Background: Phylogenetic heterogeneity across Pseudomonas genus is complemented by its diverse genome architecture enriched by accessory genetic elements (plasmids, transposons, and integrons) conferring resistance across this genus. Here, we sequenced a stress tolerant genotype i.e. Pseudomonas sp. strain RL isolated from a hexachlorocyclohexane (HCH) contaminated pond (45 mg of total HCH g−1 sediment) and further compared its gene repertoire with 17 reference ecotypes belonging to P. stutzeri, P. mendocina, P. aeruginosa, P. psychrotolerans and P. denitrificans, representing metabolically diverse ecosystems (i.e. marine, clinical, and soil/sludge). Metagenomic data from HCH contaminated pond sediment and similar HCH contaminated sites were further used to analyze the pan-genome dynamics of Pseudomonas genotypes enriched across increasing HCH gradient. Results: Although strain RL demonstrated clear species demarcation (ANI ≤ 80.03%) from the rest of its phylogenetic relatives, it was found to be closest to P. stutzeri clade which was further complemented functionally. Comparative functional analysis elucidated strain specific enrichment of metabolic pathways like α-linoleic acid degradation and carbazole degradation in Pseudomonas sp. strain RL and P. stutzeri XLDN-R, respectively. Composition based methods (%codon bias and %G + C difference) further highlighted the significance of horizontal gene transfer (HGT) in evolution of nitrogen metabolism, two-component system (TCS) and methionine metabolism across the Pseudomonas genomes used in this study. An intact mobile class-I integron (3,552 bp) with a captured gene cassette encoding for dihydrofolate reductase (dhfra1) was detected in strain RL, distinctly demarcated from other integron harboring species (i.e. P. aeruginosa, P. stutzeri, and P. putida). Mobility of this integron was confirmed by its association with Tnp21-like transposon (95% identity) suggesting stress specific mobilization across HCH contaminated sites. Metagenomics data from pond sediment and recently surveyed HCH adulterated soils revealed the in situ enrichment of integron associated transposase gene (TnpA6100) across increasing HCH contamination (0.7 to 450 mg HCH g−1 of soil). Conclusions: Unlocking the potential of comparative genomics supplemented with metagenomics, we have attempted to resolve the environment and strain specific demarcations across 18 Pseudomonas gene complements. Pan-genome analyses of these strains indicate at astoundingly diverse metabolic strategies and provide genetic basis for the cosmopolitan existence of this taxon.

Maximum Likelihood Tree (Newick Format) for 51 Pseudomonas Genomes using Azotobacter vinelandii CA as outgroup.Conserved genes (400) based phylogenetic tree of strain RL and representative Pseudomonas genotypes constructed at 1000 bootstrap with Azotobacter vinelandii CA as an out-group. Branch lengths are drawn to scale, with scale bar indicating the number of amino acid substitutions. Numbers on branches are the bootstrap values of the clusters on the right.tree_treebase.nwkMaximum Likelihood Tree (Nexus Format) for 51 Pseudomonas Genomes using Azotobacter vinelandii CA as outgroup.Conserved genes (400) based phylogenetic tree of strain RL and representative Pseudomonas genotypes constructed at 1000 bootstrap with Azotobacter vinelandii CA as an out-group. Branch lengths are drawn to scale, with scale bar indicating the number of amino acid substitutions. Numbers on branches are the bootstrap values of the clusters on the right.tree_treebase.nwk.nexAlignment Files for protein coding sequences of bacterial conserved marker genes (n=400) for 51 representative Pseudomonas genomes.Phylogenetic status of strain RL was determined using 400 conserved bacterial marker genes and representative whole genome sequences from 40 diverse Pseudomonas species as available in NCBI database (http://www.ncbi.nlm.nih.gov/genome/browse/representative/). A phylogenetic tree was hence constructed using maximum likelihood (ML) methodology on the basis of protein coding bacterial conserved gene (400) sequences [39] determined for Pseudomonas sp. RL and 50 other Pseudomonas strains with Azotobacter vinelandii being the outgroup.Alignment Files.zip