Strain and culture conditions

Two-dimensional SDS-PAGE gel electrophoresis

Approximately 4.1x10¹⁰ cells were harvested from the exponential phase in all experiments. The cells were pelleted down at 6000 rpm at 4 ºC for 10 min. Harvested cells were washed with low salt solution (3 mM KCl, 1.5 mM KH₂PO₄ , 68 mM NaCl, 9 mM NaH₂PO₄) trice. The pellet was then resuspended in rehydration buffer (9 M urea, 4% 3-([3-chloramidopropyl]dimethylammonio)-1-propane-sulfonate (CHAPS), 85 mM dithiothreitol (DTT), 0.5 mM pefabloc SC and stored at -20 ºC until next use. The cells were lysed by ultrasonication for 10 s, 5 times at 10% of maximum output (Branson sonifier 450). After 30 minutes of incubation at 37 ºC with DNase and RNase, eventually the debris was pelleted down and the proteins present in the supernatant were precipitated with acetone at -20 ºC overnight. The precipitated proteins were then resuspended in rehydration buffer and 300 µg of protein sample was loaded to 24 cm, pH 4-7 Immobiline dry strips (Amersham Biosciences) along with 1.5 µl (IPG)-buffer ph 4-7 dissolved in it for each strip. The strips were focused on an IPG-phor (Amersham Biosciences) for 1 h at 0 V, 12 h at 30 V, 2 h at 60 V, 1 h at 1000 V, and at 8000 V until approximately 75,000 Vh was reached. The strips were equilibrated in 5 ml of a solution containing 6 M urea, 50 mM Tris (pH 8.8), 30% (v/v) glycerol, 20 gL^-1 SDS and 20 gL^-1 DTT on a tilt table for 15 min. The solution was discarded and 5 ml of a second solution was added for 15 min containing 6 M urea, 50 mM Tris (pH 8.8), 30% (v/v) glycerol, 20 gL^-1 SDS and 25 gL^-1 iodoacetamide. The second dimension was performed on an EttanDalt (Amersham Biosciences) electrophoresis unit. The strips were placed on a 1.5 mm thick, 12.5% poly-acrylamide gel and sealed with 0.1% agarose in SDS- electrophoresis buffer containing 0.01% brom-phenol-blue. The gel electrophoresis was performed for 30 min at 3 W per gel followed by a further run at 20 W per gel until the end. For comparative analysis, gels were stained with coomassie blue stain.

In-gel tryptic digestion and mass spectrometry

Protein spots were excised from 2-D gels with a spot picker and placed into 96-wellmicrotiter plates, which were washed twice with TFA:acetonitrile:water (0.1:60:40). The tryptic digest was performed as reported previously with slight modifications. The samples containing the tryptic-digested proteins were mixed at a 1:1 ratio with a solution of water:acetonitrile:TFA (67:33:0.1) saturated with a-cyano-cinnamic acid. The mass spectrum was obtained on a Biflex III MALDI-TOF-MS (Bruker). The annotation of the peptide mass fingerprints was performed by the MASCOT search engine (Matrix Science). The search was done against our local E. coli database. The parameters used were, Taxonomy: All entries; Enzyme: Trypsin; Missed cleavages: 1; ppm: 100; Database: E. coli.

Analysis of two-dimensional protein gels

For comparison of protein spot densities between different strains and evolutionary conditions, gels were scanned and digitized. Image smoothing, spot detection, spot quantification, image alignment, spot matching, spot annotation, molecular weight and pI calculation, and variation analysis of the protein gels was performed using PDQuest software (Bio- Rad). For each protein spot, the annotated information along with the peak area and normalized quantity values were obtained. Along with these exported annotations, the protein spots were analysed by grouping them into various functional categories based on MultiFun and Gene Ontology terms, the classification system for cellular functions of gene products of E. coli consisting of 10 major functional categories. For each protein spot, the annotated information along with the obtained X and Y coordinates, the peak area and normalized quantity values were stored in the database. Internally these data, along with the gel images were stored by an upload function option in the database.