.2) was added to the cell suspension. Enzymatic activity was stopped after incubation at 25 ̊C for 20 min by addition of 250 μl sodium carbonate (1 M)tothe reaction mix. Resultant colour change was measured by monitoring absorbance at 400 nm. Acid phosphatase activity was expressed as a ratio of OD400to OD600 to normalize against cell density
    46. Determination of acid phosphatase activity
    47. Cells grown overnight in YNBmedium wereinoculated in fresh YNB mediumand incubated at 30 ̊C with shaking at 200 rpm. Cells were harvested when the cell density reached to an OD600of 0.6-0.8.Cells were consecutively washed with sterile MQ water and YNB without phosphate (YNB-Pi) medium. Washed cells were inoculated either in YNB orYNB-Pimediumto the initial OD600of 0.1. Cells were incubated at 30 ̊C for 3-4 h, harvested and resuspendedin 100 μlYNB-Pimedium. Radioactive P32-labelled o-phosphoric acid(Jonaki# LCP 32)was added to the cell suspension to a final concentration of 1 μCi/mlandcells were incubated for 30 min.For determining phosphate uptake, a10-12 μl cell suspensionaliquot,after every5 min,was removed and kept on ice.To this cell suspension, 500 μl ice-cold YNB-Pimediumwas addedand cells were harvested by centrifugation at 5,000 g for 5 minat 4 ̊C.These cells were washed with ice-cold YNB-Pimedium thrice and resuspendedin 100 μlPBS(1X). 10-20 μl of this cell suspension was added to5 ml scintillation fluid and β-decay counts were measured in ascintillation counter(Tri-Carb 2910 TR Liquid Scintillation Analyzer, PerkinElmer).Scintillation counts were normalized to total cell number and plotted with respect to time. Total phosphateuptake was expressed as P32c.p.m/OD600cellswhere c.p.m refers tocounts per min
    48. Phosphate uptake assay
    49. Total cellular polyphosphates were quantified viapolyacrylamide-Tris borate gel electrophoresis (PAGE-TBE).Briefly, PAGEwas performed with Tris-borate buffer (pH 8.3) todeterminethe quantityand the typeof polyphosphatesextracted fromyeaststrains. Equal amount of total RNA (20 to 100 μg) was loadedon 34%PAGE-TBE gel (22cm long, 16 cm wide and 0.8 mm thick) and electrophoresedat 500Voltsfor 20-24 hin cold-roomtill the marker dye bromophenol blue(BPB)had migrated 15-16 cm awayfrom the well.After electrophoresis, total polyphosphates werevisualizedby staining the gel with 0.05%toluidine blue staining solutionfollowed by destaining. Polyphosphates wereobserved both as asmearin the top most portion of the gel as well asdiscreet bands of long chain polyphosphatesand shortchain polyphosphatesin middle andbottom half of the gel,respectively.Polyphosphateband intensity in the gelwas quantified using ImageJ software (http://rsbweb.nih.gov/ij/)and relative amountsof long chain and short chain polyphosphatesin C. glabratacells werecalculated
    50. Quantitative analysisof polyphosphates
    51. Polyphosphates from yeast cells were extracted with phenol-chloroform solutionas described previously(Neefand Kladde,2003). Cells grown eitherovernight or to logarithmic phase in YPDmediumwere harvested by centrifugation at 1,700 rpm for 5 min. Cells were washed with 10 ml sterile MQ water and resuspendedin ice-cold 500 μl20%trichloro acetic acid (TCA) solution to the final cell densityof 100 OD. Cell suspension was transferred toa1.5 ml microcentrifuge tube. After incubation at room temperature for 5 min, cells were harvested by centrifugation at 12,000 g for 10 minat 4 ̊C and resuspendedin 250-350 μlpolyphosphate extraction buffer.Equal volume of phenol-chloroform (25:24) was addedtothe microcentrifuge tube and aqueous phase was extracted by centrifugation at 12,000 g for 8 minat room temperature. Top aqueous layer was collected with a 200 μl tip. Aqueous layer extraction was repeated once more after removal of DNA with chloroform.After centrifugation,aqueous phasecontaining RNA and polyphosphates wascollected,RNA was quantified at A260nmandstored at -20 ̊C
    52. Polyphosphate extraction
    53. were maintained in log-phase by continuous passaging in fresh YNB medium every 4 h
    54. For phosphate starvation,yeastcells grown to log-phase in YNB medium were harvested,washed with water,transferred to either regular YNBor YNB medium lackingphosphate and were grown for 16 h at 30 ̊C. Cells cultured in YNB medium
    55. Phosphate starvationof yeast cells
    56. 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
    57. Plasma membrane H+-ATPase activity assay
    58. 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
    59. 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
    60. Total membrane preparation
    61. 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
    62. Whole cell acidification assay
    63. Measurement of plasma membrane H+-ATPase activity
    64. Log-phasecells grown in YPD medium containing or lacking CaCl2and FK506 were collected, PBS-washed and loaded with ratiometric, high affinity, membrane-permeable calcium indicator, Fura-2 AM (10 M; Sigma #47989). After 30 min incubation at 30◦C, labelled cells were washed thrice with cold PBS, suspended in PBS and fluorescence was recorded at 505 nm with dual excitation at 340 and 380 nm. The ratio of fluorescence intensities between 340 and 380 nm, representing Ca-bound and Ca-free Fura-2 molecules, respectively, reflected free intracellular calcium concentrations
    65. Measurement of intracellular calciumlevels
    66. Intracellular reactive oxygen species (ROS)levels in yeast cells weredetermined usingfluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA; Sigma# D6883). Cellular esterasesremove the diacetate groups ofthe DCFH-DAand produceDCFHwhich getsreadily oxidized to highly fluorescent product 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. The fluorescent intensity of DCF corresponds to the amount ofintracellular ROSpresent in the cell.Cells grown under different environmental conditions were harvested,washed once with tissue-culture grade phosphate-buffered saline (PBS) and resuspendedin PBS to the final cell density of 1 OD. Freshly-prepared DCFH-DA (0.01M stock in DMSO) was added to the cell suspension toafinal concentration of 100 μM. Cell suspension was mixed and incubated at 30 ̊C for 30 min. After incubation,cells were washed 2-3 times with 1 ml PBS and then resuspendedin 200 μlPBS. Fluorescence intensity values wererecorded usingspectrofluorophotometer (Varioskan flash-3001, Thermo Scientific) with excitation and emission at 488and 530 nm,respectively.Fluorescenceintensityvalues obtained from probe-loaded cells were subtracted from the fluorescence intensity values obtainedfrom cells-alone samplesto remove background fluorescence
    67. Determination of intracellular reactive oxygen species (ROS)levels
    68. For determiningthe intracellular pH from fluorescence intensity values of CFDA-SE-loaded cells, anin vivocalibration curve was prepared between fluorescent intensity and pre-adjusted environmental pH values. Briefly,CFDA-SE-loaded wild-typeC. glabratacellswere incubatedwith 0.5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma# C2759) at 30 ̊C for 10 min in 50 mM CP buffer adjusted to different pH values ranging from 4.0 to 7.5, with an interval of 0.5 unit.CCCP is an ionophore which dissipates the plasma membrane pH gradient, thus, rendering the intracellular pH similar to the extracellular pH. Fluorescent intensities were determinedand a calibration curvewas plotted between the ratio of intensity at 490 to430 nm versuspH.A polynomial distribution of fluorescent intensity signal and pH was observed for CFDA-SE probe(Figure2.1)and the graphequation was used todeterminethe intracellular pHof C. glabratacells
    69. In vivointracellular pH calibration curve
    70. OD600of 0.5and transferred to a 1.5 ml microcentrifuge tube. Probe loading was carried out by adding freshly-prepared CFDA-SE solution (0.01 M stock in DMSO) tocell suspension to a final concentration of 160 μM. Cell suspension was mixed on vortex mixerfor 10 secand incubated at 37 ̊C for 1 hwith shaking at 300 rpmon thermo mixer.Cells were harvested, washed twice with 1 ml 50 mM CP buffer to remove unloaded probe,resuspendedin 250 μl CP buffer andwereincubated at 30 ̊C for 30 minwith shaking to recover from the stress induced during probe loading. Afterincubation,fluorescent intensitywasdetermined with spectrofluorophotometer (Varioskan flash-3001, Thermo Scientific) by excitation at 430 nm (pH-independent) and 490 nm (pH-dependent) with emission at 525 nm. Background fluorescence of the probe was removed by subtracting the fluorescence intensity of the probe in CP buffer from the fluorescence intensity of the probe-loaded cells
    71. Intracellular pH(pHi)in yeast cells was determinedusing fluorescent 5,(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes) asdescribed previously (Bracey et al.1998). For pHiprobe estimation,YNB medium-grown log-phase cells were inoculatedin YNB, YNB-pH 2.0 or YNB medium supplemented with acetic acid and incubated at 30 ̊C for different time points.Log-phase C. glabratacells were harvested and washed twice with 50 mM citric-phosphate (CP) buffer (pH 4.0). Washed cells were resuspendedin 1ml 50 mM CP buffer to an
    72. Measurementof intracellular pH (pHi)
    73. Following transfer,membrane was either stained with Ponceau reagent for checking the efficiency of transfer or directly processed for proteindetectionusing protein-specificantibodies.For immunoblotting, membranes were blockedwith 5% (w/v)non-fat milk solutioneitherin PBS-T or TBS-T for 2 hat RTand probedwith primary antibodies against the target proteins.For detection of CgPma1, membranes were probed with1:1000 dilution of polyclonal anti-Pma1antibody raised against S. cerevisiaePma1 (Santa Cruz #sc-33735)in PBS-T with 5% (w/v)fat-free milk for overnight at 4°C.For detection of phosphorylated form of CgSlt2,immunoblotting analysis was done withan anti-phospho-p44/42 MAPK (Thr202/Tyr204) primary antibody raised against human p44 MAPK(Cell signalingtechnology# 4370S) at a dilution of 1:6000 in TBS-T with 5% (w/v)fat-free milk for overnight at 4°C.For detection of CgCPY, membrane was incubated with polyclonal anti-CPY antibody raised against S. cerevisiaeCPY(Thermo Scientific # PA 1-27244) at a dilution of 1:10,000 in TBS-T with 5% (w/v)fat-free milk for overnight at 4°C.For CgGapdhdetection,anti-Gapdh primary antibody raised againsthuman Gapdh(Abcam # ab22555) at a dilution of 1:7000was usedin TBS-T with 5% (w/v)fat-free milk.Secondary antibodies conjugated with horseradish peroxidase(HRP)enzymewereusedin 1:10,000 dilutionto detect the immune-reactivity of primary antibodieswith the help of ECL plus Western blotting system (GE Healthcare)as per manufacturer’sinstructions
    74. Total proteins resolved bySDS-PAGE were transferred to PVDFnylon membrane by Western blotting using a Bio-Rad Mini Trans-Blot electrophoretic transfer unit in Tris-glycinetransfer bufferat 4 ̊C either at 100 Voltsfor 3 hr or 30 Voltsfor overnight
    75. Western blot analysis
    76. SDS-PAGEwas performed as described previously (Laemilli, 1970).10-40 μg protein samples were mixed with 4X SDS loading buffer and either incubated at 50 ̊C or 90 ̊C for 10 min. Denatured samples were loaded either on8%or 10%SDS-PAGEgel and run in Tris-Glycine-SDSgel running buffer at 70-100 Volts for 2-3 hin a Mini-PROTEAN®3electrophoresis unit(Bio-Rad).After electrophoresis,gels were either visualized by coomassie brilliant blue (CBB) stainingor processedfor western blotting as described below
    77. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE)
    78. Log-phase yeast cell cultures were harvested and total protein was extracted by lysingyeast cells using glass beads. Briefly,10 mllog-phase yeast culturesgrownin appropriate medium were harvested,washed once with ice-cold water and suspended in 250 μl homogenizing buffer containing 1 mM phenylmethylsulfonylfluoride(inhibitsserine proteases), 10 mM sodium fluoride(inhibit Ser/Thr and acid phosphatases), 1 mM sodium orthovanadate (inhibits Tyr and alkaline phosphatases) and 1X concentration of protease inhibitor cocktail(RocheCat # 04693159001). Cells were lysedwith glass beads by vortexing five times at high speed for 1 min with intermittent 1 min ice breaks. Unbroken cells and cell debris were removed by centrifugation at 1,000 g for 5 min at 4 ̊C. Cell lysate was collected and protein was quantified using bicinchoninic acid (BCA)protein assay kit (Thermo Scientific # 23227) as per supplier’s instructions
    79. Protein extraction
    80. Biochemical techniques
    81. hybridizations from biological replicates for each sample. Data was extracted with Feature Extraction software v 10.5 (Agilent) and normalizedwith GeneSpring GX v 11.0.1 (Agilent) software using the recommended Percentile shift Normalization to 75th percentile. Raw Data sets for this study are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=


    82. Log-phase wild-type and Cgyps1∆cells were grown in YNB and YNB-pH 2.0 medium. After 1 h incubation, yeast cells were collected, washed and were stored in RNAlater at -80°C. These frozen samples were sent to Genotypic Technology Ltd., Bangalore(http://www.genotypic.co.in) whichprovides services of global gene analysis on Agilent platform. A 8x15k GE array comprised of 60mer oligonucleotidesfor a total of C. glabrata5503 genes was used wherein average number of replicates for each probe was three. Labeling was done in single color and data is the average of two
    83. Microarray analysis
    84. .coliBW23473 electro-competent cell aliquots werethawed on ice and mixed with 1-2 lof plasmid DNA. Mixture was pulsed with the Gene Pulser®electroporation apparatus(Bio-Rad) at 1800 Volts with 25 μF and 200 Ωcurrentin a chilled0.1 cm electroporation cuvette(Bio-Rad). Immediately after successful pulsing, 1 ml LB medium was added to the cuvetteand suspension was transferred toa 1.5 ml sterile centrifuge tube. Cells wereincubated at 37°C for 1 hwith shaking and further plated onLB plates containing kanamycin(30μg/ml). Positive colonies were inoculated in LBliquid medium containing kanamycin(30μg/ml)for plasmid isolation


    85. E.coliBW23473transformation by electroporationE
    86. 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
    87. Electro-competentcell preparation
    88. the disrupted gene, BLAST N of the sequences from rescued plasmids was performedagainstC. glabrataGenolevures database (http://www.genolevures.org/blast.html
    89. Identification of disrupted locus in Tn7insertion mutants was carried out as described previously(Kaur et al.,2004). Disrupted locus in each mutant is physically marked with a mini Tn7 transposon derivative containing conditional origin of replication R6K(facilitates Tn7recovery), S. cerevisiae URA3and Klebsiella pneumoniae hphgene (confers resistance to hygromycin B)(Castaño et al.,2003). Briefly,genomic DNA was isolated fromovernight grown Tn7insertion mutants using spheroplast lysis method. After RNAse treatment, 10μgDNA was either digested withMfe1or SpeIrestriction enzymeas the Tn7 cassette lacks these enzyme sites. Followingovernight digestion, DNA wasprecipitated with 1ml ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.2).DNApellet was washed twicewith ice-cold 70% ethanol, air driedand resuspendedin sterile MQ water. DNA was recircularized withT4 DNA ligase. Resultant circular plasmid contains the Tn7cassette flanked on either side by the gene,it has disruptedin the genome of C. glabrata.This circular plasmid DNA was transformedin E.coliBW23473 strain,which contains protein Π (the product of the pir gene)required by R6Korifor replication.Transformation of circularized DNA in E.coliBW23473 electrocompetent cells was performedas described below.Plasmids fromselected transformants were isolated and sequenced with outward primers from Tn7right and left ends to sequencethe disrupted gene fragment.For identification of
    90. Tn7insertion mutant rescueand gene identification
    91. C. glabrataTn7insertion mutantlibrary was screened for reduced growth in YNB-pH 2.0 medium. Thismutant library,composed of 9,134 Tn7insertion mutants, isarrayed in 96-well microtitre plates(Castaño et al.,2003). 2 μlof each mutant strain was inoculated in 120μl YNB medium and grown overnight at 30 ̊C in an incubator with constant shakingat 120 rpm. Overnight grown cultures were 120-folddiluted with 1X PBS in a 96 well block and transferred, using a 96-well pin replicator, to YNB and YNB-pH 2.0 medium. Plates were incubated at 30°C and mutant phenotypes were recorded after 3-4days.
    92. Screening of C. glabrataTn7insertion mutants
    93. and colony purified on CAA plate. 15% glycerol stocks were made for two independent transformants and stored at -80 ̊C
    94. C. glabrataCgYPS7ORFwas cloned in a self-replicating pGRB2.2plasmidwhich contains C. glabrata CEN-ARS, S. cerevisiaeURA3gene, S. cerevisiaePGK1promoter and C. glabrataHIS3-3′ untranslated region. For cloning CgYPS7in pGRB2.2,CgYPS7ORF (1.764 kb) was PCR-amplified from the wild-type genomic DNA with high fidelity Platinum PfxDNA polymeraseusing primers carrying restriction sites for XbaIand XhoI. The1.764 kb amplifiedPCR product waspurified with QIAquick PCR purification kit (Qiagen # 28104),digested with XbaI and XhoI and cloned in the pGRB2.2plasmid at XbaI–XhoI sites in the multiple cloning site (MCS)region downstream of the PGK1promoter.Positiveclones were verified by PCR, sequencing and complementation analysesofCgyps7∆mutant. Yeast transformantsobtained by lithiumacetate methodwere selected on plates lacking uracil