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

Serial dilution plating and spotting assay

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

Growth analysis and determination of generation time

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

Strains and culture conditions

Microbiological methods

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

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

Common buffers

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

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

Bacterial media

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

Strains and plasmids

Identification of selected micro-organism

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

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

Preliminary biochemical characterization of tannases from the potent tannase producers

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

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

Gallic acid estimation (Sharma et al., 2000)

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

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

Tannin estimation (Hagerman and Butler, 1978)

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

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

Protein estimation (Lowry et al., 1951)

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

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

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

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

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

Quantitative assay

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

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

Qualitative screening for tannase producer/s

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

Screening and selection of potential tannase producers

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

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

Collection of samples

Isolation of tannase producing microorganism/s

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

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

Chemicals and Reagents

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

Ethics statement

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

Spheroplast lysis method

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

Glass bead lysis method

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

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

Spheroplast lysis method

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

Glass bead lysis method

Yeast genomic DNA isolation

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

Strains and culture conditions

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

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

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

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

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

THP-1 monocytes getdifferentiated intophagocytic macrophages upon treatment with

PMA (Phorbol myristateacetate) treatment of THP-1 monocytic cells

For cryopreservation of THP-1 and Lec-2 cells, 5-6 million cells wereresuspendedin 0.5 ml of eithercommercially procuredcell preservation medium from GIBCO(12648010)or complete medium supplemented with 10 % fetal bovine serum and 10 % DMSO.Cells were initially kept inanisopropanol bath and werelatertransferred to -70°C freezer. After 2-3 days, frozencells were transferred to liquid nitrogen container till further use. To revive the cells, frozenstockswere taken out of the liquid nitrogen container and immediately transferred to water bath set at 37°Cfor thawing. When freezing medium has thawed completely, cells were transferred to a 100 mm cell culture dishcontaining 12 ml completemedium and incubated under tissue culture conditions at 37°C and 5% CO2for 12 h. Afterincubation, medium was replaced by 12 ml fresh pre-warmed medium and incubated under tissue culture conditions till they reached 70-80% of confluencebefore splitting

Cryopreservation and revival of cell lines

Human monocytic cell line THP-1 (ATCC TIB-202TM), derived form 1 year old acute monocytic leukemia patient,wasused to perform single cell infection assays. Differentiation of these THP-1 monocytic cellsto phagocytic cells was induced by using phorbol myristateacetate (PMA)(16 nM). Lec2 (ATCC CRL-1736TM),an ovary epithelial cell line,which has beenderived fromtheChinese hamster Cricetulus griseus, wasused to determine the adherence of C. glabratacells. THP-1 and Lec-2 cells were routinely cultured and maintained in RPMI-1640 and α-MEM media, respectively, supplemented with10 % heat inactivated fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml) andstreptomycin (100 μg/ml). Cultures were maintained in cell culture incubator (Thermo scientific)at 37°C and 5% CO2. Cultures were split after every 2-3 daysandspent medium was replaced with fresh pre-warmed medium. For splitting, cells were harvested at 1,000 rpm for 2-3 min, spent medium wasdiscarded and cell pellet was resuspended in 4-6 ml of pre-warmed medium. Cell density was determined by using hemocytometer. A total of3-4 million cells were seeded in a 100 mm cell culture dishcontaining 12 mlfresh medium

Cell lines and culture conditions

Animal cell culture methods

10 mg/ml carrier DNA 5 μlAbove-mentioned reagents were added to prepare thetransformation mixture, and the volumes indicated wereused per transformation.500-1,000 ng of desired transforming DNA was added to this transformation mixture and final volume was adjusted to 360 μl with sterile water.Carrier DNA (Sonicated salmon sperm DNA, Stratagene, 201190) washeat denatured at 95⁰C for 10 min and transferred on ice before additionto the transformation mixture.43 μl DMSO was added to each transformation mixture before heat shock.Zymolyase cocktail buffer for yeast colony PCR2.5 mg/ml zymolyase (MP Biomedicals, 0832092)1.2 M SorbitolThe cocktail was prepared in sterile water

Tris-acetic acid EDTA (TAE) buffer40 mM Tris Base0.5 M EDTAFinal pHof the bufferwas adjusted to 8.5 with glacial acetic acid.TAE buffer was prepared as a 50 Xconcentrate and diluted to 0.5X concentration prior to use as agarose gel electrophoresis running buffer and to cast agarose gels.HEPES [4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid] buffer1 M HEPESFinal pHof the bufferwas adjusted to 7.5 with NaOH.HEPES was used as a buffering agent forpreparationof different pHmedium. Buffer was filter-sterilizedby usinga0.22 μm membrane filterand stored at 4°C.INOUE transformation buffer10 mM PIPES15 mM CaCl2.2H2O250 mM KCl55 mM MnCl2.4H2OFor preparation ofINOUE transformationbuffer,above-mentioned solutes were dissolved in appropriate amount in 800 ml of water and then 20 ml of 0.5 M PIPES(piperazine-1,2-bis[2-ethanesulphonic acid])(pH 6.7) was added. Final volume was adjusted to 1 litre with water, buffer was filter sterilized by usinga0.22 μm membrane filter and stored at -20°C. Stock solution of PIPES was preparedseparatelyby dissolving 15.1 gm of PIPES in 80 ml of water, pH was adjusted to 6.7 using 5 M KOH and volume was adjusted to 100 ml.Yeast transformation reagents1 M lithium acetate 36 μl50 % polyethylene glycol 240 μl

Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4Final pH of the buffer was adjusted to 7.3with 11.6 N HCland volume was adjusted to 1 Lbefore autoclaving.PBS was prepared as a 10X stock solution and diluted to 1 X concentration before autoclaving.Tris-HCl buffer0.5 M Trizma BaseFinal pHof the bufferwas adjusted to 7.6 using 11.6 NHCl.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1 mM EDTATE buffer was prepared as a 10 X concentrate and diluted to 1 X concentration before use

Common buffers

2.5 mM KCl10 mM MgCl210 mM MgSO4SOCSOB mediumwas modified to prepare the SOC medium.20 ml of sterile 1 M glucose solution was added to the autoclaved SOB medium to obtainafinal concentration of 20 mM glucosein 1 litre of medium.AntibioticsAmpicillin 60 μg/mlKanamycin 30 μg/mlStock solution of antibiotics (50 mg/ml) were prepared in sterile water. Prior to storage at -20°C,antibioticswere filter sterilizedthrougha0.22 μm membrane filter. Before pouring the plates,antibiotics were added to moderatelywarm LB-agar medium

Luria Bertani (LB)0.5% Yeast Extract1% Tryptone1% NaClSuper Optimal Broth (SOB)0.5% Yeast Extract2% Peptone10 mM NaCl

Bacterial media

All C. glabrataand bacterial strains,and plasmids used in thisstudy are listed in Table 2.1 and Table 2.2, respectively.Table 2.1: List of C. glabrataand bacterial strains used in the study

Strains and plasmids

HEK293T packaging cellswere seeded at 30-40% confluency in 60 mm dishes. After 24 h, cells were co-transfected with three plasmids required for viral production i.e. VSV-G, psPAX2 (Addgene plasmid # 12260) and pLKO.1-puro-non-targeting and shIP6K1 clones using polyethyleniminereagent(PEI) and incubated at 37°C and 5% CO2 for virion formation. After 48 h, viral particles were harvested by collecting supernatant and filtered througha 0.45 μm syringe filter unit. Viral stock was aliquoted and stored at -80°C for further use. Viral titer was approximated on the number of cells plated for the production of lentivirus. Calculations were done as per Cell Bio Labs instruction. 2 x 106cells will yield 107infectious units/mL. All necessary precautions were taken while generating lentiviral particles such as wearing mask, double gloves, and sterile filter tips. All the consumables used were bleached (1% sodium hypochlorite solution) at least 1 h before being discarded

Generation of lentiviral particle containing shRNAagainst human IP6K1-

cells convert MTT to insoluble purple formazan dye crystals which can besolubilized using detergent or DMSO and measured by colorimetry. At each time point mediacontaining MTT dye (250 μg/mL) was added to each well and incubated at 37ºC. After 2 h cells were lysed with 200 μL DMSO for 15 min on a rocker at room temperature to solubilize the formazan and absorbance was monitored at 570 nm using the EnSpire multimode plate reader (PerkinElmer). Doubling time was determined by plotting log [A570] vstime and conducting a linear regression analysis using GraphPad Prism 5

Cells were seeded in 24 well plates at 10% confluence in triplicates and allowed to grow for different lengths of time. Spent medium was replaced with fresh medium every 48 h. Cells were incubated for different lengths of time ranging from 24 h to 120 h, to allow them to grow and cell survival was monitored by the MTT assay. Metabolically active

Cell proliferation assay

Other plasmids used for lentivirus generation: VSV-G, VSV-GP (gifts from Dr. Renu Wadhwa, AIST, Japan) and psPAX2 (a gift from Dr. Didier Trono, Addgene plasmid # 12260)

Plasmids

Table 2.2:Plasmids expressing shRNAagainst mouse Ip6k2used for generating stable cells in MEFs are listed below

Lentiviral vectors (pLKO.1)encoding various shRNA sequences against human IP6K1and mouse Ip6k2were obtained from Sigma-Aldrichto generate transient and stable knockdowns. shRNA clone IDs and their representation in the thesis are given below.Table 2.1:Plasmids expressing shRNA against human IP6K1used for generating stable cells in HeLa and HCT116

Fresh overnight cultures grown in LB containing appropriate antibiotics to select for plasmids were sub-cultured 1:100(or lower dilutions for some strains)in the same medium. The cells from these cultures weretaken for microscopy at exponential phase of growth(A600 of 0.5-0.6), as such or after concentrating the cells 10-fold

Sample preparation

vii.RNA buffer II from Ambion(1-2X Xylene cyanol + Bromophenol blue)used for loading the samples. RNA isolation for Northern blotting for lacZtranscript was done aftergrowing cultures till A600of 0.6 in LB in the presence or absence of 1mM IPTG at 30oC while for lacZ-lacYʹ-tRNA(U73)Arg5or lacZʹ-tRNA(U73)Arg5transcripts, cultures were grown in LBupto A600of 0.3 and induced with 1mM IPTG for 30 min followed by RNA extraction.30ml of 10% polyacrylamide gels of 1.5mm thickness were cast in the Broviga slab vertical gel electrophoresis apparatus. Gels were polymerizedby the addition of TEMED and APS (1/100th volume of gel mix). The gel was pre-runat 300V for 15-20 minutes prior to loading.Sample preparation for gel loading was done as follows. The normalizedamounts of RNA samplesto be analyzed were mixed with the equal volumes of 2X gel loadingbuffer II(Ambion)making a final concentration of 1X. The samples were then heated at 80 degrees in a thermoblock (eppendorf) for 10 minutes and loaded on the gel when still warm. The gel was run at constant voltage of 300Vfor 3-4 hours till xylene cynol covered 2/3rddistance

The following solutions were used to cast and run denaturing PAGE gels:i.40% acrylamidestock solution ii.7.5M Ureaiii.5X TBEiv.Ammonium persulphate (APS) stock: 10% (w/v) solution made fresh v.TEMED (N,N,N′, N′-tetramethyl ethylene diamine) vi.Gel running buffer (0.5X TBE)

Denaturing polyacrylamide gel electrophoresis of RNA

For high fidelity PCR, Herculase II fusion DNA polymerase (AgilentTechnologies)was used. Approximately 0.5μg of chromosomal DNAwas used as a template in a 50μl reaction volume

The PCRs were normally performed using Taqpolymerasefrom Roche or Fermentas. Approximately 1-5ng of plasmid or 5-100ng of chromosomal DNA was used as a template in a50μlreaction volume containing 200μM of each dNTP, 20pM each of the forward and reverse primers and 1 unit of Taq DNA polymerase. For colony PCR E. coli cells from a freshly grown plate were resuspended in 10μl of sterile Milli-Q water to get a cell suspension and this was used as a template in a PCR reaction at a final volume of 50μl. The samples were typically subjected to 30 cycles of amplification with the following general conditions: Initial denaturation 95ºC5minutes Denaturation 95ºC 1 minute Annealing 55ºC 1 minute Extension 72ºC 1 minute/kb of DNA template to be amplified Final extension 72ºC 10 minutes

Polymerase Chain Reaction (PCR)

following the manufacturer’s instructions. For genomic DNA, 1ml culture was used for DNA isolationusing Qiagen or Invitrogen kits. The quality of plasmid/genomic DNApreparations was assessed following electrophoresis on 0.8% agarose gels

3ml (for high copy number)or 10 ml (for low-copy number) of cells from an overnight culture were pelleted by centrifuging for 5 minutes at 6000rpm forthe plasmid isolation which was carried out with the commercially available kits (Qiagen or Invitrogen)

Isolation of plasmid and chromosomal DNA

Growth curves were generated to compare the growth rates of E. coli test strains with control strains manually. The appropriate dilutions of the overnight cultures in desired media were made and allowed to grow at required temperature till faint turbidity was visible. At this point samples were collected every 30 minutes until stationary phase was attained. The growth curves weregenerated using Microsoft Excel or SigmaPlot software and growth rates were calculated from the slope of the graph which, in turn, was used to calculate generation time

Estimation of growth rates

β-Galactosidase assay was performed according to(Miller, 1992).Cultures were grown to A600 of 0.4-0.6 from a 1:100 dilution of overnight cultures. Around 0.1-0.5 ml of culture was made up to 1 ml with Z-buffer and lysed with the addition of 100μl of chloroform and 50μl of 0.01% SDS solution. 0.2ml of freshly prepared 4mg/ml ONPG was added to start the reaction and incubated at 28oCtill the colour of the reaction mixture turned yellow. 0.5ml of 1M Na2CO3 was added to stop the reaction and the time duration from initial addition of ONPG to the stopping of the reaction was noted. The absorbance of reaction mix was taken at 420 nm (A420) afterspinning down the mix at 12000rpm for 3 minutes. The A600of the culturesused was also noted. The enzyme’sspecific activity (in Miller units) was calculated using the following equation: β-Galactosidase specific activity (Miller units) = (1000 ×A420) / t × v ×A600Where,‘t’ is the time period in minutes and ‘v’, the volume of culture used in ml

white colonies were recovered and purified to give growth. If the mutation caused synthetic lethality then white colonies (that lack the shelter plasmid) would not be observed since plasmid loss would result in growth arrest. Therefore, lethality was inferred when either white colonies were not recoveredor were recovered but failed to purify further

To determine whether a particular mutation conferred lethality in the ppGpp0or ΔdksAbackground, an assay was devised based on the use of an unstable, easy to cure shelter plasmidpRC7, similar to that described previously(Bernhardt & de Boer, 2004). In the wild-type strain carrying pRC7, this plasmid can be lost at a frequency of 20-30% in the absence of the selection. However, this will not be seen if the plasmid loss leads to cell death. Since the plasmid pRC7 confers a lac+phenotype, in the absence of the selection plasmid loss can be visualized on X-gal IPTG containing plates as white colonies in a Δlac strain whereas the colonies that retain the plasmid will appear blue.In order to carry outsynthetic lethal screen in the ppGpp0or ΔdksAstrains, the spoT or dksAgenes cloned in pRC7 under the control of lacpromoter were used. Theseshelter plasmids,namely,pRCspoT or pRCdksA, respectivelywere transformed into the ppGpp0or ΔdksAstrain. To test the synthetic growth phenotypes, the mutations of the genes to be tested were introduced by phageP1 transductions. The resultingstrains were grown overnight in LBcontaining the antibiotic selection for the shelter plasmid and IPTG for expression of spoTor dksA, subsequently washedin minimal A medium and dilutions(usually 10−5or 10−6) of these cultureswere spreadon X-gal and IPTG containing plates without antibiotic selection for the shelter plasmid. The phenotypes of the white colonies in comparison with the blue colonies were noted. Viability of the strains was inferred when

Blue-white screening for viability or lethality phenotype

or absence of a metabolite or a particular temperature. An EOP of ≤0.01 suggests lethality of the strain on the test medium. For strains carrying IPTG-dependent plasmids, EOP was determined by growing the strains overnight in medium containing IPTG and appropriate antibiotic,and spottingserial dilutions (100or 10–1to 10–6) on +IPTG (permissive) and –IPTG (test) plates to observe growth. Theviability is scored by takingratio of the colony forming units per ml (cfu/ml = No. of colonies × dilution factor × 1000/volume of culture spotted (in μl) obtained on the –IPTG plate to that on the +IPTG plate and determinesthe EOP. Likewise, strains carrying Ts plasmids were cultured overnight at 30°C with the appropriate antibioticand the serial dilutions of this culture were spottedat two temperatures 30°C (permissive) and 42°C (non-permissive or test). The ratio of cfu/mlobtained on the test temperature to that on the permissive temperature determined the efficiency of plating at the test temperature

Efficiency of plating (EOP) is a measure of the ratio of number of colonies (obtained from a given volume of a suitable culture dilution) on a test medium to those on a control or permissive medium, and is a measure of cell viability on the former. It is a very sensitive test and is often used for determining the viability of a strain in the presence

Efficiency of plating (EOP)

C. LBON temperature-sensitivityStrains were streaked on LBON agar plates and after an overnight incubation at42°C, growth was monitored (compared to that on LBON at 30°C as control). Absence of single colony growth was taken to reflect temperature sensitivity. D. In vivotranscription termination phenotypes The rationale for each phenotype is described in the relevant section. SMG-sensitivityThe E. coli relA mutants exhibit SMG-sensitive (SMGs) phenotype,that is,growth-inhibition in the presence of serine, methionine and glycine at 1mM concentration each(Uzan & 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 & Lawther, 1989).This test was therefore used to distinguish relA+from relA−strains. 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 40μg/ml and compared with the growth on non-supplemented glucose-minimal A plates to score for SMG phenotype. galEp3assayThis assay was used to test for relief of transcriptional polarity in the rho and nusG mutants. The galEp3 (galE490*) mutation represents a 1.3kb IS2 insertion in the gal leader region (between the promoter and structural genes of the galETKM operon). The mutation causes transcriptional polarity on the structural genes due to Rho-dependent transcription termination within IS2. In this assay, the gal operon expression in a galEp3 mutant or its derivatives was monitored by usingMacConkey galactose indicator plates (with 1% galactose), where Gal+colonies are red, and Gal−colonies are white. Therefore, the depth of color serves as an indicator of relative levels of gal expression, i.e., the extent of transcriptional polarity relief

A.lacZphenotype lacZ+colonies were distinguished from lacZ–colonies on X-gal containing plate or MacConkey lactose plate. X-gal is non-inducing colourless substrate of β-galactosidase enzyme which upon hydrolysis yields dark blue indolyl group and hence the lacZ+colonies on X-gal plate appear as dark blue colonies. Similarly, on the MacConkey agar plateslacZ+colonies appear dark pink whereas lacZ–colonies remain colourless. B. UV-sensitivityTo check the UV-sensitivity of the strains qualitatively, the strains were streaked on duplicate LB-agar plates and one of the plates was UV-irradiatedwith a 15-W UV-germicidal lamp at a distance of 70cm for 30 seconds. The UV-exposed and unexposed plates were incubated overnight in the dark after wrapping with aluminium foil and then growth was scored. This test could differentiate a recA–strain (UVs) from a recA+strain (UVr)

Scoring for Phenotypes

desired temperaturefor 45 minutes and plated on an appropriate selective medium at various dilutions. An aliquot of cell suspension to which plasmid DNA was not added served as a negative control. B. Inoue method i. Preparation of high efficiency competent cells Competent cells for high efficiency transformation were prepared by the method of (Inoueet al., 1990)with few modifications. An overnight culture of the strain (routinely DH5α) was subculturedinto fresh sterile LB broth in 1:100 dilutions and grown at 18ºC to anA600of 0.55. The cells were harvested by centrifugation at 2500rpm for 10 minutes at4ºC. Thesecells wereresuspended in0.4 volumes of INOUE buffer andincubated in ice for 10 minutes. The cells were recovered by centrifugation at 2500rpm at 4ºC for 10 minutes and finally resuspended in 0.01 volume of the same buffer. Sterile DMSO was added to a final concentration of 7%. After incubating for 10 minutes in ice, the cells were aliquoted in 100μl volumes, snap frozen in liquid nitrogen and stored at –80ºC. ii. Transformation protocolFor transformation, the required number of vials wasthawed on ice and the transformation protocol as described for CaCl2method was employed

A. Calcium chloride(CaCl2)method For routine plasmid transformation, following method which is a modification of that described by(Cohenet al.,1972)was used. An overnight culture of recipient strain was subcultured 1:100 in fresh LB medium and grown till mid-exponential phase. The culture was chilled on ice for 20 minutes, and the steps thereafter performed at 4ºC. 10 ml of culture was centrifuged and pelletwas resuspended in 5 ml of 0.1M CaCl2. After 5 minutes of incubation on ice, the cells were again centrifuged and resuspended in 1ml of 0.1M CaCl2. The suspension was incubated onice for 45 minutes. To the 100μl aliquot of the cellsuspension plasmid DNA (20-200ng in less than 10μl volumes) was added, incubated for 30-40 minutes on ice and given a heat shock for 90 seconds at 42ºC. The cultures were rapidly chilled for 1 minute, mixed with 0.9ml of LB broth and incubated at

Transformation

To 2 ml of fresh overnight culture of recipient strain, 108 pfu equivalent of phage lysate was added and incubated at 37ºC without shaking for 30 minutes to facilitate phage adsorption. The unadsorbed phage particles were removed by centrifugation at 6000 rpm for 5 minutes and the pellet ofbacterial cells was resuspended in 5 ml of LB broth containing 20 mM sodium citrate to prevent further phage adsorption. This was incubated for 25-60 minutes at desired temperaturewithout shaking to allow the phenotypic expression of the antibiotic resistance gene. The mixture was then centrifuged and the pellet was resuspended in 300 μl of 0.1M citrate buffer. 100 μl aliquots were spreadon appropriate antibiotic containing plates supplemented with 2.5 mM sodium citrate. A control tube without addition of P1 lysate was also processed in the same way. In the case of selection of nutritional requirement, the infection mixture was centrifuged, resuspended in 300 μl of 0.1M citrate buffer and plated without phenotypic expression

10–6)were mixed with 0.1 ml of fresh culture grown in Z-broth. After 30 min of adsorption at 37ºC without shaking, each mixture was added on a soft agar overlay of Z-agar plates and incubated overnight at 37ºC. The phage titrewas calculated from the number of plaques obtained on the plates as follows: Phage titre(pfu) per ml = No. of plaques ×dilution factor ×1000/vol.of lysate added (in μl)

Phage P1 lysate preparation by broth method

Phage P1 transduction

0.3 ml of overnight culture of the donor strain in Z-broth was mixed with 107plaque forming units (pfu) of a stock P1 phage lysate prepared on strain MG1655. Adsorption was allowed to occur at 37ºC for 30 minutes and the lysate was prepared by broth method. To 0.3 ml of infection mixture, 8-10 ml of Z-broth was added and incubated at 37ºC with slow shakinguntil the visible lysis of the culture occurred (in 4-6 hours). The lysate was treated with 0.2ml of chloroform, centrifuged and the clear lysate wasstored at 4ºC with chloroform.Quantitation of Plaque forming units (pfu)To quantitate the titreof P1 lysate preparation, titration was done using P1 phage sensitive indicator strain such as MG1655. 100 μleach of dilution of phage (typically 10–5,

Genetic Techniques

a-Genotype designations are as in (Berlyn, 1998).b-Strains DH5α, MC4100andMG1655 were from the laboratory stock collection.FRT-indicates FLP recognition target, a sequence that is recognized by FLP recombinase and is left as a scar at the indicated sites after excision of an antibiotic marker that is flanked by the FRT sites

The E. coli strains used in this study with their genotypes are shown in the table below. Bacterial strains were routinely stored on solid agar plates at 4ºC and also as thick suspensions in 20% glycerolat –80ºC. Plasmid harbouring strains were freshly prepared by transformation of the required plasmid. The bacteriophage P1kc was used for routine transduction to move a locus from one strain to another and is referred to as P1 throughout this thesis.Bacterial Strains Table 2.1: E. coli strains used and constructed in this study:

'That colour's very good on you,' she said, smiling professio­nally.Sayako said, 'I take it and also I take it in strawberry and navy and primrose.'3The manageress inwardly rejoiced. She would now reach this week's target.4 Her job would be safe for at least another month. God bless the Japanese!Sayako walked over on stockinged feet5 to a display of suede loafers.'And these shoes to match all suits in size four,' she said. Her role model was the fibreglass mannequin6 which lolled convincingly against the shop counter, wearing the same cream suit that Sayako was wearing, the loafers that Sayako had just ordered and a bag that Sayako was about to order in navy, strawberry, cream and primrose. The mannequin's blonde nylon wig shone under the spotlights. Her blue eyes were half closed as though she were encaptured by her own beauty.She is so beautiful, thought Sayako. She took the wig from the mannequin's head and placed it on her own. It fitted perfectly.'And I take this,' she said.She then handed over a platinum card which bore the name of her father, the Emperor of Japan.

'That colour's very good on you,' she said, smiling professio­nally.Sayako said, 'I take it and also I take it in strawberry and navy and primrose.'3The manageress inwardly rejoiced. She would now reach this week's target.4 Her job would be safe for at least another month. God bless the Japanese!Sayako walked over on stockinged feet5 to a display of suede loafers.'And these shoes to match all suits in size four,' she said. Her role model was the fibreglass mannequin6 which lolled convincingly against the shop counter, wearing the same cream suit that Sayako was wearing, the loafers that Sayako had just ordered and a bag that Sayako was about to order in navy, strawberry, cream and primrose. The mannequin's blonde nylon wig shone under the spotlights. Her blue eyes were half closed as though she were encaptured by her own beauty.She is so beautiful, thought Sayako. She took the wig from the mannequin's head and placed it on her own. It fitted perfectly.'And I take this,' she said.

ZFIN: ZDB-ALT-100915-1

ZFIN_ZDB-GENO-030919-1

ZFIN_ZDB-GENO-041129-1

ZFIN_ZDB-GENO-041129-1

ZFIN_ZDB-GENO-060207-1

ZFIN_ZDB-GENO-140812-1

ZFIN_ZDB-GENO-070316-1

ZFIN_ZDB-ALT-080321-1

ZFIN_ZDB-GENO-070118-1

ZFIN_ZDB-GENO-081008-1

RRID: ZFIN_ZDB-ALT-100831-1

ZFIN_ZDB-GENO-080111-1

RRID:ZFIN_ZDB-GENO-170727-1

ZFIN_ZDB-GENO-080918-1

RRID:ZFIN_ZDB-ALT-110308-1

RRID:ZFIN_ZDB-ALT-080514-1

ZFIN_ZDB-GENO-080528–1

ZFIN_ZDB-GENO-110720–1

Finally, and for the encouragement of people troubled with accents that cut them off from all high employment,

But if the play makes the public aware that there are such people as phoneticians, and that they are among the most important people in England at present, it will serve its turn.

Pygmalion needs, not a preface, but a sequel,

IammyselfastudentofIndiandia-lects

THEMOTHER.Howdoyouknowthatmyson'snameisFreddy,prayrTHEFLOWE

Cabwhistlesblowijigfranticallyinalldirections.Pedes-triansrunningforshelterintothemarketandundertheporticoofSt.Paul'sChurch,wheretherearealreadyseveralpeople,

CoventGardenat11.15p.m.Torrentsofheavysummerrain

Pygmalion:aRomanceinFiveActs:byBernardShaw

Pygmalion:aRomanceinFiveActs:byBernardShaw

Eons upon eons ago, when human life could last up to twenty thousand years, the Blessed One Kashyapa appeared in the world.

ancient story

previous century

Adipose tissue is no longer considered to be an inert tissue that stores fat. This tissue is capable of expanding to accommodate increased lipids through hypertrophy of existing adipocytes and by initiating differentiation of pre-adipocytes. Adipose tissue metabolism exerts an impact on whole-body metabolism. As an endocrine organ, adipose tissue is responsible for the synthesis and secretion of several hormones. These are active in a range of processes, such as control of nutritional intake (leptin, angiotensin), control of sensitivity to insulin and inflammatory process mediators (tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), resistin, visfatin, adiponectin, among others) and pathways (plasminogen activator inhibitor 1 (PAI-1) and acylation stimulating protein (ASP) for example). This paper reviews some of the biochemical and metabolic aspects of adipose tissue and its relationship to inflammatory disease and insulin resistance.

Drugs may also interact with genetic pathways of disease to elicit or exacerbate symptoms of the underlying conditions. In the porphyrias,

foolish imaginations of his heart

prayed unto the Lord, yea, even with all his heart

and his whole heart was filled

But an increasing hostility on the part of the non-slaveholding States to the institution of slavery, has led to a disregard of their obligations, and the laws of the General Government have ceased to effect the objects of the Constitution.

Questions for our first 1:1

RRID:IMSR_KOMP:VG12663-1-Vlcg

It will be observed that the basis of confederation now proposed differs from that of-the-United States in several important particulars. It does not profess to be derived from the people, but would be the Constitution provided by the Imperial Parliament; thus affording the means of remedying any defect, which is now practically impossible under the American Constitution.

Fra. This is most strange,That she, who even but now, was your best object,The argument of your praise, balm of your age,The best, the dearest, should in this trice of timeCommit a thing so monstrous, to dismantleSo many folds of favour: sure her offenceMust be of such unnatural degree,That monsters it: Or your fore-voucht affectionFall into Taint; which to believe of herMust be a faith, that reason without miracleShould never plant in me.

ng some text a

Import duty on air conditioners, refrigerators and washing machines under 10-kilogram capacity has been doubled to 20 percent each from 10 percent earlier. The basic customs duty on radial tyres is now 15 percent compared with 10 percent earlier,

It appears therefore that the only alternative which now offers itself to the inhabitants of Lower Canada is a choice between dissolution pure and simple, or Confederation on one side, and representation by population on the other. And however opposed Lower Canada may be to representation by population, is there not imminent danger that it may be finally imposed upon it, if it resist all measures of reform, the object of which is to leave to the local authorities of each section the control of its own interests and institutions. We should not forget that the same authority which imposed on us the Act of Union, or which altered it without our consent, by repealing the clause which required the concurrence of two thirds of the members of both Houses in order to change the representation respecting the two sections, may again intervene to impose upon us this new change.

In order to protect local interests, and to prevent sectional jealousies, it was found requisite that the three great divisions into which British North America is separated, should be represented in the Upper House on the principle of equality. There are three great sections, having different interests, in this proposed Confederation.

To the Upper House is to be confided the protection of sectional interests ; therefore is it that the three great divisions are there equally represented, for the purpose of defending such interests against the combinations of majorities in the Assembly.

But the very essence of our compact is that the union shall be federal and not legislative. Our Lower Canada friends have agreed to give us representation by population in the Lower House, on the express condition that they shall have equality in the Upper House. On no other condition could we have advanced a step ; and, for my part, I am quite willing they should have it. In maintaining the existing sectional boundaries and handing over the control of local matters to local bodies, we recognize, to a certain extent, a diversity of interests ; and it was quite natural that the protection for those interests, by equality in the Upper Chamber, should be demanded by the less numerous provinces.

the Lieutenant-Governor, who will enjoy the right of reserving the bills of the Local Parliament for the sanction of the Governor General, will be appointed by the Governor General in Council, that is to say, by the Federal Government, and, as a matter of course, it must be expected that he will act in conformity with the views of the Federal Government. Any bill reserved by him will require to be sanctioned by the Federal Government, which may refuse such sanction if they think proper, as they undoubtedly will as regards any bill the object of which might be to give responsible government to Lower Canada, whilst all the other provinces would only have governments which were not responsible. And the militia,—it will be exclusively under the control of the Federal Government. Have the honorable the French-Canadian members, to whom I more particularly address myself at this moment, reflected on the danger to us that is contained in this provision ?

My hon. friend from Wellington admits that under the Constitution we have the power to alter the constitution of this House in so far as it relates to Canada, but he says we are not authorized to extend our action to the other provinces, in a scheme of Federal union. That is begging the question. I answer his objection that any change affecting the elective principle is a breach of trust. Besides, we do not propose to enact a system of Government embracing all British North America We have not the power to do so. We merely propose to address Her Majesty on the subject. The Imperial Parliament alone has that power ; but if we have power without a breach of trust to alter the constitution of the Legislative Council of Canada (and my hon. friend admits this), then, certainly, we cannot be guilty of a breach of trust in suggesting a change embraced in a Constitution for the various provinces.

and dissent from others, of his opinions. The essential conditions of a valid trust to express particular opinions in Parliament are then wanting. The persons nominating him to his office, do not concur as to the opinions which he is to express. How then can a trust exist which it is impossible to define. The real trust imposed on the representative is co-extensive with those obligations, which alone the trust-makers can generally confer on him,—namely, to exercise his representative power honestly and discreetly. This argument, of course, assumes that the candidate has not defined his parliamentary obligations by unconditional pledges.

That the elective members had received a sacred trust to exercise ; that they were seut here by their constituencies to represent them, and to do that only. Under these circumstances, how could they conceive they had the power to vote away the rights of their electors? That was not their mandate, and if they did, they would be doing that which they had no authority to do ; they would be doing that which they could not do without going beyond the authority con faded to them. Now, it must be frankly admitted that if the hon. gentleman’s position’ be correct, then his objection would be fatal to any elected member giving his concurrence to the scheme of the Conference. But , hon. gentlemen, let us enquire what is the position of a representative. Two elements enter into the idea of representation— namely, power and duty. A representative derives the former from his constituents acting by their majority, under the Constitution. From what source does he derive the latter ? Obviously not from his constituents, because even the majority are not agreed on all points connected with the discharge of his duty. My hon. friend (Hon. Mr. SANBORN) has spoken of the position of a representative, as being that of a trustee. I shall quote from a very able work on the British Commonwealth, in which that position is, to my mind, very fully and very satisfactorily proved to be incorrect. Cox says :— Any trust, to be obligatory in conscience, must be defined by the self-same persons who appoint the trustee, or the person who is to fulfil the trust. His powers and duties must be derived from identically the same authority, for it obviously would be contrary to morals, as it is to law, that a man would be bound in conscience to exercise, in a particular way, powers delegated to him by several others, when trey themselves, while delegating those powers, differ as to the mode in which they are to be exercised. For, which of the different ways is the trustee to choose? By whom of those who appoint him is he to be guided in preference to the rest ? At the most he is bound to exercise his trust in a particular way in those particulars only respecting which the trust makers are agreed. Let us now apply this abstract principle of equity to the relations between a representative and his constituents. Regard him as their trustee. With respect to the source of his power there is no ambiguity ; it is derived from his constituents acting by their majority. But from whom does he derive the duty of expressing this or that opinion in Parliament ? In what particular are the trust-makers agreed? The very majority who voted for him are rarely, perhaps never, all agreed on any one point on which their opinions have been compared with his. Some of them differ from him on some points, some on others, but they all voted for him, from personal consideration, or because of their agreement with him on those points which they respectively deemed most important. In the minority, also, are probably some electors who assent to some,

HON. MR. CHRISTIE—A number of the representatives in the Federal Congress who voted for it were democrats, and without their concurrence and support it could not have been carried. Besides, that was only an amendment, not a revision of the Constitution. The Constitution of the United States was not the work of a party. The revision of the Constitution of the State of New York, in 1846, was not the work of a party I t is not desirable that any Constitution should be the work of a party ; in so important an undertaking all party spirit should be laid aside. (Hear, hear.) Why ? Because men of all parties are alike interested in the formation of a Constitution, and because in the construction of such an instrument , the collective wisdom of the leading men of all parties is needed. Besides, a Constitution so framed will be more likely, as my hon. friend from Wellington has so well said, to live in the hearts and affections of the people. (Hear, hear.) To show the good sense of our neighbors on this point, they do not give the revision of a Constitution—and the work of the Conference was a revision of our Constitution—to any party, but to men specially chosen for the purpose, irom all parties ; and I think the Governor General, and the Lieutenant-Governors of the Lower Provinces acted most wisely when they selected men of all shades of political opinion to compose this Conference and to prepare this Constitution, because all party views and feelings being laid aside, the whole object and motive of the members of the body was to devise a scheme which would best tend to promote the good of their common country. (Hear , hear.)

HON. MR. CHRISTIE—The hon. gentleman, I see, has not changed the ground which he took the other day, and which is precisely as I stated it. He thinks it would have been to the public advantage if this question had been taken up and discussed by a party. In this, in my judgment, he is entirely wrong; and I say he can find no instance of a constitution having been revised by a party.

The honorable gentleman very correctly stated the manner in which the Federal Constitution may be amended, but he is in error as to the mode in which state constitutions may be revised. One of the most important of the States revised its Constitution in 1846. I refer to the State of New York. The modus operandi on that occasion was as follows :—An act was passed in the State Legislature authorizing the electors at large to choose delegates to a convention, for the express purpose of revising the Constitution. The instrument passed by the convention was then submitted to the Legislature for approval ; but the Legislature had no power to alter it. It had either to be rejected or accepted as a whole. It was so accepted, none of the details being altered. My hon. friend will see that while the Conference was composed of leading representatives of the people in the various provinces, those conventions are composed of gentlemen elected by the people for that special purpose; and that the only difference between them is in the mode of selection. However, in both cases, all political parties are represented.

Whenever a majority of the House of Representatives shall deem it necessary to alter or amend this Constitution, they may propose such alterations and amendments, which proposed amendments shall be continued to the next General Assembly, and be published with the laws which may have been passed at the same session, and if two-thirds of each house, at the next ses-

sion of said Assembly, shall approve the amendments proposed, by yeas and nays, said amendments shall, by the Secretary, be transmitted to the town, clerk in each town in this State, whose duty it shall be to present the same to the inhabitants thereof, for their consideration, at a town meeting legally warned and held for that purpose ; and if it shall appear in a manner provided by law, that a majority of the electors present at such meetings shall have approved such amendments, the same shall be valid, to all intents and purposes, as a part of this Constitution. That is the way one of the oldest states guards the rights and liberties of its people. Then here is another extract from the Constitution of the State of Mississippi, one of the new states, showing how the people there are protected against hasty innovation :— Whenever two-thirds of the General Assembly shall deem it necessary to amend or change this Constitution, they shall recommend to the electors, at the next election for members of the General Assembly, to vote for or against a convention; and if it shall appear that a majority of the citizens of the state, voting for representatives, have voted for a convention, the General Assembly shall, at their next session, call a convention, to consist of as many members as there may be in the General Assembly, to be chosen by the qualified electors in the manner, and at the times and places of choosing members of the General Assembly ; which convention shall meet within three months after the said election, for the purpose of revising, amending, or changing the Constitution.

alluded the other day to the conservative feature of the Senate in the United States, in allowing the same representation to small states as to the larger states. But this does not at all affect the general arrangement, because the large majority are large states. But while my honorable friend approves of this portion, he should have expressed an opinion on the whole system. In the United States, no change of Constitution can be effected without the consent of two-thirds of both branches of the Legislature, and that must afterwards be sanctioned by three fourths of the state governments. This is a conservative feature also.

We have had the Constitution of 1841 altered more than once—twice at least—since the union. If we find that some parts of the machinery do not work—if, after the establishment of the Confederation, we find some little error has been made—-we will then, no doubt, have power and authority also to alter it.

Smart Cities: Stockholm Royal Seaport Project Update

Urban Green Space

Last week Stefka Wiese from GreenGasDrive was with us. The startup wants to make GreenCNG available nationwide for public and private fleet customers in the CNG filling station network. It should be made climate neutral, especially from waste biogas. In addition, GreenGasDrive advises on upcoming investment decisions and utilization optimization

The DIGINET-PS research project involves the development and implementation of a connected urban infrastructure test field along a stretch of Straße des 17. Juni between Ernst-Reuter Platz and the Brandenburg Gate, designed to advance and validate automated and autonomous driving and its broad range of individual areas under real-life traffic circumstances. The test infrastructure is intended to provide central, regional and supra-regional companies and R&D institutes with the opportunity to field test automated driving within an authentic urban setting. This will establish Germany’s first pilot project to build a digital test field for automated driving in highly complex traffic situations. DIGINET-PS will contribute to implementing Berlin’s smart city strategy in the area of mobility and will also support the state’s Digital Agenda.

The ELEKTRA research project seeks to develop an energy-efficient, hybrid-powered inland push boat. The project will demonstrate hydrogen and fuel cell technologies in combination with accumulators on inland waterway vessels in order to test the technology under operating conditions. Focus AreasElectric heavy-cargo shipsBZ drive

The research project eMobility-Scout deals with the conceptualization, implementation and testing of a cloud-based IT solution for the operation of electric vehicles and for sharing charging infrastructures among different businesses. In doing so, eMobility-Scout is considering the interest shown by many companies and institutions for simple and smart integration of electric vehicles into their fleets. Solutions for diverse areas of application in commercial mobility are being developed and tested, while taking logistical, economic and ecological aspects into account. Experienced project partners and affiliates, as well as renowned end users, are contributing extensive knowledge to the project. The results of the research project for IKT EM III should also gain substantial recognition in the public sphere.

A fully integrated logistics concept to establish environment and climate friendly supply chains in urban districts is going to evolve from the project. Co-creation processes, which are supported by online tools, are being used to develop the concept. The development and testing of sustainable business and deployment models is the goal of Distribut(e) and this will be addressed through the development of a shared e-logistics system in Klausenerplatz and at Mierendorff-Insel. The system will relate to a specific district (Kiez) in Berlin. A digital dialogue platform will be set up for the purpose of ordering local goods and for the booking of cargo bikes. Other low-threshold service offerings will be developed using Urban Design Thinking and will be integrated into the operating model.Focus Areas Integrated shared e-logistics at Mierendorff-Insel and at KlausenerplatzUrban Design Thinking: Formats for the co-creation process for sustainable urban district logisticsEfficiency enhancement in the delivery of goods in the ‘last mile’Mobile and online participation: Promotion of local production chains and activation of local stakeholders such as small to medium sized companies and local residentsIdentification of new sharing and value-in-use conceptsHigher standards in terms of the transparency, sovereignty, security and efficiency of data on the online platform

if any Legislative Councillor shall, for two consecutive sessions of Parliament, fail to give his attendance in the said Council, his seat shall thereby become vacant.

4

Charlotte's Web

I am very careful to make sure the books I have come from reputable publishers, I read everything before I put it on the shelf, and I am watching out for your kids,”