appropriate secondary antibody (conjugated with horse-radish peroxidase) diluted in 5% fat free milk solution (in PBST) and incubated for 45 minutes at room temperature. After incubation the membrane was washed and processed for the detection of protein bands using ECL-plus detection reagent (Amersham Biosciences) followed by detection of signal on X-ray film (Hyperfilm-ECL, Amersham Biosciences)

Site Directed Mutagenesis

The proteins were resolved using denaturing SDS-PAGE gel and after completion of the run, the gel was over laid on a nitrocellulose paper cut to the size of gel and kept in the blotting cassette in the presence of blotting buffer. Finally the cassette was put in the mini transblot apparatus (Bio Rad) and blotting was done for 4 hours at a constant voltage of 60 V. Then the membrane was taken out and rinsed in PBS containing 0.1% Tween - 20 (PBST) for 5 minutes by gentle shaking. Later the membrane was immersed in 5% non-fat milk solution in PBST with gentle shaking for 1 hour at 37°C. The membrane was washed off from the traces of the fat free milk with PBST and the membrane was over laid with primary antibody diluted in PBST for 3 hours at 4°C with shaking. After incubation the membrane was washed with PBST and layered with

Immunobloting

For adherent cells from which lysates have to be prepared , culture medium was removed and cells were washed with ice cold 1X PBS twice and then scraped with cell scraper in Cell Lysis buffer. Cells were rotated at 4°C for 30min at cold room and centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected and protein concentration was estimated using BCA assay. For standard western, 50-70μg of protein was loaded on to the gel

Lysate Preparation for Immuno- blotting

DTT. Then contents were then mixed and 1μl (200 units) of M-MLV was added. The mixture was then incubated at 37°C for 50 minutes. The reaction was stopped by incubating the mixture at 70°C for 15 minutes. The cDNA thus prepared was then used as a template for PCR. The expression of the investigated genes was determined by normalizing their expression against the expression of actin or GAPDH gene

Semi-quantitative RT-PCR

μg of total RNA was reverse-transcribed using poly-T oligonucleotide and M-MLV reverse transcriptase (Invitrogen) according to manufacturer’s protocol. Briefly, a 20μl reaction volume was made for 1μg of RNA. In a microcentrifuge tube, 1μl oligo (dT)(500μg/ml) , 1μg total RNA, 1μl 10mM dNTP mix and sterile water was added to afinal volume of 13μl. The mixture was then incubated at 65°C for 5 minutes ad quickly chilled on ice. To this mixture were added 4μl of 5X first strand buffer and 2μl of 0.1M

Total RNA was isolated by TRIzol method using the manufacturer’s protocol. Briefly, medium was removed, from 35mm dish and 1ml to TRIzol was added directly to the dish and kept at room temperature for 5 minutes. The cells were harvested by pipetting up and down three four times and transferred to a 1.5ml microfuge tube. For each 1mlTRIzol, 200μl of chloroform was added and tubes were shaken vigorously for 10 seconds to completely dissociate the nucleoprotein complexes, followed by vortexing for about 30 seconds. The mixture was kept for 3-5 minutes at room temperature and then centrifuged at maximum speed for 10 minutes. The upper aqueous phase was transferred into a fresh microcentrifuge tube and 500 μl of isopropanol was added and this was incubated at -20°C for 1 hour. The RNA was pelleted by centrifugation at maximum speed for 30 minutes at 4°C. The supernatant was decanted and the pellet washed with 1ml of 70% ethanol followed by a second wash with 1ml of 90% ethanol and centrifugation at maximum speed for 10 minutes. The supernatant was removed and the pellet air-dried for about 5 minutes and re-solubilized in 30-50 μl RNase free deionized (DEPC-treated Milli-Q) water and aliquots were stored at -70°C

Total RNA isolation from cultured mammalian cells

Transient transfection of plasmid DNA in culture cells was performed using Lipofectamine 2000 according to manufacturer’s protocol. Briefly, forty million cells were seeded in a 35mm tissue culture dish, one day before transfection. Transfection was performed 18-24 hrs after seeding the cells. 4μg DNA was mixed in 50μl of Opti-MEM in one eppendorf tube. In another tube, 5μl of Lipofectamine 2000 was diluted in 50μl Opti-MEM and incubated at room temperature for 5 minutes. After five minutes, DNA and Lipofectamine 2000 were mixed together and complexes, incubated for 30 minutes at room temperature. Meanwhile, the adherent cells were washed twice with PBS and 1ml of Opti-MEM was added. 100μl of complexes were then added to each dish containing cells and medium. After 6hrs, the medium containing complexes was removed and complete medium was added and transgene expression was accessed 24-48 hrs after transfection

Transient transfections in adherent cells

The quantity and purity of nucleic acids was determined by measuring the absorbance at 260 and 280 nm. The concentration of nucleic acids was calculated by taking 1 OD 260= 50 μg/ml for DNA, 40 μg/ml for RNA and 33 μg/ml for single stranded oligonucleotides. The purity of nucleic acids was checked by their A260/A280 ratio

Spectro-photometric estimation of nucleic acids

ethanol has dried. The pellet was resuspended in 20 μl of milliQ water and 20 μg/ml RNase added. The tube was incubated at 50°C for 45 min. the tube was vortexed for few seconds. Quality of the plasmid DNA was then accessed by running 1% agarose gel.

stored. To prepare competent cells pre-inoculum was prepared. A single bacterial colony was picked from LB agar plate, inoculated into 3 ml LB medium, and incubated overnight at 37°C temperature with shaking at 200 rpm. 1% of this pre-inoculums was sub cultured in 100 ml LB-broth and incubated at 18°C with shaking until OD at 600nm reached 0.5 - 0.6 (approx.). Culture was kept on ice for 10 min. with constant shaking.Cells were pelleted by centrifugation at 2000 g at 4°C for 8 min. Pellet was resuspended in 40 ml of ice-cold TB buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000 g at 4°C for 8 min. Pellet was resuspended in 8 ml of TB buffer in which final concentration of DMSO was 7% and left on ice for 10 min. 100 μl aliquots were made and snap frozen in liquid nitrogen and stored at -80°C

All the salts (10 mM PIPES, 15 mM CaCl2.2H2O, 250 mM KCl, 55 mM MnCl2.2H2O) except MnCl2 were dissolved in milliQ water and pH was adjusted to 6.7 with 1N KOH. MnCl2 was dissolved separately in mill Q water. MnCl2 was added drop wise while stirring (MnCl2 if added directly will give a brown color to the solution and precipitate out, hence it needs to be dissolved separately). Solution was then filter sterilized and

Overnight Grown culture was pelleted by centrifugation at 10,000g at 4°C for 3 min and the supernatant was discarded. Pellet was resuspended in 250 μl of ice-cold alkaline lysis solution 1. 300 μl of alkaline solution 2 was then added and the tube was inverted gently 3-4 times and incubated at room temperature for 5 min. 350 μl of ice cold solution 3 was added and mixed by inverting the tube rapidly for 3 or 5 times. Suspension was incubated on ice for 10 min. Bacterial lysate was spun at 10,000g for 12 min at 4°C. Supernatant was transferred to a fresh tube. 0.4 volume of phenol: chloroform was added to the supernatant and the contents mixed. It was then spun at 10,000g at 4°C for 12 min. Aqueous phase was taken out in a fresh tube and 0.6 volume of isopropanol was added, mixed properly and incubated at room temperature for half an hour followed by spinning at 10,000g at RT for 20 min. Supernatant was discarded. Pellet was washed with 70% ethanol. The tube was stored at room temperature until the

Ultra Competent Cells Preparation

Preparation of Plasmid DNA by alkaline lysis

Experimental Procedures

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

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

Activation of CM10 (weak cation exchange ) array

d. 3 μl of sample prepared in low stringency buffer was added to the spot activated with low stringency buffer and incubated in humid chamber for 30 min. and removed using whatman strips. (same protocol was repeated for the samples prepared in high stringency buffer on spots activated with high stringency buffer). e. Stringent washings were given to each spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH7.0 for 30 sec and removed using Whatman filter paper strips. f. 1-2 μl of SAP matrix was added to each spot and allowed to dry. g. The chip was then placed in the SELDI machine

One set of cell extracts was prepared in low stringency buffer by mixing cell extracts and low stringency buffet in 1:1 ratio and another in high stringency buffer. b. 10 μl of low stringency/high stringency buffer was added to the spots on the chip and incubated in a humid chamber for 5 min. c. Buffer was removed using Whatman strips without touching the spot surface. This step was repeated once

Activation of LSAX (strong anion exchange ) array

Activation of H50 protein chip array

b. 5 μl of ACN + TFA (25% ACN in PBS + 0.1% TFA) was added to the spot surface and removed after 30 sec. c. 5 μl of cell lysate sample was then spotted on the chip and kept in a humid chamber for 30 min. d. Stringent washes were given by spotting 5 μl water on the spot surface for 30 sec and removing using Whatman filter paper strips. This was followed with a 25% ACN wash or three washes with 25% ACN or 50% CAN or 75% ACN. e. Washing was performed by spotting 5 μl of water for 30 sec followed by removal using Whatman filter paper strips. f. Dried chip at room temperature. g. 1-2 μl of SAP matrix (5 mg of matrix + 200 μl ACN + 200 μl of 1% TFA) was then spotted on the chip surface and allowed to dry. h. The chip was then placed in the SELDI machine

5 μl of water was added to each spot on the chip and removed after 30 sec using Whatman filter paper strips. Care was taken not to touch the spot surface. This step was repeated once

5 μl of 0.1% TFA was applied to the spots on the SEND array and removed after 30 sec using Whatman paper (care was taken not to touch the spot surface). b. 5 μl of cell lysate sample was spotted on the SEND array and incubated in a humid chamber for 10 min. Removed after 30 min. c. 5 μl of 0.1% TFA was then added and removed after 30 sec. d. 2 μl of 25% ACN in 0.1% TFA was added to the spots and allowed to dry. e. The chip was then placed in the SELDI machine

Activation of SEND arrays for peptide analysis

Trypsinization: The decolourized bands were dried in a vacuum dryer for 1 hr until the gel pieces were completely dry. 5 μl of 0.1 μg/μl trypsin and 25 μl of 25 mM NH4HCO3 (pH 8.0) were then added to the dried gel pieces. The tubes were sealed with parafilm and kept in a water bath at 37 ̊C, overnight. Care was taken that the gel pieces in the tubes did not dry up. If the gel pieces got dried, 25 μl of NH4HCO3 was added on top. Peptide extraction: A 1:1 mixture of ACN:5% TFA in water was added (30 μl) to overnight tryptic digests and kept for 30 min. The elutant was removed in a separate low binding tube. The extraction step was repeated once more. The elutant was then dried in a vacuum dryer (1-2 hr) and reconstituted in 5 μl of 25% ACN in 0.1% TFA

Destaining of gel bands: The protein bands of differentially expressed proteins were cut out from the gel and put in low binding microfuge tubes. 150 μl of 50:50 Acetonitrile:Ammonium bi carbonate pH 8.0 (NH4HCO3) was then added and kept under shaking for 30 min. Coloured liquid was discarded and the washing step repeated until the bands decolourised

Destaining of gel, trypsinization and peptide extraction

12% resolving gel (for 25 ml)Water = 8.2 ml 30% Acrylamide = 10.0 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.01 ml 5% stacking gel (for 10 ml)Water = 6.8 ml 30% Acrylamide = 1.7 ml 1.5 mM Tris (pH 6.8) = 1.25 ml 10% SDS = 0.1 ml 10% APS = 0.1 ml TEMED = 0.01 ml

A double cylinder gradient former was used with 12% poly acrylamide gel mix in the inner cylinder and a 3% polyacrylamide gel mix in the outer cylinder that was stirred using a magnetic bead on a magnetic stirrer. A pump was connected to the flow tube and the flow rate adjusted at 5-8 to cast a 12-3% gradient gel. A 5% stacking gel was used. After the protein samples were run on the gradient gel, it was stained in instant blue over night under shaking. 3% resolving gel (for 25 ml)Water = 15.68 ml 30% Acrylamide = 2.5 ml 1.5 mM Tris (pH 8.8) = 6.3 ml 10% SDS = 0.25 ml 10% APS = 0.25 ml TEMED = 0.02 ml

Casting a gradient SDS-polyacrylamide gel

1μl of the cell lysate was mixed with 200 μl of 5X Bradford reagent and 800 μl of water. O.D was measured at 595 nm. Standard curve of BSA was plotted using various dilutions of BSA protein by Bradford method. Protein estimation of the cell lysate samples was performed using the standard curve equation y=0.0695x + 0.0329 μg/μl

Protein estimation by Bradford method

microfuge tubes and snap frozen in liquid nitrogen and were stored at ─80 ̊C. Protein estimation was performed simultaneously with one of these aliquots

The strains were grown to stationary phase in 500 ml LB supplemented with ampicillin (100 μg/ml) overnight. Cells were pelleted at 2100g for 30 min at 4 ̊C and dissolved in 5 ml of 1X PBS with 2X protease inhibitor and 3 mM DTT. Cells were lysed using French Press at 1500 psi for three cycles. The lysed cells were pelleted at 20,000g for 45 min at 4 ̊C. Clear supernatant was collected in sterile 2 ml

Preparation of cell extracts

These experiments were undertaken in the laboratories of Dr. Sylvie Rimsky and Dr. Malcolm Buckle at the Ecole Normale Superioure, Cachan, Paris (France)

Methods for SELDI (Surface Enhanced Laser Desorption/Ionization)

the membrane and sandwiched between 1 piece of buffer-soaked Whatman paper from Biorad on each side. The sandwich was placed between graphite electrodes with the membrane towards the anode. The transfer was done for 12-16 hr using a voltage of 40 V in the cold room. Protein transfer was viwed using Ponceau S staining. Blot was dipped in the Ponceau S stain under shaking and washed using PBST. After transfer, the membrane was blocked with 5% non-fat milk in PBST (1X PBS with 0.1% Tween-20) for 2 hr at room temperature. The membrane was then washed thrice with PBST under shaking and incubated with the primary antibody (1:1000 dilution in PBST) for 12-16 hr. The membrane was again washed thrice under shaking with PBST and incubated with 1:20000 dilution alkaline phosphatase conjugated anti-goat IgG secondary antibody (in PBST) for 2 hr. The membrane was once again washed thrice as described above and the signal developed using ECL kit from Amersham on X-ray films in a dark room. The reactive protein bands appeared as black bands upon gentle shaking at room temperature in 1X developer solution. The reaction was stopped by dipping and shaking the film in 1X Fixer solution followed by washing in water

The protein samples separated on SDS-PAGE were transferred to PVDF (polyvinyledene difluoride) membrane (Amersham, Buckinghamshire, UK) electrophoretically by a semi-dry method using BioRad apparatus. The gel and the membrane (pre-wetted with methanol) were wetted with transfer and the gel was placed in contact with

Western blotting with anti-Rho/anti-NusG antibody

(ii) Stacking gel buffer: 1.0 M Tris-Cl pH 6.8 (iii) Resolving gel buffer: 1.5 M Tris-Cl pH 8.8 (iv) SDS stock: 10% (w/v) solution (v) Ammonium persulphate (APS) stock: 10% (w/v) solution made fresh (vi) Gel running buffer (1X) (vii) Loading dye (6X): (viii) Lysis buffer (RIPA) Gels of 1.5 mm thickness were cast in the Biorad small gel apparatus. Resolving gel of 10% (10 ml) was made by mixing 4.2 ml 10% acrylamide, 3.1 ml water, 2.5 ml of 1.5 M Tris-Cl pH 8.8 and 0.1 ml of 10% SDS. Stacking gel (2 ml) was made by mixing 0.33 ml of 30% acrylamide, 1.4 ml of water, 0.25 ml of 1 M Tris-Cl pH 6.8 and 0.02 ml of 10% SDS. Gels were polymerized by the addition of TEMED (N,N,N′, N′-tetramethyl ethylene diamine) and APS (1/100th volume of gel mix). Sample preparation for gel loading was done as follows. Mid log and late log phase 10 ml cultures were centrifuged at 26000g and the cell pellet was resuspended in 0.5 ml RIPA buffer. Cells were sonicated on ice for 1 min at output power of 5 to get a cleared lysate. The culture lysate was centrifuged at 26000g to recover the clear supernatant. Total cell protein was quantified in the lysates using BCA kit reagents (BioRad) using the manufacturers protocol. Appropriate volume of cell lysate was mixed with the loading dye in a final concentration of 1X and loaded onto the gel. The gel was run at constant voltage of 60 V for stacking and 80 V for resolving gel

The method followed was as described in Sambrook and Russell (2001). The following solutions were used to cast and run SDS-PAGE gels. (i) Acrylamide stock: 29% (w/v) acrylamide and 1% N,N′-methylene bisacrylamide

Sodium dodecyl sulphate-polyacrlyamide gel electrophoresis (SDS-PAGE)

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits on an automated sequencer following the manufacturer's instructions by either CDFD or an outsourced sequencing facility

DNA sequencing

Inverse PCR is a technique to amplify unknown regions flanking the site of transposon insertion using the primers designed from the known sequence from one end of the transposon element. Genomic DNA was digested with a 4-base recognition restriction enzyme, Sau3A1 followed by intramolecular ligation set up at high dilutions. These ligated molecules were then used as templates for the PCR performed with a pair of divergently-oriented primers designed from one end of the transposon named AH1-AH2. The PCR product thus obtained was sequenced with the same set of primers to identify the junction sequence at the site of transposon insertion and hence the identity of the gene disrupted in each case. Typical PCR conditions used were as follows:- Annealing 55°C 2 min Elongation 72°C (1 min/kb of DNA template to be amplified) Denaturation 95°C 2 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C

Inverse PCR

Denaturation 95°C 1 min After 30 cycles of PCR, the final elongation step was carried out again for 10 min at 72°C

The PCRs were normally performed using a PCR amplification kit from Fermentas/Sigma (USA), following the company's protocols. Approximately, 10 ng of chromosomal or 1-2 ng of plasmid DNA was used as template in a 50 μl reaction volume containing 0.2 mMM of each dNTP, 20 picomoles each of forward and reverse primer and 0.5 units of Taq DNA polymerase. In some cases, freshly streaked E.coli cells from a plate were resuspended in 50 μl of sterile Milli Q water to get a cell suspension (~ 109 cells/ml) and 10 μl from this was used as the source of DNA template. The samples were subjected to 30 cycles of amplification and the typical conditions of PCR were as follows (although there were slight modifications from one set of template/primers to another): The initial denaturation was done at 95°C for 3 min and the cycle conditions were as given below. Annealing 55°C 1 min Elongation 72°C (1 min/kb of DNA template to be amplified)

Polymerase chain reaction (PCR)

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

Preparation of ultracompetent cells

Overnight cell culture raised in LB medium was subcultured 1:100 in LB with 20 mM MgCl2. When the A600 reached 0.4-0.6, the culture was centrifuged at 2800g for 5 min at 4 ̊C. To the cell pellet 0.4 volumes of ice-cold TBF-I buffer was added and incubated on ice for 15 min. The cell suspension was centrifuged at 2800g for 5 min at 4 ̊C and the cells recovered were dissolved in 0.04 volume of ice-cold TBF-II buffer and kept on ice for 45 min. 100 μl aliquots of these competent cells were used for transformation using the normal transformation protocol

TBF method for preparation of high competency cells

For routine plasmid transformations, where high efficiency is not required, the following method which is a modification of that described by Sambrook and Russell (2001) was used. An overnight culture of the recipient strain was subcultured in fresh LB and grown till mid-exponential phase. The culture was chilled on ice for 15 min, and the steps hereafter were done on ice or at 4°C. The culture was centrifuged, and the pellet was resuspended in one third volume of cold 0.1 M CaCl2. After 15 min incubation on ice, the cells were again recovered by centrifugation, and resuspended in one tenth volume of cold 0.1 M CaCl2. The suspension (0.1 ml) was incubated on ice for 1 h after which DNA was added (~10-100 ng of DNA in less than 10 μl volume). The mixture was again incubated on ice for 30 min, and then heat shocked for 90 seconds at 42°C. Immediately 0.9 ml of LB broth was added to the tube and incubated at 37°C for 45 min for phenotypic expression of the antibiotic marker before being plated on selective medium at various dilutions. A negative control tube (with no plasmid DNA addition) was also routinely included in each of the experiments

Calcium chloride method

Transformation protocols

Typically, 100-200 ng of DNA was used in each ligation reaction. The ratio of vector to insert was maintained between 1:3 and 1:5. The reactions were usually done in a 10 μl volume containing ligation buffer (provided by the manufacturer) and 0.05 Weiss units of T4 DNA ligase, at 16°C for 12-16 hr

Ligation of DNA

DNA fragments to be used for ligation were eluted from the agarose gel, after the electrophoresis. The gel piece containing the desired band of DNA was sliced out and the DNA was purified from the gel using the purification kits available for this purpose. The efficiency of elution was determined by checking a small aliquot of DNA sample on the gel

Purification of DNA fragments by elution from the gel

0.5-1 μg of DNA was used for each restriction enzyme digestion. 2-4 units of the restriction enzymes with the appropriate 10X buffers supplied by the manufacturers were used in a total reaction volume of 20 μl. The digestion was allowed to proceed for 6 h or 10min. (for FAST digest enzymes) at the temperature recommended by the manufacturer. The DNA fragments were visualized by ethidium bromide staining following electrophoresis on 1-1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments

Restriction enzyme digestion and analysis

The DNA samples were mixed with the appropriate volumes of the 6X loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 30% glycerol in water) and subjected to electrophoresis through 1-1.5% agarose gel in either 1X TBE or 1X TAE buffer. The gel was stained in 1 μg/ml of ethidium bromide solution for 30 min at room temperature and the bands were visualized by fluorescence under UV-light

Agarose gel electrophoresis

Quiagen/HiPura following the manufacturer's protocols

The rapid alkaline lysis method of plasmid isolation, as described by Sambrook and Russel (2001), was followed with minor modifications. Bacterial pellet from 3 ml of stationary-phase culture was resuspended in 200 μl of ice-cold solution I (50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA pH 8.0 containing 1 mg/ml lysozyme) by vortexing. After 5 min incubation at room temperature, 400 μl of freshly prepared solution II (0.2 N NaOH, 1% SDS) was added and the contents were mixed, by gently inverting the tube several times. This was followed by the addition of 300 μl of ice-cold solution III (5 M potassium acetate, pH 4.8) and gentle mixing. The tube was incubated on ice for 5 min and centrifuged at 20,0000g for 15 min at 4°C. The clear supernatant was removed into a fresh tube and, if required, was extracted with an equal volume of phenol:chloroform mixture. The supernatant was precipitated with either two volumes of cold 95% ethanol or 0.6 volumes of isopropanol at room temperature for 30 min. The nucleic acids were pelleted by centrifugation, washed with 70% ethanol, vacuum dried, and dissolved in appropriate volume of TE buffer. If required, the sample was treated for 30 min with DNase free RNase at a final concentration of 20 μg/ml. The plasmid DNA was checked on a 0.8% agarose gel and stored at −20°. The plasmid DNA thus isolated was suitable for procedures such as restriction digestion, ligation, and preparation of radiolabeled probes. Plasmid isolation was also done with any of the commercially available kits from

Isolation of plasmid DNA

and the aqueous phase transferred to a fresh tube. The aqueous phase was further extracted successively, first with phenol:chloroform:isoamyl alcohol (25:24:1) and then with chloroform:isoamyl alcohol (24:1). DNA was precipitated from the clear supernatant by the addition of 0.6 volumes of isopropanol. The chromosomal DNA was either spooled out or pelleted at this stage, washed with 70% ethanol, air-dried, and dissolved in suitable volume of TE buffer

The method as described in the manual Current Protocols in Molecular Biology was followed for preparation of chromosomal DNA. Cells from 1.5 ml stationary phase culture were recovered by centrifugation and resuspended in 567 μl of TE buffer. To this, 30 μl of 10% SDS, and 3 μl of proteinase K (20 mg/ml) were added in that order and the cell suspension mixed and incubated at 37°C for 1 h. Next, when the suspension looked cleared, 100 μl of 5 M NaCl was added, thoroughly mixed, followed by the addition of 80 μl of CTAB/NaCl (10% cetyltrimethylammonium bromide in 7 M NaCl) and vigorous mixing (by inverting the microfuge tube). The suspension was incubated at 65°C for 10 min, brought to room temperature, extracted with an equal volume of chloroform-isoamyl alcohol (24:1 v/v)

Extraction of chromosomal DNA from bacterial cells

Molecular and genetic techniques

The method followed is essentially the same as described by Jin et al. (1992). Overnight bacterial cultures grown in minimal A medium supplemented with 0.4% glycerol and 0.5% Casamino acids with the appropriate antibiotic were subcultured 1:100 in the same medium in a volume of 20 ml (0.2% arabinose was added for induction of the plasmid-borne gene downstream of Para, wherever required) at 37 ̊C. Cultures were induced with 1 mM IPTG at A600=0.3. 1 ml samples were aliquoted at time intervals of 0 sec, 20 sec, 40 sec, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min and 6 min into 1 ml of 0.1 mg/ml ice cold chloramphenicol and the samples were put on ice. After sampling the cultures were incubated at 37 ̊C for 15 min. 0.5 ml of this culture was then taken in duplicate tubes for β-galactosidase assays

RNA polymerase elongation rate measurement

high osmolarity conditions (Gowrishankar, 1989; Csonka, 1989) for β-galactosidase assay

Assays for determination of β-galactosidase enzyme activity in cultures were performed as described by Miller (1992) after permeabilizing the cells with SDS/chloroform, and the activity values were calculated in Miller units, as defined therein. For determination of proU activity from a proU::lac fusion that contains the proUpromoter cloned upstream of the lacZYA genes (as in plasmid pHYD272), cultures used were grown in LBON or K-medium (low osmolarity medium) since proU is also induced under

β-Galactosidase assay

Enzyme assays

a very sensitive test and is often used for determining the viability of a strain in the presence 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 the appropriate antibiotic and plating an appropriate dilution (10─5 and 3X10─6) on +IPTG (permissive) and –IPTG (test) plates to observe growth. The ratio of the number of colonies obtained on the –IPTG plate to that on the +IPTG plate determined the efficiency of plating. The colonies from the test plate (that is, ─IPTG plate) were also subsequently subcultured on the same medium (─IPTG) to determine viability of the strain. Likewise, strains carrying Ts plasmids were cultured overnight at 30 ̊C with the appropriate antibiotic and dilutions of this culture (10─5 and 3X10─6) were plated at two temperatures 30 ̊C (permissive) and 37 ̊C or 39 ̊C (non-permissive or test). The ratio of the number of colonies obtained on the test temperature to that on the permissive temperature determined the efficiency of plating at the test temperature. Viability of the strain was subsequently confirmed after subculturing from the test plate as stated above

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

Efficiency of plating

Strains were grown overnight in LB containing 0.4% maltose and 10 mM MgSO4,subcultured and grown to early stationary phase in the same medium. 100 μl of the culture was mixed with 2.5 ml of soft agar and overlaid on LB agar plates supplemented with 0.4% maltose and 10 mM MgSO4. Serial dilutions of λcI857 lysate were prepared (in LB) and 10 μl were spotted from each dilution (and the undiluted) on the soft agar lawn and allowed to dry. The plates were incubated at the appropriate temperature overnight, and the plating efficiency determined

Determination of λ-plating efficiency

The lacZ U118 is an amber nonsense mutation(Am) that confers Lac─phenotype and also polarity of the downstream lacYA genes in the operon due to premature Rho-dependent transcription termination within the untranslated region of lacZ. Melibiose is a sugar which can only be utilized in a lacZ (Am) strain at high temperature (39 ̊C, when the native melibiose permease is inactive) if the downstream gene lacY encoded permease is transcribed and translated. Therefore, in lacZ (Am) strains, growth on minimal melibiose plates (0.2%) at 39°C reflects transcriptional polarity relief at the lac locus, and the same was scored after streaking the relevant strains on such medium

lacZ (Am) assay

dependent transcription termination within the untranslated region of trpE. Anthranilate is a precursor of tryptophan, which is the product of trpE-encoded anthranilate synthase. Therefore, in trpE(fs) strains, growth on minimal glucose plates supplemented with anthranilate (100 μg/ml) reflects transcriptional polarity relief at the trp locus, and the same was scored after streaking the relevant strains on such medium

The trpE9777 is a frameshift (fs) mutationconfers Trp auxotrophy and also polarity on the downstream trpDCBA genes in the operon due to premature Rho-

trpE(fs) assay

The galEp3 (galE490∗)mutation represents a 1.3 kb 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 galEp3mutant or its derivatives was monitored by one of two means. In the first, MacConkey galactose indicator plates (with 1% galactose) were used, 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. In the second method, growth of strains on minimal-galactose (0.2%) was used as a test for Gal+ phenotype

galEp3 assay

In vivo transcription termination assays

Strains were streaked on LBON agar plates and after an overnight incubation at 42°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. Whenever needed the phenotype was also quantitatively assessed by plating dilutions of cultures on LBON agar plates and the drop in plating efficiency was scored after overnight incubation at 30°C and 42°C

LBON(Ts) phenotype

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)

SMG resistance

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

Lac phenotype

Scoring for phenotypes

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

Selection for Tets colonies on medium described by Maloy et al

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

Preparation of λ lysate by propagation from a single isolated plaque

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

Preparation of lambda (λ) lysates

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

Obtaining transposon insertions near gene 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

Generation of random Tn10 insertions into the genome of E.coli

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

P1 transduction

To quantitate the P1 phage lysate preparations, titration was done using a P1-sensitive indicator strain such as MG1655. 100 μl each of serial dilutions of the phage (typically 10−5, 10−6) were mixed with 0.1 ml of the fresh culture grown in Z-broth. After 15 min adsorption at 37°C without shaking, each mixture was added in a soft agar overlay to Z agar plates, and incubated overnight at 37°C. The phage titre was calculated from the number of plaques obtained on the plates

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

Broth method

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

Phage P1 lysate preparation

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

Densitometry

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

Electrophoretic mobility shift assay (EMSA)

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

Primer extension analysis

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

Radiolabelling of oligonucleotides and PCR products

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

DNA sequencing

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)

Molecular techniques

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

Native Isoelectric Focusing

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

Gel-filtration chromatography

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

Sodium dodecyl sulphate-polyacrlyamidegel electrophoresis (SDS-PAGE)

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

Protein estimation

Overexpression and purification of ArgPand ArgPdproteins

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

Biochemical techniques

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

Ligation of DNA

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 PCR products

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

Agarose gel electrophoresis

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

Isolation of chromosomal DNA

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

Isolation of plasmid DNA

Recombinant DNA techniques

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

Microarray details

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

β-galactosidase assay

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)

Test for thialysine resistance

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

Test for ArgR+/–phenotype

Test for canavanine (CAN) sensitivity

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

A. lacphenotype

Scoring for phenotypes

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

Preparation of high efficiency competent cells

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

A. Calcium chloride method

Transformation

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,

Phage P1 transduction