This test was therefore used for two purposes: (i) to distinguish relA+ from relA− strains, and (ii) as a qualitative measure of transcriptional polarity relief at the ilv locus. Growth in the presence of amino acids Serine, Methionine, and Glycine (SMG) was scored on glucose-minimal A plates supplemented with each of the amino acids at 100 μg/ml and compared with the growth on non-supplemented glucose-minimal A plates to score for SMG phenotype

The E. coli relA mutants exhibit SMG-sensitive (SMGS) phenotype i.e. growth-inhibition in the presence of Serine, Methionine and Glycine at 1 mM concentration each (Uzan and Danchin, 1978) and is proposed to be a consequence of transcriptional polarity exerted by a frameshift mutation in the ilvG gene on the expression of downstream genes of the ilvGMEDA operon (Lopes et al., 1989). It was observed in another study that the rho and nusG mutants that are defective for transcription termination conferred SMG-resistant (SMGR) phenotype in a relA1 strain (Harinarayanan and Gowrishankar, 2003)

Lac+ colonies were distinguished from Lac− on MacConkey-lactose plates or on Xgal indicator plates. Xgal is a non-inducing colourless substrate of β-galactosidase enzyme which upon hydrolysis yields dark blue indolyl moieties and hence, the Lac+ colonies on Xgal indicator plates are seen as dark blue colonies. Xgal was prepared as a stock solution of 5 mg/ml in dimethyl formamide and used at a final concentration of 25 μg/ml. On MacConkey-lactose medium (pH around 7.1) on the other hand, Lac+ strains can utilize the lactose sugar present in the medium to lower the pH of the medium to 6.8, resulting in a pink coloured colony while Lac─ strains are unable to utilize lactose to give a white colour

colonies come up after 48 hr. A freshly grown overnight culture of the TetR strain was washed once with an equal volume of citrate buffer and resuspended at 10- or 100-fold dilution in the same buffer. 0.1 ml aliquots were then spread on Maloy plates. Colonies were obtained at a frequency of ~ 4 x 10−5/plated cell. The colonies from the selection plate were purified on medium of the same composition and then scored for the Tets phenotype

The method described by Maloy and Nunn (Maloy and Nunn, 1981) was followed for obtaining spontaneous TetS mutants of a TetR strain. Freshly grown cells of the TetR strain of O.D 0.7-0.8 was washed once with an equal volume of citrate buffer and resuspended at 10- or 100-fold dilution in the same buffer. 0.1 ml aliquots were then spread on Maloy agar plates and TetS colonies, which came up after 48 hr of incubation at 37 ̊C with a frequency of 5-8 big colonies/106-107 cells plated were purified on the same medium. This is not a clean selection since in a background lawn of slow growing TetR colonies, few faster growing TetS

A single plaque of λ contains approximately 105-106 pfu/ml. The method of propagation of λ from a single plaque was as follows. The contents of a single isolated plaque were drawn into a 1-ml pipette tip and dispensed into 0.2 ml of LB broth. After addition of a drop of chloroform, the contents were vortexed and centrifuged. The clear supernatant was mixed with 50 μl of λ-sensitive cells and incubated for 20 min at room temperature for adsorption. 10 ml of Z-broth supplemented with 5 mM MgSO4 was then added to the infection mixture, and incubated at 37°C with shaking until lysis. The lysate thus obtained usually contained 109pfu/ml

The method used was essentially the same as that described for preparation of P1 lysate, except that the λ-sensitive C600 cells used for infection were grown in LB broth containing 0.4% maltose and 10 mM MgSO4. The lysate thus prepared was checked for supE+revertants by plaquing on both supE (C600) and supE+strains (MG1655) using various dilutions of the lysate. To be used for experimental purposes, a phage titre of the order of 1010 to 1011 on the supE strain and around four orders of magnitude lower on the supE+strain, indicating very less frequency of supE+ revertants in the lysate is ideal

Obtaining transpositions near a gene of interest was achieved in a two-step procedure. The population (pool) of cells carrying random transpositions (described in the previous Section) at different places on the chromosome was used to prepare a P1 phage lysate. This lysate was then used to infect a suitable recipient strain and transductants were sought in a simultaneous (double) selection for two markers, namely the antibiotic marker on the mini-transposon and selection for the phenotype of the gene or mutation which was intended to be linked with the antibiotic marker. The transductants so isolated were purified and further P1 phage preparations were made on these individual clones. By retransducing with these lysates into the same recipient cells and observing the segregation of phenotypes after selection for the transposon marker, the cotransduction values were obtained between the transposon insertion and the gene (or mutation) of interest

the phage (λ1098 for Tn10dTet transpositions and λNK1324 for Tn10dCm transpositions) at a multiplicity of infection (moi) of 0.05 in the presence of 5 mM MgSO4. This mixture was incubated for 15 min at 37°C to allow for phage adsorption. The unadsorbed phage was then removed by centrifugation and the pellet was resuspended in 10 ml of LB broth containing 5 mM sodium pyrophosphate. It was incubated without shaking at 37°C for 30 min for phenotypic expression. The rest of the mixture was diluted into 100 ml of LB broth with 5 mM sodium pyrophosphate carrying the required antibiotic and amplified overnight by growth at 30°C. This population of cells was used as a source of random transposon insertions. The λ lysates used for the transposition experiments carry amber mutations, and were propagated on a supE strain C600 by the protocol described below in section 2.14

The method used was essentially the same as that described by Miller (Miller, 1992). The strain to be used for obtaining random Tn10dTet or Tn10dCm insertions was grown overnight in Z-broth containing 0.4% maltose. The culture was then diluted 50-fold in the same medium and grown to an A600 of 0.8. Two ml of the culture was infected with 107 pfu of

To 2 ml of the fresh overnight culture of the recipient strain grown in Z-broth, 108 pfu of P1 lysate was added and incubated at 37°C without shaking for 15 min to facilitate phage adsorption. The unadsorbed phage particles were removed by centrifugation at 4000 rpm for 5 min and the pellet of bacterial cells was resuspended in 5 ml of LB broth containing 20 mM sodium citrate to prevent further phage adsorption. This was incubated at 37°C for 30 min with slow shaking to allow for phenotypic expression of the antibiotic resistance gene. The mixture was then centrifuged, and the pellet was resuspended in 0.3 ml of citrate buffer. 100 μl aliquots were plated on appropriate antibiotic containing plates supplemented with 2.5 mM sodium citrate. A control tube without the addition of the P1 lysate, was processed in the similar way as described above. In case of selection for nutritional requirements, the infection mixture was centrifuged, washed once in 5 ml of citrate buffer and plated without phenotypic expression

To 0.3 ml of infection mixture, 10 ml of Z-broth was added and incubated at 37°C with slow shaking until growth followed by the visible lysis of the culture occurred (in ~ 4-6 h). The lysate was treated with 1 ml of chloroform, centrifuged and the clear lysate was stored at 4°C with chloroform

0.3 ml of overnight culture of the donor strain in Z-broth was mixed with 107 plaque forming units (pfu) of a stock P1 lysate prepared on strain MG1655. Adsorption was allowed to occur at 37°C for 15 min and the lysate was prepared in the following ways

Most chemicals were obtained from commercial sources. The sources for some of the fine chemicals used in this study are given below. 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 routinely from Himedia. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4 DNA ligase, DNA polymerases for PCR amplification and DNA size markers were obtained from companies including New England Biolabs and Fermentas. Quiagen or HiPura Kits used for plasmid isolation, purification of DNA fragments. The oligonucleotide primers used in this study were mainly synthesized on order by Ocimum Biosolutions or MWG Biotech Pvt. Ltd

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

Antibiotics were used at the following final concentrations (μg/ml): Rich media Minimal media Ampicillin (for plasmids) 100 50 Ampicillin (chromosome) 30 30 Chloramphenicol (for plasmids) 50 25 Chloramphenicol (chromosome) 25 25 Kanamycin 50 25 Nalidixic acid 50 - Rifampicin 100 - Streptomycin 50 100 Streptomycin 100 200 Spectinomycin 50 100 Tetracycline 15 8 Trimethoprim (for plasmids) 60 30 Chloramphenicol 0.1mg/ml The 10 mg/ml chloramphenicol stock in ethanol was used to make 0.1mg/ml solution in water

NP-40 1% Tris 50 mM Sodiun deoxycholate 0.5% SDS 0.1% pH adjusted to 8.0 Running buffer for Western blotting Glycine 14.4g/l Tris base 3.05g/l SDS 1.0g/l Transfer buffer for western blotting Glycine 14.4g/l Tris base 3.03g/l The above salts were dissolved in 800ml of miliQ water and 200ml of methanol was then added. The buffer was chilled before use. PBST for Western blot 10X PBS (1000 ml) Sodium chloride 80 g Potassium chloride 2 g Disodium hydrogen phosphate 14.1 g (Na2HPO4) Potassium dihydrogen phosphate 2.49 g (KH2PO4) 1 l of 1X PBS + 1 ml of Tween-20

TBF-I buffer (200ml) Potassium acetate 0.588 g Calcium chloride 0.249 g Manganese chloride 1.98 g Rubidium chloride 2.418 g 15% Glycerol 30 ml pH adjusted to 5.8 with 1M acetic acid TBF-II buffer (100 ml) MOPS 0.209 g Calcium chloride 1.102 g Rubidium chloride 0.120 g 15% Glycerol 15 ml pH adjusted to 6.5 with 1M potassium hydroxide Acrylamide solution (30%) Acrylamide 29 g Bis-acrylamide 1 g H2O 100 ml Non denaturing polyacrylamide gel (12%) 30% acrylamide 38.6 ml H2O 40.6 ml TBE 20 ml 10% APS 0.7 ml RIPA buffer (Radio Immuno Precipitation Assay buffer): RIPA buffer for bacterial cell lysis Sodium chloride 150 mM

Water to 1000 ml MacConkey lactose agar: MacConkey Agar Base (Difco) 51.5 g Lactose 1% Water to 1000 ml Maloy agar: Tryptone 5 g Yeast extract 5 g NaCl 10 g NaH2PO4 10 g Chlorotetracycline (12.5 mg/ml) 4 ml Water 1000 ml Bacto-agar 15 g After autoclaving, the following solutions were added, ZnCl2 (20 mM) 5 ml Quinaldic acid (10 mg/ml) 10 ml Citrate buffer: (0.1 M; pH 5.5) Citric acid (0.1 M) 4.7 volumes Sodium citrate (0.1 M) 15.4 volumes TBE and TAE buffers: TBE: 90 mM Tris-borate, 2 mM EDTA (pH 8.0) and TAE: 40 mM Tris-acetate, 2 mM EDTA (pH 8.0) were used as standard electrophoresis buffers. TBE and TAE were prepared as 10X and 50X concentrated stock solutions, respectively, and used at 1X concentration

cto-agar 15 g LBON agar: LBON medium1000 mlBacto-agar 15 gLB soft agar: LB medium 100 ml Bacto-agar 0.6 gK-Medium: KH2PO4 1.0 mM FeSO4 0.5 mg/l (NH4)2SO4 1.5 mM MgCl2 0.08 mM Casamino acids 5 g/l Thiamine 2 mg/l pH was adjusted to 7.0 with Tris free base. K-medium is low osmolarity (70 mOsm) medium (Kennedy, 1982). Z broth: LB medium 100 ml CaCl2 (0.5 M) 0.5 ml MacConkey agar: MacConkey Agar (Difco) 51.5 g Water to 1000 ml MacConkey galactose agar: MacConkey Agar Base (Difco) 51.5 g Galactose 1%

Glucose/Glycerol-minimal A 19 amino acid medium: This medium is essentially the same as glucose/glycerol-minimal A medium described above except that all the 19 amino acids (except tryptophan) were added after autoclaving in a final concentration of 40 μg/ml from autoclaved 4mg/ml amino acid stock solutions. Minimal agar: Contained 1.5% Bacto-agar (Difco) in minimal A Medium. The plates were poured after mixing double strength minimal A medium with 4% agar (in water) that had been autoclaved separately. Wherever required, to test polaity relif at lacZ(am) or trpE(fs), meliobose (0.2%) was replaced for glucose and anthranilate at 100 μg/ml (4 mg/ml stock prepared in DMF) was replaced for tryptophan respectively. LB medium: Tryptone 10 g Yeast extract 5 g NaCl 10 g Water to 1000 ml pH adjusted to 7.0 - 7.2 with 1 N NaOH. LBON medium: Tryptone 10 g Yeast extract 5 g Water to 1000 ml pH adjusted to 7.0 - 7.2 with 1 N NaOH LB agar: LB medium 1000 ml

All the media and buffers were sterilized by autoclaving for 15 minutes at 121°C. Media and buffers used in this study are described below. Glucose/Glycerol-minimal A medium: K2HPO4 10.5 g KH2PO4 4.5 g (NH4)2SO4 1 g Sodium citrate, 2H2O 0.5 g Water to 1000 ml After autoclaving the following solutions were added. MgSO4 (1 M) 1 ml Glucose (20%) 10 ml Or Glycerol (80%) 5ml Vitamin B1 (1%) 0.1 ml Amino acids and bases, when required, were added to a final concentration of 40 μg/ml. When growth on other carbon sources was to be tested, glucose was substituted by the appropriate sugar at 0.2%; when used as carbon source, the final concentration of Casamino acids was 0.5%

aStrain DH5α, MC4100 and MG1655 was from our laboratory stock collection. Strains described earlier include GJ3107, GJ3110, GJ3161, GJ3168, GJ3171 (Harinarayanan and Gowrishankar, 2003), and RS353 and RS445 (Chalissery et al., 2007). Strain GJ5147 is an Ilv+ derivative of GJ3073 (Chalissery et al., 2007). Strains GJ6504, GJ6509, GJ6511, GJ6516, GJ6520 and GJ6524 were constructed by S. Aisha (unpublished). Strains GJ5108, GJ5146, GJ5153 were constructed by K. Anupama (unpublished). b Genotype designations are as described in Berlyn (1998). cK7906 strain is described in Zheng and Friedman (1994). d MDS42 strain is as described in Posfai et al. (2006)

Table 2.1 : List of E. coli K-12 strains

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

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

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

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

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

The bacteriophage P1kc was from our laboratory collection and is referred to as P1 throughout this thesis. Phage λcI857 was also from our laboratory collection. Other bacteriophages that were used in this study included the following: (i) λNK1098 carries a Tn10 transposon with a tertracycline (Tet) ressistance marker. (ii) λNK1324 carries a mini-Tn10 transposon Tn10dCm with a chloramphenicol (Cm)-resistance marker, Cmr. The lambda phage vectors above (Kleckner et al., 1991) were used to make random transposon insertions in the chromosome either for the purpose of insertional mutagenesis or for tagging antibiotic resistance markers to point mutations

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

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%

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

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

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

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

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

Site directed mutagenesis

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

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

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

Isolation of total cellular RNA

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

For amplification of short length (100-200-bp)DNA fragmentsor that do not

Polymerase chain reaction (PCR)

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

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

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

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

Protein concentrations were estimated by the method of Bradford (1976). The A595wasmeasured after complexation with Bradford reagent. Bovine serum albumin was usedas standard against whichthe unknown protein concentrations were estimated

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

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

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

PCR products were purified using the PCR Purification Kit (Qiagen) as per the manufacturer's instructions

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

Purification of DNA by gel elution

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

Restriction enzyme digestion and analysis

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

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

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

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

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

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

β-galactosidase assay was performed according to Miller (1992). An overnight grownculture of the bacterial strain was sub-cultured in glucose Minimal A medium

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)

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

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

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

NaCl-sensitivity testing

agar platesLac+colonies will appear dark pink colonies whereas Lac–will remain colourless

Competent cells for high efficiency transformations were prepared by a method ofInoue et al. (1990) with few modifications. An overnight culture of the strain (routinelyDH5α) was sub-cultured into fresh sterile LB-brothin 1:100 dilutions and grown at 18ºC to an A600of 0.55. The cells were harvested by centrifugation at 2500 rpm for 10-min at 4ºC. This was re-suspended in 0.4 volumes of INOUE buffer and incubated inice for 10 min. The cells were recovered by centrifugation at 2500 rpm at 4ºC for 10-min and finally re-suspended in 0.01 volume of the same buffer. Sterile DMSO wasadded to a final concentration of 7%. After incubating for 10-min in ice, the cells werealiquoted in 100 μl volumes, snap frozen in liquid nitrogen and stored at –70ºC

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

the infection mixture was centrifuged, washed in 5 ml of citratebuffer and plated without phenotypic expression

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,

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

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

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,

Antibiotics were used at the following final concentrations in various media as given inTable 2.4.Table 2.4Concentrations of antibiotics (μg/ml)

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

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

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%

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

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

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

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

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

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

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

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

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

For TEM, C. glabrata cells were digested with zymolyase 20T for 3 h at 30◦C, centrifuged at 1,000 g and washed with YPD medium. Cell fixation was performed as described for SEM and dehydrated samples were embedded in araldite 6005 resin. After complete polymerization at 80 ̊C for 72 h, ultra-thin (50-70 nm) sections were preparedwith a glass knife on Leica Ultra cut (UCT-GA-D/E-1/00)microtomeand mounted on copper grids. Aqueous uranyl acetate-stained and Reynolds lead citrate-counterstained samples were viewed under Hitachi H-7500 transmission electron microscope

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

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

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

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

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

Cell wall β-glucan measurement was carried out as describedpreviously with some modifications(Kapteynet.al.,2001). Briefly, cell wall fractions were washed multiple times with 1 N NaCl. Washed cell walls were boiled twice in 50 mM Tris-HCl(pH 7.8) containing 2% SDS, 100 mM Na-EDTA and 40 mM β-mercaptoethanol for 5 min to remove non-covalently linked proteins and other contaminants. SDS-treated cell wall fraction was collected and rinsed thrice with water. For β-glucan isolation, cell wallswere extracted three times, each for 1 h, in 0.5 ml 3% NaOH at 75 ̊C and centrifuged at 1,200 g.All 3% NaOH supernatant fractions were saved for isolation of mannan as described below. 3% NaOH-extractable cell wall pelletwasneutralized twice in 100 mM Tris-HCl (pH 7.5) and once in 10 mM Tris-HCl (pH 7.5) and digested with 5 mg/ml zymolyase-20T in 10 mM Tris-HCl (pH 7.5) for 14-16 h at 37 ̊C. This treatment liberates approximately 90-95% glucose into the supernatant. Total glucan content in the cell wall was measured by estimating glucose from both the solubilised supernatant and zymolyase-20T insoluble pellet fractions with phenol-sulphuric acid carbohydrate estimation method using purified glucose as the standard

Yeast cell wall was isolatedas describedpreviously(De Groot et al., 2004). Briefly, cells grown underdifferent environmental conditions were harvested at 5,000 g for 5

Cells grown to log-phase in YPD medium were spotted on CAAmedium and overlaid with a nitrocellulose filter. Cells were allowed to grow at 30 ̊C for 18-20 h. After incubation, the filter was washed with water to remove cells and membrane-bound CPY was detected by immunoblotting withpolyclonal anti-CPY antibody (Thermo Scientific) at a dilution of 1:15,000

CPY activity was measured as described previously (Jones,2002). A 2.5 mg/ml stock solution of CPY-specific substrate N-benzoyl-L-tyrosine p-nitroanilide(BTPNA, prepared in dimethyl formamide) was diluted 5 times with 0.1 M Tris-HCl (pH 7.5). 100 μl diluted substrate solution was added to a 96-well plate containing 25 μl cell suspension (5 x 107cells). After 18 h of incubation at 37 ̊C, plate contents were clarified by centrifugation and colour formation was quantified by absorbance at 405 nm. Background absorbance measured using BTPNA-free cell cultures was subtracted from BTPNA-loaded cell cultures and absorbancevalues were normalized to total number of viable cells to enumerate total cellular CPY activity

ammonium molybdate, respectively, to the assay buffer.For specific inhibition of vacuolar membrane H+-ATPaseactivity, vacuolar membrane fractions were incubatedwith 1-2.5 μM bafilomycin for 5 minprior to the activity assay.ATPase activity was initiatedby adding ATP to the assay buffer to afinal concentration of 5 mM and incubating the reactionat 30 ̊C for 30-60 min.Reaction was stopped by adding an equal volumeof a stop-developing solution (1% (w/v)SDS, 0.6 M H2SO4, 1.2%(w/v)ammonium molybdate and 1.6%(w/v)ascorbic acid). Amount of inorganic phosphate (Pi) liberated was measured at A750nmafter 10 minincubation at room temperature. Standard curve prepared with 0-50 micromoles of KH2PO4 was used for the determination of total Pi. The ATPase activity of the vacuolarmembrane H+-ATPase was expressed in micromoles of Pireleased per milligram protein per min

Vacuolar membrane H+-ATPase activitywas measured inbothcrude membrane fraction and purifiedvacuolar membrane fraction asdescribed previously(Woolfordet al.,1990).Activity inthe crude membrane fractions was carried out with 2.5-10 μgprotein in 50 μl assay buffer (5 mM MgCl2, 25 mM MES/Tris-HCl(pH 6.9)and 25 mM KCl). For activity inthe purified vacuolar membrane fraction, a totalof300 μl reactionmix was setup with of 2.5-10 μgprotein samples.Residual activities from other ATPases such as mitochondrial ATPases, plasma membrane H+-ATPase and phosphataseswere inhibited by adding 2 mM NaN3, 200 μM NaVO4and 0.2 mM

Vacuole membraneswere isolatedwith slight modifications of Cabrera’s method(Cabrera et.al.,2008). Log-phase, YPD medium-grown cells wereinoculated in 1 lt YPDmedium to an initialOD600of 0.1. Cells were incubated at 30 ̊C with shaking at 200 rpm till the cell density reached to OD600of 0.8-1.0.Cells were harvested by centrifugation at 5,000 g and washed once with 30 ml 2% ice-cold glucose solution. Cells were incubated in 15 ml solution containingglycine-NaOH(50 mM; pH10)andDTT(2 mM) at 30 ̊C for 10 min. After incubation, cells were normalized to adensity of1000OD600and resuspendedin 15 ml spheroplasting buffer containing 10-15mg of zymolyase20T.Cells were incubated at 30 ̊C for 45-60 minor till the spheroplasting was completed.Spheroplasts werecollected by centrifugation at 4,500 rpmfor 5 minat 4 ̊C, washed gently with15 ml 1.2 M sorbitol solutionandresuspendedin 3.5 ml 15%ficoll solution made in PS buffercontaining 1X protease inhibitor cocktail. This suspension was homogenized on ice with 20-25 strokes in a loose-fitting Dounce homogenizer. Homogenate was transferred to an ice-cold,ultra-clear Beckman ultracentrifuge tube, overlaid witha gradient of3 ml 8%ficoll solution, 2.5 ml 4%ficoll solutionand 2.5 ml PS buffer lacking ficoll and centrifuged at 1,10,000g(30,000 rpm)for 90 minat 4 ̊Cin a pre-cooled Beckman ultracentrifuge with SW41-Ti swinging bucket rotor.Centrifugation was carried out with slow acceleration and deceleration settings.White creamy vacuole membrane layer wascollected from the interfaceof 0and4% ficoll gradientwithout mixing the layers.Total protein concentration in thevacuole fraction was estimated using BCAprotein assay kit as described earlier

Crude fractionation of total membraneswas carried outviadifferential centrifugation asdescribed previously (Moranoand Klionsky,1994)with slight modifications. Cells grown tolog-phase in YPDmedium werecollected, washed,normalizedto 10 OD600and resuspendedin 1 ml spheroplast buffer containing 1-2mg of zymolyase20T (MP Biomedicals).Following incubation at 30 ̊Cfor 30-45 min,spherolplastswerecollected by centrifugation at 800 g for 3 minat 4 ̊C and resuspendedin 1 mlice-cold Tris-EDTA (pH 7.5). Spheroplastswere lysed with 100 μl 0.5mm glass beads on a vortex mixer with 10 secpulsegiven thricewith intermittent ice-breaks.Cellsuspension was centrifuged at 800 g for 5 minat 4 ̊C to pellet unbrokenspheroplastsdown andthesupernatant was centrifuged at 15,000 g for 5 minat 4 ̊C to obtainthemembrane fraction pellet.Pellet was washed once with ice-cold Tris-EDTA (pH 7.5), resuspendedin 50 μl of the samebuffer and stored at -20 ̊Ctill further use. Protein concentration of pellet fraction was estimated using BCAprotein assay kit with BSA as thestandard

A calibration curve of fluorescence intensity values versuspH was prepared for BCECF-AM-loaded wt cells by incubatingcellsin YPD medium containing 50 mM MES, 50 mM HEPES, 50 mM KCl, 50 mM NaCl, 0.2 M ammonium acetate, 10 mM NaN3, 10 mM 2-deoxyglucoseand5 μM carbonyl cyanide m-chlorophenylhydrazone, titrated to five different pH values in the range of 4.0-8.0. Fluorescence intensity values were measured by excitation at 440and 490 nm with emission at 535 nm and a graph was plotted between the ratio of intensity at 490 to 440 nm versuspH. Similar to pHi calibration curve, a polynomial distribution of fluorescent intensity signal and pH was observedfor BCECF-AMprobe

fluorescence by excitation at 440 (pH-independent) and 490 nm (pH-dependent) with emission at 535 nm. Ratio offluorescence intensity at 490 to440 nm was used tocalculatethe vacuolar pH. Background fluorescence was removed by subtracting the fluorescence intensity values of cells without BCECF-AM from the fluorescence intensity values of the probe-loaded cells

Vacuole pH inyeast cells was determined asdescribed previously (Padilla-López and Pearce, 2006). Briefly, log-phase,YPD medium-grown yeast cells were harvested and suspended in 200 μl YPD medium containing 50 μM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Invitrogen # B1150) to the final cell density of 4 x 107 cells. Cells were incubated at 30 ̊C for 30 min at room temperaturefollowed by three washeswith YPD medium. Washed cells were resuspended in 1 ml YPD medium and 200 μl cell suspension was used for recording

Vacuolar morphology of C. glabratacells was examinedby staining vacuoleswith FM4-64 (Molecular Probes, Invitrogen). FM4-64 is a lipophilic dye that exhibits long wavelength red fluorescence when boundto lipids. FM4-64 binds to the plasma membrane and follows the endocytic pathway to reach the vacuole(Vida and Emr, 1995).Log-phase,YPDmedium-grown cells were harvested and washed with 1X PBS. 1 ODcells were resuspendedin 50 μl YPDmedium containing 30 μM FM4-64 andincubated at 30 ̊C for 30-45 min. After incubation, cells were washed thricewith YPD mediumand resuspendedin 100 μl of the samemedium. Cells were observed under confocal laser scanning microscope(Zeiss LSM 510 Meta)with 63X objective lens,2.5X final zoom, pinhole set at 108 μm and emission filterset to LP 565nmto capture fluorescence image.Along with the fluorescenceimage, aphase contrastimage was alsocaptured for each sample

cellswere collected and washed with chilled sterile water.1 OD600cells were resuspendedin 20 μl chilled10%TCA solution containing 8 mM EDTA (pH 8.0) and incubated at room temperature for 15-20 min.Followingincubation, cellsuspension was centrifuged at 12,000 rpm for 5 minat 4 ̊Cand supernatant was transferred to a fresh 1.5 ml microcentrifuge tube. 10 μl of this supernatant fraction was diluted 75-foldwith ATPassay mix dilution buffer provided with the kit. 50 μl of diluted suspension was added to anequal volume of ATPassay mix (Sigma # FLAAM) which containedfirefly luciferase and luciferin with MgSO4, EDTA, DTT and BSA inTricine buffer.Luminescence was measured inluminometer (Varioskan flash-3001,Thermo Scientific). Total ATP was quantified usingpurified ATP as the standardand expressed in moles/OD cells

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

Estimation of total glycogen in cells was performed asdescribed previously (Parrou et al., 1997) with slightmodifications.Briefly, YPD medium-grown C. glabratacells were harvested, washed once with 1 ml ice-cold waterandresuspendedin 250 μl sodium carbonate(0.25 M)solution. After incubation at95 ̊C for 4 hin water bath with occasional stirring, cell suspension was cooled and pH of the suspension was adjusted to 5.2 by adding 150 μl 1 M acetic acid. Tothis suspension,600 μl 0.2M sodium acetatewas added and cell suspension was incubated with 1-2 U/ml of α-amyloglucosidase from A.niger(Sigma #A7420)at 57 ̊C for overnight with constant agitation.Resultant glucose liberated by α-amyloglucosidase digestion was collected in the supernatant fraction and quantifiedby phenol-sulphuric acid methodof carbohydratedetermination.For quantification, commercially available purified glucose was used as a standard and total glycogen incells was expressed as μg/2 x 107cells tonormalizeagainstcell density

Trehalose from C. glabratacells was extracted by trichloro acetic acid (TCA)solutionas described previously (Lillie et al.,1980). Cells grown in YPDmediumwere collected at different time pointsof growth and washed thrice with ice-cold sterile water. Cells were immediatelystored at-20 ̊Ctill further use.For trehalose isolation, 10-20 OD600cells were thawed in 500 μl TCA (0.5 M) solutionon ice and incubated at room temperaturefor 1 h.Supernatant fraction was collected by sedimenting cells at 14,000 rpm for 5 minat 4 ̊C.TCA extractionwas repeated withcells once more and the resultingsupernatant was mixed with the earlier fraction.Extractedtrehalose was measuredby phenol-sulphuric acid methodof carbohydratedeterminationwithcommercially available purified trehalose(Becton, Dickinson and Co.) as a standard.Total trehalosecontent was normalized to the cell densityand expressed as μg/2 x 107cells

Quantitative measurement of periplasmic acid phosphatase activity in phosphate-starved C. glabratacells was performedas mentioned previously (Orkwis et al., 2010). A total of 0.5 OD600YNB-grown and phosphate-starved cells were collected, washed thrice with cold water and oncewith cold 0.1 M sodium acetatebuffer (pH 4.2). Washed cells were resuspendedin 500 μl sodium acetate (0.1 M)and incubated at 30 ̊C with constant stirring. After 10 min incubation, 500 μl freshly-prepared solution of 20 mM p-nitrophenyl phosphate in 0.1 M sodium acetate(pH 4.2) was added to the cell suspension. Enzymatic activity was stopped after incubation at 25 ̊C for 20 min by addition of 250 μl sodium carbonate (1 M)tothe reaction mix. Resultant colour change was measured by monitoring absorbance at 400 nm. Acid phosphatase activity was expressed as a ratio of OD400to OD600 to normalize against cell density

Determination of acid phosphatase activity

Cells grown overnight in YNBmedium wereinoculated in fresh YNB mediumand incubated at 30 ̊C with shaking at 200 rpm. Cells were harvested when the cell density reached to an OD600of 0.6-0.8.Cells were consecutively washed with sterile MQ water and YNB without phosphate (YNB-Pi) medium. Washed cells were inoculated either in YNB orYNB-Pimediumto the initial OD600of 0.1. Cells were incubated at 30 ̊C for 3-4 h, harvested and resuspendedin 100 μlYNB-Pimedium. Radioactive P32-labelled o-phosphoric acid(Jonaki# LCP 32)was added to the cell suspension to a final concentration of 1 μCi/mlandcells were incubated for 30 min.For determining phosphate uptake, a10-12 μl cell suspensionaliquot,after every5 min,was removed and kept on ice.To this cell suspension, 500 μl ice-cold YNB-Pimediumwas addedand cells were harvested by centrifugation at 5,000 g for 5 minat 4 ̊C.These cells were washed with ice-cold YNB-Pimedium thrice and resuspendedin 100 μlPBS(1X). 10-20 μl of this cell suspension was added to5 ml scintillation fluid and β-decay counts were measured in ascintillation counter(Tri-Carb 2910 TR Liquid Scintillation Analyzer, PerkinElmer).Scintillation counts were normalized to total cell number and plotted with respect to time. Total phosphateuptake was expressed as P32c.p.m/OD600cellswhere c.p.m refers tocounts per min

Total cellular polyphosphates were quantified viapolyacrylamide-Tris borate gel electrophoresis (PAGE-TBE).Briefly, PAGEwas performed with Tris-borate buffer (pH 8.3) todeterminethe quantityand the typeof polyphosphatesextracted fromyeaststrains. Equal amount of total RNA (20 to 100 μg) was loadedon 34%PAGE-TBE gel (22cm long, 16 cm wide and 0.8 mm thick) and electrophoresedat 500Voltsfor 20-24 hin cold-roomtill the marker dye bromophenol blue(BPB)had migrated 15-16 cm awayfrom the well.After electrophoresis, total polyphosphates werevisualizedby staining the gel with 0.05%toluidine blue staining solutionfollowed by destaining. Polyphosphates wereobserved both as asmearin the top most portion of the gel as well asdiscreet bands of long chain polyphosphatesand shortchain polyphosphatesin middle andbottom half of the gel,respectively.Polyphosphateband intensity in the gelwas quantified using ImageJ software (http://rsbweb.nih.gov/ij/)and relative amountsof long chain and short chain polyphosphatesin C. glabratacells werecalculated

Polyphosphates from yeast cells were extracted with phenol-chloroform solutionas described previously(Neefand Kladde,2003). Cells grown eitherovernight or to logarithmic phase in YPDmediumwere harvested by centrifugation at 1,700 rpm for 5 min. Cells were washed with 10 ml sterile MQ water and resuspendedin ice-cold 500 μl20%trichloro acetic acid (TCA) solution to the final cell densityof 100 OD. Cell suspension was transferred toa1.5 ml microcentrifuge tube. After incubation at room temperature for 5 min, cells were harvested by centrifugation at 12,000 g for 10 minat 4 ̊C and resuspendedin 250-350 μlpolyphosphate extraction buffer.Equal volume of phenol-chloroform (25:24) was addedtothe microcentrifuge tube and aqueous phase was extracted by centrifugation at 12,000 g for 8 minat room temperature. Top aqueous layer was collected with a 200 μl tip. Aqueous layer extraction was repeated once more after removal of DNA with chloroform.After centrifugation,aqueous phasecontaining RNA and polyphosphates wascollected,RNA was quantified at A260nmandstored at -20 ̊C

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

For phosphate starvation,yeastcells grown to log-phase in YNB medium were harvested,washed with water,transferred to either regular YNBor YNB medium lackingphosphate and were grown for 16 h at 30 ̊C. Cells cultured in YNB medium

Plasma membrane H+-ATPase activitywas measured inthe total membrane fraction as described previously (Nakamura et al., 2001).5μg totalmembrane fraction was incubated at 30 ̊C in 120 μl reaction buffer containing 10 mM MgSO4and 50 mM KCl in 50 mM MES (pH 5.7) with 5mM adenosine tri-phosphate (ATP). To eliminate possible contribution of residual ATPases, viz.,vacuolar ATPases, mitochondrial ATPases or non-specific phosphatases, 50mM KNO3, 5mM NaN3and 0.2mM ammonium molybdate were used, respectively, in the assay mixture. Reaction was stoppedafter 30 minby adding 130μl stop-developing solution containing 1% (w/v) SDS, 0.6M H2SO4, 1.2%(w/v)ammonium molybdate and 1.6%(w/v) ascorbic acid. Amount of inorganic phosphate (Pi) liberated was measured at A750nmafter 10 minincubation at room temperature. A standard curve prepared with0-50 μmolesof KH2PO4 was used fordetermination of total Piamount.ATPase activity of the plasma membrane was expressed in micromoles of Pireleased per milligram protein per min. ATPase activity was also determined in the presence of plasma membrane H+-ATPase inhibitor diethylstilbestrol (DES,Sigma# D4628),wherein total membrane fraction was incubated with 0.2mM DES for 5 min, prior to the enzymatic measurement

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

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

To assess the activity of plasma membrane proton pump, CgPma1, in cells grown in differentexternal pH environment,whole cell acidification assaywas carried out.This assay is a measurement of glucose-responsive proton pump activityin live cellsand is based on a decrease inthe pH of a weakly-buffered solutionupon extrusion of H+ions from thecell. The amount of change in the pH of the medium represents a crude measurement of the activity of functional plasma membrane proton pump in live cells. Whole cell acidification assay was conductedwithcellsgrown in YNB pH 5.5 and YNB pH 2.0medium as described previously (Martinez-Munoz and Kane, 2008) with slight modifications.After growth at30 ̊C for 2 h, cells were harvested, washed and resuspended(1.5-3.0 mg wet weight/ml) in 15ml MES/TEA (1mM; pH 5.0) buffer. Cell suspension was kept at 25 ̊C with continuousagitation. Extracellular pH of the buffer solution was recorded at 1 mininterval for 20 minwith the help of a pH meter(BT-600, BoecoGermany). To activate plasma membrane proton pumping, glucose and KCl were added to a final concentration of 40mM after 3 and 8 minincubation, respectively. Plasma membrane proton pump activitywas plotted as a change in the pH of the extracellular solutionversustime

Log-phasecells grown in YPD medium containing or lacking CaCl2and FK506 were collected, PBS-washed and loaded with ratiometric, high affinity, membrane-permeable calcium indicator, Fura-2 AM (10 M; Sigma #47989). After 30 min incubation at 30◦C, labelled cells were washed thrice with cold PBS, suspended in PBS and fluorescence was recorded at 505 nm with dual excitation at 340 and 380 nm. The ratio of fluorescence intensities between 340 and 380 nm, representing Ca-bound and Ca-free Fura-2 molecules, respectively, reflected free intracellular calcium concentrations

Intracellular reactive oxygen species (ROS)levels in yeast cells weredetermined usingfluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA; Sigma# D6883). Cellular esterasesremove the diacetate groups ofthe DCFH-DAand produceDCFHwhich getsreadily oxidized to highly fluorescent product 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. The fluorescent intensity of DCF corresponds to the amount ofintracellular ROSpresent in the cell.Cells grown under different environmental conditions were harvested,washed once with tissue-culture grade phosphate-buffered saline (PBS) and resuspendedin PBS to the final cell density of 1 OD. Freshly-prepared DCFH-DA (0.01M stock in DMSO) was added to the cell suspension toafinal concentration of 100 μM. Cell suspension was mixed and incubated at 30 ̊C for 30 min. After incubation,cells were washed 2-3 times with 1 ml PBS and then resuspendedin 200 μlPBS. Fluorescence intensity values wererecorded usingspectrofluorophotometer (Varioskan flash-3001, Thermo Scientific) with excitation and emission at 488and 530 nm,respectively.Fluorescenceintensityvalues obtained from probe-loaded cells were subtracted from the fluorescence intensity values obtainedfrom cells-alone samplesto remove background fluorescence

For determiningthe intracellular pH from fluorescence intensity values of CFDA-SE-loaded cells, anin vivocalibration curve was prepared between fluorescent intensity and pre-adjusted environmental pH values. Briefly,CFDA-SE-loaded wild-typeC. glabratacellswere incubatedwith 0.5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma# C2759) at 30 ̊C for 10 min in 50 mM CP buffer adjusted to different pH values ranging from 4.0 to 7.5, with an interval of 0.5 unit.CCCP is an ionophore which dissipates the plasma membrane pH gradient, thus, rendering the intracellular pH similar to the extracellular pH. Fluorescent intensities were determinedand a calibration curvewas plotted between the ratio of intensity at 490 to430 nm versuspH.A polynomial distribution of fluorescent intensity signal and pH was observed for CFDA-SE probe(Figure2.1)and the graphequation was used todeterminethe intracellular pHof C. glabratacells

OD600of 0.5and transferred to a 1.5 ml microcentrifuge tube. Probe loading was carried out by adding freshly-prepared CFDA-SE solution (0.01 M stock in DMSO) tocell suspension to a final concentration of 160 μM. Cell suspension was mixed on vortex mixerfor 10 secand incubated at 37 ̊C for 1 hwith shaking at 300 rpmon thermo mixer.Cells were harvested, washed twice with 1 ml 50 mM CP buffer to remove unloaded probe,resuspendedin 250 μl CP buffer andwereincubated at 30 ̊C for 30 minwith shaking to recover from the stress induced during probe loading. Afterincubation,fluorescent intensitywasdetermined with spectrofluorophotometer (Varioskan flash-3001, Thermo Scientific) by excitation at 430 nm (pH-independent) and 490 nm (pH-dependent) with emission at 525 nm. Background fluorescence of the probe was removed by subtracting the fluorescence intensity of the probe in CP buffer from the fluorescence intensity of the probe-loaded cells

Intracellular pH(pHi)in yeast cells was determinedusing fluorescent 5,(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes) asdescribed previously (Bracey et al.1998). For pHiprobe estimation,YNB medium-grown log-phase cells were inoculatedin YNB, YNB-pH 2.0 or YNB medium supplemented with acetic acid and incubated at 30 ̊C for different time points.Log-phase C. glabratacells were harvested and washed twice with 50 mM citric-phosphate (CP) buffer (pH 4.0). Washed cells were resuspendedin 1ml 50 mM CP buffer to an

Following transfer,membrane was either stained with Ponceau reagent for checking the efficiency of transfer or directly processed for proteindetectionusing protein-specificantibodies.For immunoblotting, membranes were blockedwith 5% (w/v)non-fat milk solutioneitherin PBS-T or TBS-T for 2 hat RTand probedwith primary antibodies against the target proteins.For detection of CgPma1, membranes were probed with1:1000 dilution of polyclonal anti-Pma1antibody raised against S. cerevisiaePma1 (Santa Cruz #sc-33735)in PBS-T with 5% (w/v)fat-free milk for overnight at 4°C.For detection of phosphorylated form of CgSlt2,immunoblotting analysis was done withan anti-phospho-p44/42 MAPK (Thr202/Tyr204) primary antibody raised against human p44 MAPK(Cell signalingtechnology# 4370S) at a dilution of 1:6000 in TBS-T with 5% (w/v)fat-free milk for overnight at 4°C.For detection of CgCPY, membrane was incubated with polyclonal anti-CPY antibody raised against S. cerevisiaeCPY(Thermo Scientific # PA 1-27244) at a dilution of 1:10,000 in TBS-T with 5% (w/v)fat-free milk for overnight at 4°C.For CgGapdhdetection,anti-Gapdh primary antibody raised againsthuman Gapdh(Abcam # ab22555) at a dilution of 1:7000was usedin TBS-T with 5% (w/v)fat-free milk.Secondary antibodies conjugated with horseradish peroxidase(HRP)enzymewereusedin 1:10,000 dilutionto detect the immune-reactivity of primary antibodieswith the help of ECL plus Western blotting system (GE Healthcare)as per manufacturer’sinstructions

Total proteins resolved bySDS-PAGE were transferred to PVDFnylon membrane by Western blotting using a Bio-Rad Mini Trans-Blot electrophoretic transfer unit in Tris-glycinetransfer bufferat 4 ̊C either at 100 Voltsfor 3 hr or 30 Voltsfor overnight

SDS-PAGEwas performed as described previously (Laemilli, 1970).10-40 μg protein samples were mixed with 4X SDS loading buffer and either incubated at 50 ̊C or 90 ̊C for 10 min. Denatured samples were loaded either on8%or 10%SDS-PAGEgel and run in Tris-Glycine-SDSgel running buffer at 70-100 Volts for 2-3 hin a Mini-PROTEAN®3electrophoresis unit(Bio-Rad).After electrophoresis,gels were either visualized by coomassie brilliant blue (CBB) stainingor processedfor western blotting as described below

Log-phase yeast cell cultures were harvested and total protein was extracted by lysingyeast cells using glass beads. Briefly,10 mllog-phase yeast culturesgrownin appropriate medium were harvested,washed once with ice-cold water and suspended in 250 μl homogenizing buffer containing 1 mM phenylmethylsulfonylfluoride(inhibitsserine proteases), 10 mM sodium fluoride(inhibit Ser/Thr and acid phosphatases), 1 mM sodium orthovanadate (inhibits Tyr and alkaline phosphatases) and 1X concentration of protease inhibitor cocktail(RocheCat # 04693159001). Cells were lysedwith glass beads by vortexing five times at high speed for 1 min with intermittent 1 min ice breaks. Unbroken cells and cell debris were removed by centrifugation at 1,000 g for 5 min at 4 ̊C. Cell lysate was collected and protein was quantified using bicinchoninic acid (BCA)protein assay kit (Thermo Scientific # 23227) as per supplier’s instructions

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=

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

.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

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

the disrupted gene, BLAST N of the sequences from rescued plasmids was performedagainstC. glabrataGenolevures database (http://www.genolevures.org/blast.html

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

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.

and colony purified on CAA plate. 15% glycerol stocks were made for two independent transformants and stored at -80 ̊C

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

stranded DNA. Final reaction volume was adjusted to 20 μl with DEPC-treated waterandamplificationreaction was carried out usingthese parameters: initial denaturation at 95 ̊C for 5 min followed by 40 cycles of denaturationat 95 ̊C for 30 sec, annealing at 55 ̊C-57 ̊C for 30 sec, elongation at 72 ̊C for 40 sec and final extension at 72 ̊C for 10 min. Transcript levelswerequantified with an end-point value known as Ct (cycle threshold). The Ctdefines the number of PCR cycles required forthe fluorescent signal of SYBR green dye to cross more than the background level. The Ctvalue isinversely proportional to the amount of nucleic acid product. Ctvalues were obtained during exponential phase of amplification and used forcalculation of relative-fold change in gene expression after normalization to Ctvalues ofeither housekeeping gene ACT1 (gene encoding actin)orTDH3 (gene encoding Gapdh)with the help of the following formula. Fold change in expression = 2-∆∆Ct∆∆Ct= ∆Cttreated -∆Ctuntreated∆Cttreated = Ctvalue forgene of interest under test/treatedcondition -Ctvalue forinternal controlgene(ACT1/TDH3) under test/treatedcondition∆Ctuntreated = Ctvalue forgene of interest under untreatedcondition -Ctvalue forinternal control (ACT1/TDH3)gene under untreatedcondition

Todeterminethe expression level of a specific gene, quantitative real-time polymerase chain reaction (qRT-PCR/qPCR)was performed oncDNA usinggene specific primers. Primers for qPCR weredesigned in such a way so as to get amplification products in a size range of 150 to 300 bp. Optimalprimer and cDNA concentrationswere standardized and qPCR was performed in ABI Prism 7000/7500 Real time PCR Machine (Applied Biosystems). Briefly, 0.4 μl cDNA was mixed with 0.1 to 0.2 picomolesof gene specific forward and reverse primers and 10 μl 2X MESA GREEN qPCR™Mastermix Plus containing SYBR green dye (Eurogentec) in awell of a96-well PCR plate (Axygen). SYBR green is a dye that specifically binds to double

1 μg good quality RNA was treated with DNase I (amplification grade, Invitrogen) to remove DNA contamination and used for complementary DNA (cDNA) synthesis using reverse transcriptase enzyme and oligo-dT primers.SuperScript®III First-Strand Synthesis System (Invitrogen) was used to carry out cDNA synthesis reaction according to the manufacturer’s instructions. cDNA was stored at -20 ̊C

autoclavable plastic items to removeRNase contamination. RNA was isolated from C. glabratacells using hot phenol extraction strategy.Log-phase cells well harvested at 5,000 g for 5 min at 4 ̊C, resuspended in 1 ml ice-cold DEPCwater and transferred to a 2 ml microcentrifuge tube. Cells were spun down at 6,000 g for 3 min at 4 ̊C and resuspended in 350μl AEsolution. 50 μlSDS and 400 μl acid phenol wereadded tothe above tubeand mixed well by vortexing. Tubes were incubated at 65 ̊C for 15minwith continuousmixing. After incubation, tubes were kept on ice for 5 min and centrifuged at 12,000 rpm for 5 min at 4 ̊C. Aqueous phase was collected and re-extracted with an equal volume of cholroform. Total RNA was precipitated at -20 ̊C with1/10thvolume of 3 M sodium acetate (pH 5.2) and 2.5 volume of ice-cold 100% ethanol and collected by centrifugation at 12,000 rpm for 5 min at 4 ̊C. RNA pellet was washed with ice-cold 70% ethanol and resuspendedin 100 μl commercially available DEPC-treated water (Sigma # 95284). RNA concentration was measured byrecordingabsorbance at 260 nm. Purity of RNA sample was checked by A260nm/A280nmratio where ratio of >1.8 was considered as good quality RNA. RNA integrity was checked by gel electrophoresis on 8% agarose gel made in DEPC-treated TAE buffer

All solutions were made in RNase free diethylpyrocarbonate(DEPC)water. Microcentrifuge tubes and tips employed for RNA workwere autoclaved twice and kept at 70 ̊C for overnight before use. RNaseZap®(Ambion) was sprayed on non-

Themethod was used for isolation of good quality genomic DNA that wasused to map Tn7insertionin C. glabratamutants.Briefly,10 mlsaturated yeast culturewasharvested, resuspendedin 1 ml sterile water and transferred toa2 ml microcentrifuge tube. Cells were pelleteddown by centrifugation at 4,000 rpm for 5 min. Supernatant was discarded and the pellet was resuspendedin 500 μl freshly prepared solutioncontaining100mM EDTAand 5% β-mercaptoethanol andincubated at 42 ̊C for 10 min. After incubation,cells were spun down at 5,000 rpm for 1 minand resuspendedin 500μl freshly-prepared BufferB. One tip full of lyticase(Sigma # L4025) was added and cellsuspension was incubated at 37 ̊C for 1 h. Following incubation,cell suspension was spun down at 6,000 rpm to recover spheroplasts.Spheroplasts weregently resuspendedin 500μl BufferCand DNA was twice extracted with 500μl phenol:chloroform:isoamyl alcohol (25:24:1)solution.Aqueous layer was collected in a new 2ml microcentrifuge tube and DNA was precipitated with 1ml ethanol and 1/10thvolume of 3M sodium acetate (pH 5.2)by centrifugation at 13,000 rpm for 5 min. Pellet was resuspendedin 200 μl TE containing 0.3 μl of RNase Cocktail™and incubated at 37 ̊C for 30 min.After incubation, 300 μl additional TE was added and DNAwas re-precipitated withethanol and 3 M sodium acetateas described above. Pellet was washed with 70% ethanol anddried under air. DNA pellet was finally suspended in 100 μl TE and stored at -20 ̊C

phenol:chloroform:isoamyl alcohol (25:24:1)was added to the tube and mixed thoroughly.Aqueous phase was collected after centrifugationat 12,000 rpm for 3 minand was transferred toanew 2 ml microcentrifuge tube.1 ml absoluteethanol was added to the aqueous phase and DNA was precipitated by centrifugation at 12,000 rpm for 8 minat 4 ̊C.DNA pellet was washed with chilled 70%ethanol and dried under air. DNA pellet was resuspendedin 50 μl TE containing 0.3 μl of RNase Cocktail™(Ambion®# AM2286)and incubated at 50 ̊C for 20 min. 200 μl additional TE was added to the above suspension and DNA was stored at -20 ̊C

In this method of genomic DNA extraction,yeast cells werelysed by mechanical disruption with glass beads. Briefly, yeast cells were harvested after overnight growth in YPD medium, resuspendedin 500 μl waterand transferred toa2 ml microcentrifuge tube.Cells were pelleteddown at 10,000rpm for 1 min. Resulting supernatant was discarded and the pellet was resuspendedin 500 μl Buffer A. The tube was incubated at 65 ̊C for 15 min. After incubation, 500 μl ofphenol:chloroform:isoamyl alcohol (25:24:1) and 0.5 gm of acid-washed glass beads (Sigma # G8772) were addedto the tube. Cells were lysed by three cycles of high speed vortexing withintermittent ice breaksfor 45 secand pelleteddown at 12,000 rpm for 3 minat 4 ̊C.Uppermost aqueous phase was transferred to a 2 ml microcentrifuge tube,500 μl of

This quick extraction method was used to isolate genomic DNA which was used as templateto amplify gene of interestor toverify the knock-out. C. glabratacells were grownovernight to saturation in 10 mlYPD medium at 30 ̊C.Cells were harvested at 4,000 rpm for 5 min, resuspendedin 400 μl Buffer Acontaining 50 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1%Triton X-100 and 1%SDSand weretransferred to a2 ml microcentrifuge tube. Equal volume ofphenol-chloroform solution was added to the abovesuspensionfollowed byvortexingfor 2-3 minand incubationat 42 ̊C for 30 minwithcontinuous agitation at 800 rpm on thermomixer (Eppendorf). Cell debris was removed bycentrifugation at 12,000 rpm for 5 minand aqueous fraction(~ 350 μl)was transferred to a new 2 ml microcentrifuge tube.0.3 μl RNaseCocktail™(Ambion® # AM2286) containing RNase A (500 U⁄ml) and RNase T1 (20,000 U⁄ml) was added and tubes were incubated at 37 ̊C for 30 min. DNA was precipitated with 2.5 volumesof chilled ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.2).DNA pellet was washed with chilled 70%ethanol and semi-dried under air.Pellet was suspendedin 100μlTE (10 mM Tris-HCland 1 mM EDTA; pH 8.0)and stored at -20 ̊C.DNA concentration was determined by recordingabsorbance at 280 nmin Nanodrop (Nanodrop ND-1000, Thermo Scientific).

Based on the subsequent use, DNA from C. glabratacells was extracted using three different methodologie

C. glabratayeast cells were grown overnight in 5 ml YPD medium at 30 ̊C. An aliquot from the overnight culture was inoculated in 10 ml fresh YPD medium to an initial OD of 0.1. Cells were incubated at 30 ̊C till the cultureOD600was between 0.4 and 0.6. Cells were harvested in a sterile 50 ml centrifuge tube and washed twice with sterile Milli-Q(MQ)water. Washed cells were suspended in 100 μl of 100 mM LiOAc, mixed thoroughly and transferred to a sterile 1.5 ml microcentrifuge tube. A transformation mix containing 240 μlpolyethylene glycol(PEG) (50% (w/v)), 36 μl LiOAc(1 M), 25μl ultrapure single-stranded salmon sperm DNA (2 mg/ml) (Clonetech) was added to 50 μl cell suspension. 50 μltransforming DNA (1μg circular plasmid DNA) was added to the above suspension. Whole mixture was vortexed gently and incubated at 30 ̊C for 45 min. 43 μl DMSO was added to the tubeand incubated at 42 ̊C for 15 min. Cells were collected after centrifugation at 5,000 rpm for 1 min and suspended in minimal medium containing 0.6% Bacto-Casamino acid. Transformation mixture was plated on CAA plates and transformants were selected for uracil prototrophy

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

E. coli DH5α ultra-competent cells were transformed with plasmid DNA by heat shock at 42 ̊C for 90 sec as described previously in Molecular Cloning-A Laboratory Manual (Sambrook and Russell,2001). Bacterial transformants were selected on LB agarmediumcontaining appropriate antibiotics. Transformants obtainedwere colony purified on LB plates containing antibiotics.Presence of the desired insertwas first verified by colony PCR followed by PCRusing extracted plasmid DNA as template

suspension was kept on ice for 10 min and 50 μl volume was aliquoted to chilled sterile microcentrifuge tubes. Cellswere immediately snap-frozen in liquid nitrogen and stored at -80 ̊C

A single colony of E.coli DH5-α strain was inoculated in 10ml LB medium and incubated at37 ̊C for overnight. 4 ml of thisovernight culture was inoculated in 2 lt SOB medium and incubated at 18 ̊C till theOD600reaches to 0.5. Cells were harvested by centrifugation at 2,500 g for 10 min at 4 ̊C and washed gently in 80 ml ice-cold Inoue transformation buffer. Cells were collectedby centrifugation at 2,500 g for 10 min at 4 ̊C and gently resuspended in 20 mlice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. Cell

All experiments in this studywere performed with log-phase cellsunless otherwise mentioned. For obtaining log-phase cells, overnight YNB-or YPD medium-grown yeast cellswerere-inoculated in fresh YNB or YPD medium to an initial OD600of 0.1-0.2.Cells were incubated at 30 ̊C with shaking at 200 rpmtill the OD600reached to 0.4-0.6 OD. After incubation, log-phase cellswere collected bycentrifugation at 4,000 rpm for 3 min,washed once with the same medium and usedforfurtheranalysis

C. glabratastrains were grown overnighteither in YPDor YNBliquid mediumat 30 ̊C with shaking at 200 rpm. Cells were harvested and suspended in 1X PBS to a final OD600of 1.0.Five 10-fold serial dilutions of cell suspension wereprepared in PBS and3-4μlwasspotted on YPD/YNBplates containing various test compoundsusing a multi-channel pipette.Plates were incubated at 30 ̊C and growth profileswererecorded after2-4days

Yeast cell viability was measured by plating appropriate dilutions of cell cultureonYPD plates at various time intervalsduringgrowth.Cell suspension was diluted in1X PBS. YPD plates were incubated at 30 ̊C for 2-3 daysand total colony forming units(CFUs)were calculated by counting the number of coloniesthat appeared onYPDplatesand dividing that number by anappropriate dilution factor

preparedin appropriate solvents, sterilizedby autoclaving or filtrationand stored at appropriate temperature

For growth analysisof C. glabratastrains, a single colony from YPD or YNBagar mediumwas inoculated in appropriate liquid medium and incubated at 30 ̊C with shaking at 200 rpmfor 14-16 h. This overnight grown culture was used toinoculatetest medium to an initial OD600of 0.1to 0.3.Optical density/Absorbance of the cell suspensionwas measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) at600nmat regular time-intervals up to a period of 96 h.Absorbance values were plotted with respect to time. Generation time of yeast strains wascalculated fromthe logarithmic (log) phase of cellgrowth. Growth profilesbetween 4 (t1)and 8 h(t2)time interval wereconsideredfor calculationof generation time usingfollowing formula. Generationtime(G)= (t2-t1) x {log (2)/ [log (Bf/Bi)]}G= Generation time in ht1=Initial timepoint taken for analysist2 = Final timepoint taken for analysisBf= Number of cells at time t2(calculated on the basis of OD600values, wherein1 OD600of C. glabratacorresponds to 2 X 107cells.)Bi= Number of cells at time t1(calculatedas mentioned above)Severalyeast strains used in this study were analysed for their susceptibility to variouschemical compounds,drugsand metal ions. For this purpose, stock solutions were

mM final concentration) and pH was adjustedto the desired valueby addition of HCl or NaOH. Medium was sterilized by autoclaving.YNBagar plates ofdifferent pHwereprepared by mixing equal volume of separately autoclaved 4% bacto-agar solution and2X varied pH-adjusted-YNB liquidmedium.All routine sterilization of mediumand solutionswas either carried outby autoclaving at 121 ̊C for 15-20 minat highpressure condition(15 psi)or filtration with 0.2 μmpolyvinylidene fluoride(PVDF) membranefilter unit (Millex®-GV, Millipore).Both yeast and bacterial strains were stored as frozen 15% glycerol stock at -80 ̊Cfor extendedlifetime

C.glabratastrains were maintainedeither on rich YPDor synthetically-defined YNB medium. C.glabratacells were routinely culturedat 30 ̊Cwith shaking at 200 revolutions per min(rpm)unless otherwise mentioned. Forgrowthexperiments, C. glabratastrains were freshly revived on YPDmediumfrom glycerol stocks.Escherichia coliDH5α bacterial strainwasused for plasmid transformation and propagationpurposes and maintained on LB medium.E.coliBW23473 bacterialstrainwas used to rescue Tn7transposon cassette from C. glabrataTn7insertion mutantsand maintained on LB medium. Bacterial strainsharboring plasmids were maintained on LBagar plates supplemented withappropriate antibiotics.For plasmid isolationpurpose,bacterial strains were grown overnight in liquid LB brothcontainingappropriate antibiotics at 37 ̊C with shaking at 200 rpm. Forpreparation of the solid medium, 2%bacto-agar was added to the mediumand autoclaved. To prepare medium of different pH, YNB mediumwas either buffered with citrateor HEPESbuffer (100

Oligonucleotides/primers used in this study were designed using either free online-tool Primer3 (http://frodo.wi.mit.edu/) or Gene Runner software (http://www.generunner.net/). Oligonucleotides used in this study were commercially

synthesized from MWG Biotech Pvt. Ltd., Bangalore. All primers used in this study are listed in Table 2.3.Table 2.3: List of primers used in this study

10mM EDTA0.1% SDS 1 M ureaToluidine blue staining solution:0.05% Toluidine blue20% Methanol2% GlycerolSolution was prepared in H2O.Destaining solution for polyphosphate gels:20% Methanol2% GlycerolSolution was prepared in H2O.Spheroplast buffer:50 mM Potassium phosphate (pH 7.5)0.6M Sorbitol0.2 X YPD mediumPS(PIPES-Sorbitol)buffer:10 mM PIPES-KOH (pH 6.8)200mM Sorbitol1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use

Citric-Phosphate buffer:0.5 M citric acid0.5 M dibasic sodium phosphatepH was adjusted to 5.0 with phosphoric acid and filter-sterilized.MES/TEA buffer:1 mM MES(2-(N-morpholino)ethanesulfonic acid)pH was adjusted to pH 5.0 with TEA(triethanolamine).Plasma membrane suspension buffer:50 mM Tris-HCl(pH 7.5)0.1mM EDTA0.1 mM Dithiothreitol 20% GlycerolPolyphosphate extraction buffer:50 mM HEPES (pH 7.2)

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

15% Acetic acidTris-Borate Saline(TBS):25 mM Tris150 mM NaClpH was adjusted to 7.4withHCl.This was prepared as 10 X stock solution and used at 1 X concentration.Blocking and wash buffers(PBS-T and TBS-T):5% Fat-free milk 0.1% Tween-20 Volume was made to 100 ml either with 1 X PBS(PBS-T)or 1 X TBS(TBS-T)

0.02% Bromophenol blue 2% DTT This was prepared as a 4 X stock solution and used at a 1 X concentration.SDS-PAGE running buffer:0.25 M Tris-HCl (pH 8.0) 1.92 M Glycine 1% SDS This was preparedas a 10 X stock solution and used at a 1 X concentration.Coomassie brilliant blue (CBB) staining solution:50% Methanol10% Acetic acid0.1% Coomassie brilliant blue-R250Western blotTransfer buffer:0.25 M Tris-HCl (pH 8.0) 1.92 M Glycine 1% SDS Thiswas preparedas a 10 X stock solution and used ata 1 X concentration.1X Transfer buffer (1litre):200 ml of methanol 100 ml of 10 X transfer buffer 700ml of waterPonceau 3S staining solution:0.25% Ponceau 3S40% Methanol

SDS-PAGE30% Acrylamidesolution29 g Acrylamide1 g Bis-acrylamideDissolved in 100 ml H2O.10% Sodium Dodecyl Sulfate (SDS):10 g SDS in 100 mlH2OResolving gel mix (12%) (15 ml): 4.89 ml H2O6 ml 30% acrylamide:bisacrylamide (29:1) mix3.8 ml 1.5 M Tris-HCl (pH 8.8) 150 μl 10% SDS 150 μl 10% APS 10 μl TEMEDStacking gel mix (3 ml):1.689 ml H2O500 μl 30% acrylamide:bisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8) 30 μl 10% SDS 30 μl 10% APS 10 μl TEMEDSDS loading buffer:130 mM Tris-HCl (pH 8.0) 20% (v/v)Glycerol 4.6% (w/v) SDS

Whole cell lysis buffer(Homogenizing buffer):50 mM Tris-HCl(pH 7.5)2 mM EDTA10 mM sodium fluoride*1 mM sodium orthovanadate*1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use

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

15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O pH was adjusted to 6.7 with 1 N KOH. MnCl2needsto beaddedseparately,drop by drop with stirring, tothe buffer. PIPES goes into solutionwhenpH is greater than 6.7. The solution, after pH adjustment to 6.7 was filter-sterilized and stored at -20ºC.Reagents for yeast transformation:1 M Lithium acetate (LiOAc)50% Polyethylene glycol10 mg/ml Carrier DNADimethylsulfoxide (DMSO)

INOUE transformation buffer:For bacterial DH5α ultra-competent cells preparation10 mM PIPES (free acid)

10 mM Tris-HCl (pH 8.0)1 mM EDTA Tris-Acetic acid EDTA (TAE) buffer:40 mM Tris base 0.5 M EDTApH was adjusted to 8.5 with glacial acetic acid.This was prepared as a 50 X stock solution and used at a 1 X concentration. Tris-Borate EDTA (TBE) buffer:90 mM Tris-borate 2 mM EDTA (pH 8.0) pH was adjusted to 8.3withHCl.This was prepared as a 10 X stock solution and used at a 1 X concentration.Both TAE and TBE were used asstandard gel electrophoresis buffers.HEPES buffer:This was used to prepare YNB medium of different pH.1M HEPESpH was adjusted to 7.5withNaOH.Bufferwas filter-sterilized and stored in an amber-coloured bottle. Citrate buffer(0.1M, pH 5.5):4.7 volume of 0.1 M Citric acid 15.4 volume of 0.1 M Sodium citrate

Phosphate-Buffered Saline (PBS):137 mM NaCl 2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3.This was prepared as a 10 X stock solution andused at a 1 X concentration.Tris-HCl buffer:0.5 M TrizmaBase pH was adjusted to7.6 using concentrated HCl.This was prepared as a 10 X stock solution andused at a 1 X concentration.Tris-EDTA (TE)buffer:

0.67% Yeast Nitrogen Base2% DextroseYeast Carbon Base (YCB):1.17% Yeast CarbonBase1% DextroseCAA:0.67% Yeast Nitrogen Base 2% Dextrose0.6% Casamino acids Plates weremade by adding 2% agar

Yeast Extract-Peptone-Dextrose (YPD):1% Yeast extract2% Peptone 2% DextroseYeast Nitrogen Base (YNB)

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

Bacterial medium

antibodies,anti-mouse IgG andanti-rabbit IgG conjugated with horseradish peroxidase (HRP) were obtained from Cell Signaling Technology, USA

All chemicals were purchasedfrom commercial sources. Mediacomponents for bacterial and yeast growthwere obtained from BD (Becton, Dickinson and Company, USA). Other chemicals were purchased from Sigma-Aldrich Co., USA. Materials used in recombinant DNA experiments were primarily obtained from New England Biolabs, Invitrogen, Bangalore Genei and MBI Fermentas. SuperScript™ III first-strand synthesis system was purchased from Invitrogen.MESA GREEN qPCR MasterMix Plus for SYBR®Assay was purchased from Eurogenetec. Kits used for plasmid isolation, PCR product purificationand DNAgelextractionwerefrom Qiagen.Radioactive chemical, ortho-P32-phosphoric acid,wasprocured from BRIT-Jonaki, CCMB, Hyderabad.Anti-Pma1 polyclonal antibody raised against S. cerevisiaePma1 was purchased from Santa CruzInc.,USA. Anti-phospho-p44/42 MAPK (Thr202/Tyr204) was purchased from Cell Signaling Technology, USA. Anti-CPY polyclonal antibody raised against S. cerevisiaeCPY was procuredfrom Thermo Scientific. Anti-Gapdh antibody raised against human Gapdh was purchased from Abcam. Secondary

All C. glabratastrains and plasmids used in this study are listed in Tables 2.1 and 2.2, respectively.Table 2.1: List of yeast and bacterial strains used in this study

activated TLC silicagel-60plate and transferred to theTLC chamber. After the solvent had migratedupwards (1.5 cm fromthetop), TLC plate was removed, air dried behind perspex shield, wrapped with cling plastic wrap and was exposed to phophorimager screenfor 2 h. Phosphorimager screen was scanned usingaFugi-FLA 9000 scanner

To resolvephospholipids,a TLC chamber was prepared by pouring50 ml developing solution and sealing the chamber with aluminium foil so that developing solution can generate vapor. TLC silicagel-60plate(Merck)was incubated at 80ºCfor 4 h for activation. After 30 min of TLC chamber preparation, phospholipidsextracted from C. glabratacells werespotted at thebottom (1.5 cm fromthelowerend) of the

PI-3kinase reaction was set up ina total volume of50μlin a 1.5 ml microcentrifuge tube as described below.PI-3 kinase reaction buffer = 25 μlSpheroplast lysate = 20 μl (equivalent to 10 μg protein)Sonicated phosphatidyl inositol = 5 μlReaction mix was incubated at 25ºC for 20 min and enzyme reaction was stopped by adding 80μlHCL (1N) solution. To extract phospholipids, 160 μl chloroform:methanol (1:1) was added to the reaction mix withcontinuous mixing. Organic phase containing phopholipidswas separated fromaqueous phase by centrifugation at 7,500g for 4 min at 4ºC and transferred to a new vial. Using vacuum evaporator apparatus, solvent was evaporated and phospholipidsweredissolved in 10 μl chloroform

PI-3 kinase reaction set up and phopsholipid extraction

10 mg phosphatidylinositol-sodium salt(from Glycine max)was dissolved in 2 ml chloroform to prepare a 5 mg/ml stock solution. This solution was prepared in a small glass vial aschloroformis known to reactwith polypropylene. Small aliquots of stock solution were madeand stored at -20ºC till further use. To avoid spillage due to vapor pressure, vials containing phosphatidylinositol-sodium salt solutionwereopened very carefully.To prepare sonicated phosphatidylinositolfor one PI-3 kinase reaction, 2 μlof the stock phosphatidylinositolsolution (10 μg) wastransferredtoanew1.5 ml microcentrifuge tube. Using vacuum evaporator apparatus, chloroformwas evaporated from the solution and phosphatidylinositol-sodium saltwas resuspended in 5 μl sonication buffer.For sonication, a total of 20 pulses, each of 30 sec with30 sec resting time weregiven on ice

A single colonyof desired C. glabratastrainwas inoculated in YPD-liquid mediumand grown for 14-16 h. 50 μl overnight culture was inoculated inYPD-liquid mediumfor 4 h. Log-phase-grownyeast cells were harvested,washedwith PBSandwereinoculated atinitial OD600of 2 and 4,into YNB-dextrose and YNB-sodium acetate liquid medium,respectively.After 4 hincubation,yeast cells were harvested by centrifugation at 2,500g for 5 minand treated with 1.2 M zymolyasefor 1 hto obtain spheroplasts.Post zymolyase treatment, spheroplasts were resuspended in 100 μl resuspension bufferandanequal amount of 0.25 mm glass beadswasadded to lyse the spheroplasts. Using bead beater apparatus, spheroplasts were lysed and protein concentration in spheroplast lysateswas determined usingbicinchoninic acid assay (BCA) method and samples were stored at -20ºC till further use

In vitroPI-3 kinase reactions wereset up to measure PI-3P synthesized as described earlier(Whitman et al., 1988)

Colony blot assay was performed to analyse secretion of carboxypeptidase Y(CPY)as described previously (Roberts et al., 1991). Single colony of a C. glabratastrain was inoculated in YPD medium andculture was grown till stationary phase. 0.1 OD600equivalent cellsfrom this culture were spotted on CAA medium,overlaidwith a nitrocellulose membrane and plate was incubated at30 ̊C for 18-20 h.Afterincubation, nitrocellulose membranewas washed with water to remove cells and membrane-bound CPYwas detected by immunoblotting with polyclonal anti-CPY antibody at a dilution of 1:10,000

was washed three times with PBS to remove non-adherantC. glabratacellsand Lec-2 cells were lysed in 5% SDS. Lysates were transferred totubes containing scintillation fluidand radioactive counts obtained were considered as ‘output values’. Percentage adherence wasdetermined using following formula

Adherence of C. glabratacells toLec-2 epithelial cells wasmeasured as described previously(Cormack et al., 1999).Lec2cells were seeded ina 24-well tissue culture plate at a seeding density of 5X105cells per well and allowed to adhere for 12 h. After 12 h,medium supernatant was discarded by inverting the plate in a reservoir and cells were washed thrice with PBS. Lec2 cells were fixed in 3.7% para-formaldehyde for 15 minfollowed by 2 PBS washes. PBS containing antibiotics, penicillin and streptomycin,was added toeach well of the 24-well plate and Lec-2 cellswere stored at 4°C.For adherence measurement,strains were taken out either on YPD or CAA mediumandgrown at 30°C for 2 days. Single colony of a C. glabratastrain wasinoculated in 10 ml CAA medium ina 100 ml culture flaskand allowed to grow at 30°C for 16-20 h. 100 μlyeast culture wasreinoculated in fresh 5 ml CAA liquid medium in a 15 ml polypropylene tube. 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN labelmix(JONAKI, India) was added to thetube and cultures were grown at 30°C for 16-20 h for radiolabeling of C. glabratacells. C. glabratacells from 1 ml culture were harvested and washed threetimes with PBS to remove residual S35(Met:Cys-65:25) labeling mix from medium supernatant. Next,cells were resuspended in 1 ml PBS. OD600was measured and cell suspensions of 0.5 OD600were prepared. PBS was aspirated out of the wells of 24-well plate containing fixed Lec-2 cells. 200 μl of S35(Met:Cys-65:25)-labeled C. glabratacell suspensions were added to each well. To determine the total amount of radioactivity present in labeled C. glabratacell suspension, 200 μl of S35(Met:Cys-65:25)-labeledC. glabratacell suspensions were transferred to a scintillation vial containing scintillation fluid. Radioactive counts present in this fraction were considered as ‘input values’. For measurement of yeast adherence to Lec-2 cells, plates were centrifuged at 1,000g for 5 min and incubated for 30 min at room temperature. Following incubation, each wel

Adherence assay

20 mg protein samples, isolated from RPMI-grown and macrophage-internalized yeast, were usedto measure KDAC activityusing HDAC Fluorimetric Assay/Drug Discovery Kit (EnzoLifeScience) as per manufacturer’s instructions

For protein extraction, yeast cells were suspended in 50-100μl protein extraction buffer containing 320 mM (NH4)2SO4, 200 mM Tris-Cl (pH 8), 20 mM EDTA (pH 8), 10 mM EGTA (pH 8), 5 mM MgCl2, 1 mMDTT, 10% glycerol and protease inhibitorsand disrupted using glass beads.Cell lysate was centrifuged at 7,500g and4oC for 15 min. 30 μg of total protein was resolved on a 15% SDS-PAGE gelat 32 mA till the dye front reachedthe bottom. Resolved proteins were transferred to Hybond-P membrane at 350 mA for 1.5 h in the cold room.Transfer of the proteins was visually confirmed by examining marker’s lane and membranes wereincubated in a small box for 2 h in 5% fat free milkprepared in 1X TBST for blocking. Blocking solutions were discarded and primary antibody, appropriately diluted in 5% fat free milkprepared in 1X TBST,was added to the box containing membrane. After overnight incubation in primary antibody, membranes were washed thrice with 1X TBST for 10 min. Membranes wereincubated for 2 h inappropriate secondary antibodydiluted in 5% fat free milkprepared in 1X TBST. Blots were either developedby chemiluminescence based ECL-Plus western detection system orChemidocTMgel imagingsystem. CgGapdhwas used as a loading control. To exclude the possibility of any contribution of THP-1 proteins tocell extracts prepared frommacrophage-internalized yeast, two control experiments wereperformed. First, we probedthe blots with antibodies specific for mammalian tubulin and actin.As expected, we neitherdetectedanysignal for mammalian actin nor formammalian tubulin. In the second control experiment, we treated macrophage lysates with proteinase-K prior to the yeast pellet disruptionand probed yeast lysates for different histone modifications.This proteinase-K treatmentdid not alter the epigenetic signature of C. glabratacells.Together, these data indicate that yeast protein samples were devoid ofany mammalian protein contamination