Supplementary Material
1. Method for the Proteomics:
1.1 Protein Extraction:
Single colony of Pseudomonas migulae S10724 strain was inoculated in 500 ml of broth and grown overnight in “Nutrient broth and Burk’s medium” representing two different conditions - low temperature nitrogen sufficient condition (NSC) and low temperature nitrogen fixing condition (NFC), respectively. Culture was harvested at 10,000 rpm for 10 min and pellet was washed twice with Normal saline solution. Pellet was then dissolved in 4ml of 0.1M Phosphate buffer saline (chilled) and sonicated for 3 min in ice. One mM PMSF (100μl) was added to the sonicated pellet. Cell suspension was centrifuged for 45 min at 10,000 rpm. The supernatant was lyophilized and were send to Sandor Proteomics Pvt Ltd, Hyderabad for further analysis[8].
1.2 Two dimensional gel electrophoresis and gel image analysis:
2D gel electrophoresis was done at Sandor Proteomics Pvt Ltd, Hyderabad using Bio-Rad 2D gel electrophoresis unit. Protein samples were solubilised in isoelectric focusing (IEF) buffer containing 7 M urea, 2 M thio-urea, 4% (w/v) CHAPS, 1% (w/v) dithiothreitol (DTT) and 0.2% (v/v) ampholytes pH 3-10 (Bio-Rad, Hercules, US). The 17 cm immobilized pH gradient (IPG) strips (pH 3-10 or 4-7, Bio-Rad, Hercules, US) were passively rehydrated for 16 h with 300 µL of cell extract samples containing 1-2 mg of protein. IEF was performed in a Protean IEF cell system (Bio-Rad, Hercules, US) with up to 50,000 VH at a maximum voltage of 10,000 V. Strips were equilibrated for 15 min in equilibration buffer I (30%, v/v, glycerol, 6 M urea, 1% DTT, a trace of bromophenol blue) and for 15 min in equilibration buffer II (equilibration solution I with DTT replaced by 4% iodoacetamide). In the second dimension, IPG strips were run vertically onto SDS-PAGE 12% gels using PROTEAN® II xi 2D Cell (Bio-Rad, Hercules, US). Gels were stained with 0.1% Coomassie Brilliant Blue R-250 (Hi-media, US) and scanned with a computer-assisted G-800 densitometer (Bio-Rad, Hercules, US). The pI and Mw of remaining 58 protein spots were calculated by analyzing the gels manually[8]. Computational study of these spots was carried out with the help of ExPASy software package of proteomic analysis based on pI and Mw. Swiss-Prot/TrEMBL databases were searched with the help of Tagident tool for their functional analysis [8].
1.3 MALDI-TOF-MS analysis and MASCOT Database Searches
MALDI-TOF-MS Analysis was done at Sandor Proteomics Pvt Ltd, Hyderabad using Bruker Daltonics - Ultraflex™ III Mass Spectrometer with spectra internally calibrated using trypsin auto-digestion products. The obtained peptide masses were searched on the MascotTM PeptideMass Fingerprint database (Matrix Science). The data that were obtained were used in the determination of the identity of the proteins using the Mascot search tool (
2. Identification of bacterial isolates
Identification of bacterial isolates was performed by sequencing 16S rDNA of bacterial isolates. The genomic DNA was isolated as described previously [1]. The PCR assay was performed using Applied Biosystems, model 9800 (Foster, California, USA) with 50ng of DNA extract in a total volume of 25μl. The PCR master mixture contained 2.5 μl of 10X PCR reaction buffer (with 1.5 M MgCl2), 2.5 μl of 2 mM dNTPs, 1.25 μl of 10 pm/μl of each oligonucleotide primer 8F (5’- CCA GAGTTT GAT CMT GGC TCA G -3’) and 1391R (5’- GACGGGCGGTGTGTRCA -3’) [2, 3, 4, 5], 0.2 μl of 5U/ μl Taq DNA polymerase and 15.76 μl of glass-distilled PCR water. Initially denaturation accomplished at 94°C for 3 min. Thirty-two cycles of amplification consisted of denaturation at 94°C for 30s, annealing at 55°C for 30s and extension at 72°C for 1.30 min. A final extension phase at 72°C for 10 min was performed. The PCR product was purified by PEG-NaCl method [11]. Briefly, all samples was mixed with 0.6 times volume of PEG-NaCl, 20% [PEG (MW 6000) and 2.5 M NaCl] and incubated for 20 min at 37°C. The precipitate was collected by centrifugation at 3,800 rpm for 20 min. The pellet was washed with 70% ethanol, air dried and dissolved in 15 μl sterile distilled water. The thermocycling for the sequencing reactions began with an initial denaturation at 94°C for 2 min, followed by 25 cycles of PCR consisting of denaturation at 94°C for 10s, annealing at 50°C for 10s, and extension at 60°C for 4 min using primers 704F (5’- GTAGCGGTGAAATGCGTAGA-3’) [2,3,4,5] and 907R (5’- CCGTCAATTCMTTTGAGTTT-3’) [4,5]. The samples were purified using standard protocols described by manufacturer (Applied Biosystems Foster City, USA). To this, 10 μl of Hi-Di formamide was added and vortexed briefly. The DNA was denatured by incubating at 95°C for 3 min, kept on ice for 5-10 min, and was sequenced in a 3730 DNA analyzer (Applied Biosystems, Foster City, USA) following the manufacturer’s instructions. The obtained sequences were analysed using Sequence Scanner (Applied Biosystems) software. The rDNA sequence contigs were analysed using online databases viz. NCBI-BLAST [6] to find the closest match of the contiguous sequence.
Phylogenetic Analyses
The obtained partial 16SrDNA gene sequences were aligned using BioEdit® and edited in DAMBE® [7,12]. The evolutionary history was inferred using the Neighbor- Joining method [9]. The percentage of replicate trees in which the associated taxa are clustered together in the bootstrap test (1,000 replicates) is recorded next to the branches (Fig 2a). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree using Molecular Evolutionary Genetics Analysis (MEGA, Version 5) software [10].
Figure 2a. Neighbour- joining inferred tree based on 16S rRNA gene sequences showing the evolutionary relationship of bacterial isolates found growing at 10°C to 15°C and utilising atmospheric nitrogen with previously characterized species. Type strains are indicated by ‘T’. Bar length represent the number of the base substitution per site. Bootstrap values expressed as percentage of 1000 resamplings of the neighbour-joining dataset are showed at nodes.
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