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Present study was designed to isolate and characterize the persistent organic pollutants (POPs) metabolizing bacteria from tannery effluents and petrol contaminated soil samples collected from gasoline stations of Bahawalpur, Pakistan. For the isolation of individual bacteria capable of metabolizing different POPs methods of selective enrichment culture and serial dilution were employed. Followed by isolation, growth behavior, corresponding POPs removal efficiencies, biochemical characteristics and antibiotics resistance profiles were documented. For molecular analysis, 16S rRNA gene was amplified and sequenced. Retrieved sequences were analyzed through BLAST analysis and to reflect relationship phylogenetic trees were constructed using software MEGA version 7. To detect the possible metabolic pathways responsible for POPs degradation, the isolates with high removal efficiencies were selected. Extracellular and intracellular metabolites of the selected bacteria were extracted and subjected to GC-MS and LC-MS analysis. Effect of selected carbon source on proteins production of bacteria was elucidated through LC-MS analysis of the intracellular proteins extracted from various bacterial isolates. The 16S rRNA metagenome sequencing was performed in order to confirm the POPs metabolizing potential of isolated bacteria among the bacterial community of tannery effluent and petrol contaminated soil. Shotgun metagenome sequencing helped to determine the possible genes and pathways associated with POPs degradation. Total forty-one bacteria were isolated during present study of which twenty-eight were toluene metabolizing, four were naphthalene metabolizing and nine were petrol metabolizing bacteria. The top BLAST similarity analysis revealed similarity of isolates with Brevibacillus agri strain NBRC 15538 (n = 27), Bacillus paralicheniformis strain KJ-16 (n = 4), Burkholderia lata strain 383 (n = 6), Burkholderia pyrrocinia strain LMG 14191 (n = 1) and Brevibacillus formosus strain NBRC 15716 (n = 3). All isolates were found sensitive to teicoplanin and linezolid and resistant to oxacillin and chloramphenicol. However, two bacteria, IUBT9 and IUBT26, exhibited sensitivity to oxacillin. Present study bacteria were examined to have considerable toluene removal potential (28-93%), naphthalene removal potential (70-83%) and petrol removal potential (41-69.5%). Biochemical characterization revealed presence of many valuable enzymes like arginine dehydrolase, naphthylamidase, glucosaminidase and phosphatase and ability of isolates to ferment variety of sugars like esculin, mannitol, sorbitol and inulin. GC-MS and LC-MS based analysis of bacterial metabolites confirmed degradation potential of the isolated bacteria to metabolize toluene, naphthalene and petrol through not only the earlier reported metabolic pathways but also some novel metabolic pathways which have not been reported so far. LC-MS analysis of bacterial proteins revealed the presence of enzymes involved in the degradation of toluene, synthesis of secondary metabolites, central dogma of gene expression, chemotaxis, spore development, transport of ions and central carbon metabolism further confirming the utilization of the provided carbon sources (toluene, naphthalene, petrol) by present study bacteria.
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