Sampling site
Samples were collected from the supraglacial site of the East Rathong glacier, located between latitudes 27°33'36"N and 27°36'40"N, longitudes 88°06'03"E and 88°07'38"E [44]. It is a south-east facing, debris-free and summer-nourished glacier in the West district of Sikkim that forms the dominant glacier in Eastern Himalayas. The area experiences a cool and wet climatic condition, and snowfall is not unusual even during the monsoon season [45]. The sample collection was done in the month of May. Ice meltwater samples were collected in sterile 250 ml amber wide mouth bottles (Tarson, India), and transported in ice buckets with ice packs. Samples were stored at 4°C until analysis.
Isolation and identification of bacteria
The ice melt-water and the water samples of the glacier surface were enriched in sterile distilled water in shaking conditions for 2 h at 10°C. Subsequently, tenfold serial dilutions of each samples were made to plate in triplicate on R2A agar medium (pH 7.0; Himedia, India) and Antarctic Bacterial Medium (ABM) plates [peptone (0.5%, w/v), yeast extract (0.2%, w/v) and agar (2%, w/v) and incubated at 10° C for 10-15 days. Viable bacteria obtained in the agar plates were counted as colony forming units (CFU). Unique morphotypes from each plate were purified and maintained on ABM plates. The pure cultures obtained were preserved using 20 % glycerol at -80° C for further studies.
Growth parameters were checked at different temperatures (4, 20, 28, 37, 50°C), pH (1, 3, 5, 7, 9, 11, 13) and varying salt concentrations (1-6%) in ABM agar plates.
Molecular characterization
16S rRNA gene sequencing and phylogenetic analysis
Genomic DNA was extracted from each of the isolates using the CTAB method, as given by Chen and Kuo [46]. The 16S rRNA gene was amplified by PCR using universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5'-GGTTACCTTGTTACGACTT-3'). PCR was performed with 20μl reaction mixtures containing approximately 50 ng of template DNA, 2 μM forward primer, 2 μM reverse primer, and 1X GoTaq Green PCR Master Mix (Promega, US). DNA amplification was carried out in G-Storm Thermocycler (Somerset, United Kingdom) with an initial denaturation step of 94° C for 5 minutes, followed by 30 cycles of denaturation at 94° C for 1 min, annealing at 55° C for 1 min, and extension at 72° C for 2 min and then a final extension step of 72° C for 5 min. The PCR products were purified prior to sequencing by treatment of ExoSAP-IT solution as per manufacturer’s instructions (Affymetrix, US).
Purified PCR products were subjected to cycle sequencing using the forward, reverse and internal primers with Big Dye Terminator cycle sequencing kit v.3.1 (Applied Biosystems, US) protocol as described earlier [47]. The sequencing reaction was performed with 5 μL reaction mixture containing approximately 50 ng template DNA and 1 pmol of sequencing primers. Post reaction cleanup was performed using Montage Sequencing Reaction clean up kit (Millipore, US) using a Vacuum Pump Assembly (Millipore, US). Cleaned samples after cycle sequencing were bi-directionally sequenced using an automated Genetic Analyzer ABI 3130XL (Applied Biosystems, US).
The generated sequences were used to perform Basic Local Alignment Search Tool (BLAST) [48] analysis to determine the nearest phylogenetic neighbors against the database of type strains. For phylogenetic analysis, 16S rRNA gene sequences of the neighbors were obtained from the GenBank database (NCBI) and Molecular Evolutionary Genetics Analysis software (MEGA version X) was used for phylogenetic analyses [18]. The sequences of identified phylogenetic neighbors were aligned using Clustal W inbuilt with MEGA X. E. coli K12 MG1655 was used as the outgroup organism. Maximum likelihood method using Kimura 2-parameter model was employed to construct the Phylogenetic trees with 1000 bootstrap replications to assess nodal support in the tree.
Genotyping by ERIC-PCR
The isolates that showed similar phenotypes were compared through enterobacterial repetitive intragenic consensus (ERIC)-PCR. Briefly, genomic DNA was extracted and subjected to PCR amplification using the primers ERIC1 (5’-ATGTAAGCTCCTGGGGATTCAC-3’) and ERIC2 (5’-AAGTAAGTACTGGGGTGAGCG-3’), as given by Khosravi et al. [49]. The thermocycling conditions consisted of first denaturation cycle at 95 °C for 7 min followed by 30 cycles, including denaturation at 94 °C for 1 min, annealing for 1 min at 52 °C for ERIC- PCR, extension at 65 °C for 8 min, one final extension cycle at 65 °C for 16 min, and hold at 4 °C. The amplified products were subjected to electrophoresis on 1.5% agarose gel, stained with 0.5 μg/μl ethidium bromide (Qiagen, Germany) and analyzed under UV light in a gel documentation system (Syngene G-BOX transilluminator, US). The banding profiles were observed. The visible bands were converted into a binary matrix and used to construct a dendrogram using the Jaccard similarity index and the unweighted pair group method (UPGMA) with the aid of software Past, version 3.25.
Multilocus Sequence Analysis (MLSA)
The eleven strains belonging to the species of Pseudomonas were studied for their phylogenetic relationship by multilocus sequence analysis of five housekeeping genes. The genes selected were gyrB, ileS, nuoD, recA, and rpoD, based on the criteria that they are present as single copies in the genome, and are homologous and ubiquitous in the studied taxa [10]. The primers used for PCR amplification of the housekeeping genes were based on previous studies [50] (Supplementary Table S2). The PCR condition for all genes consisted of: an initial denaturation step of 2 min at 94°C, followed by 35 cycles of 30s at 94°C, 20s at 54°C, and 2 min at 72°C, and a final extension of 7 min at 72°C. After Sanger sequencing, the partial sequences obtained for these five genes were submitted in GenBank (NCBI). Based on 16S rRNA gene similarity, seventeen closely related Pseudomonas species were taken and the partial sequences of these five housekeeping genes were retrieved from the complete genomes of corresponding type strains of Pseudomonas from the GenBank database. Multiple sequence alignment of the nucleotides was performed with CLUSTAL W and the sequences were trimmed manually for subsequent phylogenetic analyses. Sequences were translated to amino acid and the open reading frame was determined in MEGA X. For phylogenetic analysis, the partial sequences of the five protein-coding genes were concatenated into a single alignment using MEGA X in the order: gyrB-ileS-nuoD-recA-rpoD. Partitionfinder v2.1.1 [51] was used to determine the best-fit partitioning schemes and substitution models of molecular evolution. Maximum likelihood (ML) trees were constructed with the same partitioning schemes and model using RAxMLGUI v1.5 [52] through the CIPRES Science Gateway for individual and concatenated datasets [53].
Kimura 2-Parameter (K2P) genetic distances [31] were calculated between the eleven Pseudomonas strains and their nearest neighbours taken from the NCBI database using MEGA, Version X.
Nucleotide polymorphism
Gene parameters, such as GC content, number of polymorphic sites, parsimony-informative sites, synonymous and non-synonymous sites, percentages of mean sequence similarities, number of nucleotide differences per site (Θ), nucleotide diversity per site (π) and Tajima's D statistic for individual gene sequences and the concatenated sequence were computed using MEGA X. The confidence of the branches of the ML tree was based on 1000 bootstrap replicates. E. coli K12 MG1655 was used as outgroup.
Physiological characterization
Tolerance to UV-C
For the radiation resistance test, the colony count method corresponding to UV-C irradiated aliquots and non-irradiated control was performed as described previously [54], with minor modifications. The bacterial strains were grown in ABM broth till cell O.D. 600 of ̴ 1.0 was attained. After centrifugation at 8,000 rpm for 5 minutes, the pellet was washed and re-suspended in normal saline. UV-C exposures of 150, 300 and 450 Jm-2 were given to the cell suspensions. Serial dilutions of UV irradiated as well as non-irradiated bacterial cultures were made and spread plated. After incubation for 3-5 days at 20°C, the number of colony-forming units was determined and the survival percentage was calculated. For comparison, a mesophilic type strain Pseudomonas aeruginosa MTCC 2453 and a radioresistant type strain Deinococcus radiodurans MTCC 4465 were subjected to the same conditions of UV-C exposure.
Tolerance to freezing
Tolerance to freezing was checked for 96 hrs. Cultures were grown till the stationary phase in ABM broth and culture tubes in triplicates were placed in -20°C freezer. After every 24 h of freezing, tubes were removed, thawed for 1 h at 20°C and 100 μL of the thawed culture was serially diluted in normal saline. The diluted culture was spread on ABM agar and incubated at 20°C for 2-4 days. The average of the triplicate colony counts was used for determining the survival percentage. Plates of unfrozen culture served as 0-time point control and a mesophilic type strain Pseudomonas aeruginosa MTCC 2453 was subjected to the same condition of freezing.
Production of extracellular cold-active enzymes
Screening of the isolates for extracellular hydrolytic activities
The isolates were screened for the production of hydrolytic enzymes such as amylase, cellulase, lipase, and protease at 10°C by spotting the cultures on their selective media containing specific substrate. Protease activity was assayed in Skim-milk agar. A clear zone around the colony indicated the production of proteases [55]. Lipase activity was checked on the enrichment medium Tributyrin agar. The colonies with clear hydrolysis zones were marked as lipase producers [56]. Carboxymethyl cellulose (CMC) agar was used for the detection of cellulase activity. On flooding the plates with Gram’s iodine, the zone of clearance around the bacterial colonies represented positive cellulase producers [57]. Amylase activity was checked on starch agar plates. Amylolytic isolates were selected by flooding the starch agar plates with Gram’s iodine solution. Isolates with distinct clear zone around the colonies were identified as amylase producers [58].
Quantitative estimation of extracellular Protease Activity
Extracellular protease activities of eleven bacterial isolates were determined by a previously described method [59]. In brief, eleven bacterial isolates were grown in Antarctic Bacterial Medium (0.2% yeast extract, 0.5% peptone) broth for 12h at 20°C. The bacterial growth was checked at 600 nm. 1% (v/v) bacterial culture was seeded into the protease production medium (K2HPO4 0.1%, KH2PO4 0.05%, CaCl2 0.02%, MgSO4.7H2O 0.05%, Glucose 1%) with 1% skim milk as substrate and incubated at 20°C, 120 rpm. After 48h of incubation, the cultures were centrifuged at 10,000 rpm for 15 mins at 4°C. The cell-free supernatant was used as a source of enzyme. For the enzymatic assay, the reaction mixture containing 100 μl of the enzyme and 400 μl of 1% casein solution in 50 mM Tris buffer (pH 8) was incubated for 10 min at 5 and 15°C. The reaction was terminated by adding 0.5 ml of trichloroacetic acid (1.2 M) and centrifuged for 10 min at 6000xg. 500 μl of the filtrate was mixed with 1 ml of 400 mM Na2CO3 solution and 50 μl Folin- Ciocalteu’s reagent. The amount of tyrosine released was determined spectrophotometrically at 660nm against the enzyme blank. The control was treated in the same way, except TCA was added before enzyme addition. One unit of protease activity was equivalent to the amount of enzyme that required releasing 1 μg of tyrosine/ml/min under standard assay conditions.
Statistical Analysis
All experiments were conducted in triplicate, and the results were presented as the mean ± standard deviation. Statistical analysis for significant differences was performed by one-way ANOVA followed by Dunnett's multiple comparison test (at p < 0.05 statistical significant differences) using the software GraphPad Prism7 (GraphPad Software, Inc., San Diego, USA).
Nucleotide sequence accessions numbers
The accession numbers obtained from NCBI GenBank for the 16S rRNA gene sequences against each strain are provided in Supplementary Table S1, and those for partial housekeeping gene sequences lie from MN238515-MN238591.