Abstract
Pseudomonas asiatica strain PNPG3 demonstrated broad-spectrum heavy-metal tolerance, with minimum inhibitory concentrations (MICs) of 800, 400, 4, and 6 µg/mL (ppm) for arsenite, cadmium (Cd), cobalt (Co), and nickel (Ni), respectively. When exposed to 1 mM arsenite, strain PNPG3 retained approximately 89% of its p-nitrophenol (PNP) degradation efficiency relative to the PNP-only baseline, mineralizing 86% of 0.5 mM PNP within 66 h and releasing 0.41 mM nitrite, indicating strong catabolic resilience under combined PNP–arsenite stress. Genome analysis identified a distinct arsenic (As) tolerance and biotransformation gene cluster on contig 1, comprising coordinated transport, regulatory, and metabolic components, including an ArsR/SmtB family transcriptional regulator and an ArsJ-associated glyceraldehyde-3-phosphate dehydrogenase, suggesting the presence of a specialized and potentially novel As detoxification mechanism. Comparative genomics further revealed conservation of key abiotic and biotic stress response genes, along with metabolic pathways supporting degradation of styrene, dioxins, polycyclic aromatic hydrocarbons, cyanate, and diverse aromatic xenobiotics. Chromate reductase (ChrR) and arsenate reductase (ArsC), key enzymes involved in the biotransformation of Cr (VI) to Cr (III) and arsenate [As(V)] to arsenite [As(III)], respectively, were modeled, characterized, and validated, followed by docking analyses to elucidate heavy-metal interactions at their active sites. Molecular dynamics simulation (MDS) indicated that the ArsC–arsenate complex exhibited higher structural stability and compactness, with limited conformational fluctuations, implied greater robustness of arsenate reduction compared to Cr(VI) reduction under in situ metal stress conditions. Overall, the phenotypic robustness and genomic potential of strain PNPG3 underscore its strong capacity for heavy-metal tolerance, PNP biodegradation, and arsenate biotransformation, highlighting its promise for scalable bioremediation applications.
Data availability
The datasets generated and/or analysed during the current study are available in the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India (accession number MTCC 13,126), and in the National Center for Biotechnology Information (NCBI) repository, with raw sequencing reads deposited in the Sequence Read Archive under accession number SRR18163134 and the whole-genome shotgun sequence available under accession number JALLKV000000000.
References
Tchieno, F. M. M. & Tonle, I. K. p-Nitrophenol determination and remediation: An overview. Rev. Anal. Chem. 37, 20170019 (2018).
Kuang, S. et al. Effects of p-nitrophenol on enzyme activity, histology, and gene expression in Larimichthys crocea. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 228, 108638 (2020).
Stavrakakis, I., Remmas, N., Melidis, P. & Ntougias, S. Effect of the oxidative phosphorylation uncoupler para-nitrophenol on the activated sludge community structure and performance of a submerged membrane bioreactor. Water 13, 3222 (2021).
Rodrigues, C. S. et al. Degradation of p-Nitrophenol by activated persulfate with carbon-based materials. J. Environ. Manage 343, 118140 (2023).
Jaiswal, S. & Shukla, P. Alternative strategies for microbial remediation of pollutants via synthetic biology. Front. Microbiol 11, 808 (2020).
Sengupta, K., Swain, M. T., Livingstone, P. G., Whitworth, D. E. & Saha, P. Genome sequencing and comparative transcriptomics provide a holistic view of 4-nitrophenol degradation and concurrent fatty acid catabolism by Rhodococcus sp. strain BUPNP1. Front. Microbiol. 9, 3209 (2019).
Alam, S. A. & Saha, P. Evidence of p-nitrophenol biodegradation and study of genomic attributes from a newly isolated aquatic bacterium Pseudomonas asiatica strain PNPG3. Soil Sediment Contam Soil Sediment Contam. Int. J. 32, 994–1011 (2022).
Alam, S. A. & Saha, P. Chemotactic response of p-nitrophenol degrading Pseudomonas asiatica strain PNPG3 through phenotypic and genome sequence-based in silico studies. 3 Biotech 13, 408 (2023).
Abbas, S. et al. Molecular characterization of heavy metal-tolerant bacteria and their potential for bioremediation and plant growth promotion. Front. Microbiol 16, 1644466 (2025).
Jan, A. T. et al. Heavy metals and human health: mechanistic insight into toxicity and counter defense system of antioxidants. Int. J. Mol. Sci. 16, 29592–29630 (2015).
Akhter, M., Tasleem, M., Alam, M. M. & Ali, S. In silico approach for bioremediation of arsenic by structure prediction and docking studies of arsenite oxidase from Pseudomonas stutzeri TS44. Int. Biodeterior. Biodegradation 122, 82–91 (2017).
DesMarias, T. L. & Costa, M. Mechanisms of chromium-induced toxicity. Curr. Opin. Toxicol. 14, 1–7 (2019).
Genchi, G., Sinicropi, M. S., Lauria, G., Carocci, A. & Catalano, A. The effects of cadmium toxicity. Int. J. Environ. Res. Public Health 17, 3782 (2020).
Alam, S. A. & Saha, P. Azoreductases of Pseudomonas sp. PNPG3: A bioinformatics and molecular simulation study for azo dye detoxification. J. Biomol. Struct. Dyn. https://doi.org/10.1080/07391102.2025.2562136 (2025).
Alam, S. A. & Saha, P. Biodegradation of p-nitrophenol by a member of the genus Brachybacterium, isolated from the river Ganges. 3 Biotech 12, 1–10 (2022).
Alam, S. A., Khan, B., Karmakar, D., Mandal, R. & Saha, P. Integrative bioinformatics and chemotactic insights into p-nitrophenol bioremediation by halotolerant aquatic Pseudomonas sp. strain PNPBRP5 (2). Arch. Microbiol. 207, 1–12 (2025).
White, G. F., Snape, J. R. & Nicklin, S. Biodegradation of glycerol trinitrate and pentaerythritol tetranitrate by Agrobacterium radiobacter. Appl. Environ. Microbiol. 62, 637–642 (1996).
Nurk, S., Meleshko, D., Korobeynikov, A. & Pevzner, P. A. metaSPAdes: A new versatile metagenomic assembler. Genome Res. 27(5), 824–834 (2017).
Aziz, R. K. et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genom. 9, 75 (2008).
Tatusova, T. et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 44, 6614–6624 (2016).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Pellegrinetti, T. A. et al. PGPg_finder: A comprehensive and user-friendly pipeline for identifying plant growth-promoting genes in genomic and metagenomic data. Rhizosphere 30, 100905 (2024).
Artimo, P. et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 40, 597–603 (2012).
Geourjon, C. & Deleage, G. SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Bioinform 11, 681–684 (1995).
Klausen, M. S. et al. NetSurfP-2.0: Improved prediction of protein structural features by integrated deep learning. Proteins: Struct., Funct., Bioinf. 87, 520–527 (2019).
Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: A web-based environment for protein structure homology modelling. Bioinform 22, 195–201 (2006).
Colovos, C. & Yeates, T. O. Verification of protein structures: Patterns of nonbonded atomic interactions. Protein Sci. 2, 1511–1519 (1993).
Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: A program to check the stereochemical quality of protein structures. Appl. Crystallogr. 26, 283–291 (1993).
Jendele, L., Krivak, R., Skoda, P., Novotny, M. & Hoksza, D. PrankWeb: A web server for ligand binding site prediction and visualization. Nucleic Acids Res. 47, 345–349 (2019).
Liu, Y. et al. CB-Dock2: Improved protein–ligand blind docking by integrating cavity detection, docking and homologous template fitting. Nucleic Acids Res. 50, 159–164 (2022).
Laskowski, R. A. & Swindells, M. B. LigPlot+: Multiple ligand–protein interaction diagrams for drug discovery. J. Chem. Inf. Model 51, 2778–2786 (2011).
Wu, Y., Tepper, H. L. & Voth, G. A. Flexible simple point-charge water model with improved liquid-state properties. J. Chem. Phys. 124, 024503 (2006).
David, C. C., Jacobs, D. J. Principal component analysis: a method for determining the essential dynamics of proteins. In Protein dynamics: Methods and protocols 193–226. Totowa, NJ: Humana Press (2013).
Sengupta, K., Maiti, T. K. & Saha, P. Degradation of 4-nitrophenol in presence of heavy metals by a halotolerant Bacillus sp. strain BUPNP2, having plant growth promoting traits. Symbiosis 65, 157–163 (2015).
Tempestti, J. C. M. et al. Detoxification of p-nitrophenol (PNP) using Enterococcus gallinarum JT-02 isolated from animal farm waste sludge. Environ. Res. 231, 116289 (2023).
Chakraborti, D. et al. Groundwater arsenic contamination in the Ganga River Basin: A future health danger. Int. J. Environ. Res. Public Health. 15, 180 (2018).
William, V. U. & Magpantay, H. D. Arsenic and microorganisms: Genes, molecular mechanisms, and recent advances in microbial arsenic bioremediation. Microorganisms 12, 74 (2023).
Khan, Z., Nisar, M. A., Hussain, S. Z., Arshad, M. N. & Rehman, A. Cadmium resistance mechanism in Escherichia coli P4 and its potential use to bioremediate environmental cadmium. Appl. Microbiol. Biotechnol 99, 10745–10757 (2015).
Moore, C. M., Gaballa, A., Hui, M., Ye, R. W. & Helmann, J. D. Genetic and physiological responses of Bacillus subtilis to metal ion stress. Mol. Microbiol. 57, 27–40 (2005).
Tasleem, M., El-Sayed, A. A. A., Hussein, W. M. & Alrehaily, A. Bioremediation of chromium-contaminated groundwater using chromate reductase from Pseudomonas putida: An in silico approach. Water 15, 150 (2022).
Mitra, P., Singha, S., Roy, P., Saha, D. & Chatterjee, S. A molecular docking study between heavy metals and hydrophilic Hsp70 protein to explore binding pockets. J. Proteins Proteomics 15, 413–428 (2024).
Yan, G. et al. Genetic mechanisms of arsenic detoxification and metabolism in bacteria. Curr. Genet. 65, 329–338 (2019).
Fekih, I. B. et al. Distribution of arsenic resistance genes in prokaryotes. Front. Microbiol. 9, 2473 (2018).
Butcher, B. G., Deane, S. M. & Rawlings, D. E. The chromosomal arsenic resistance genes of Thiobacillus ferrooxidans have an unusual arrangement and confer increased arsenic and antimony resistance to Escherichia coli. Appl. Environ. Microbiol. 66, 1826–1833 (2000).
Ji, G. & Silver, S. Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258. J. Bacteriol. 174, 3684–3694 (1992).
Neyt, C., Iriarte, M., Thi, V. H. & Cornelis, G. R. Virulence and arsenic resistance in Yersiniae. J. Bacteriol. 179, 612–619 (1997).
Li, X. & Krumholz, L. R. Regulation of arsenate resistance in Desulfovibrio desulfuricans G20 by an arsRBCC operon and an arsC gene. J. Bacteriol. 189, 3705–3711 (2007).
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SAA-Formal Analysis, designing of experiments, wet lab and docking, MDS Investigation, Methodology, Validation, and Writing, Review and Editing; PS-contributed reagents/materials, and reviewed drafts of the paper; DK- prepared heatmap, molecular docking; TN- initial wet lab experiment; RM, SB- reviewed drafts of the paper.
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Alam, S.A., Karmakar, D., Nayek, T. et al. Genomic and structural elucidation of multi-heavy metal tolerance in the p-nitrophenol-degrading bacterium Pseudomonas asiatica strain PNPG3. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40113-5
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DOI: https://doi.org/10.1038/s41598-026-40113-5
