ABC  Vol.3 No.6 , December 2013
Isolation and molecular characterization of a novel pseudomonas putida strain capable of degrading organophosphate and aromatic compounds

A bacterial strain designated in this study as POXN01 was found to be capable of degrading the synthetic organophosphorus pesticides paraoxon and methyl parathion. The strain was initially isolated through enrichment technique from rice field soil near Harlingen, Texas. Phylogenetic analysis based on 16S rRNA, gyrB and rpoD gene alignments identified the POXN01 isolate as a new strain of Pseudomonas putida, which is closely related to the recently discovered nicotine-degrading strain Pseudomonas putida S16. While being unable to metabolize nicotine, the POXN01 isolate was observed to actively proliferate using monocyclic aromatic hydrocarbons, in particular toluene, as nutrients. Search for the genetic determinants of paraoxon catabolism revealed the presence of organophosphorus-degrading gene, opd, identical to the one from Sphingobium fuliginis (former Flavobacterium sp. ATCC 27551). Assimilation of aromatic compounds likely relies on phc ARKLMNOPQ gene cluster for phenol, benzene and toluene catabolism, and on benRABCDKGEF cluster for benzoate catabolism. The observed versatility of POXN01 strain in degradation of xenobiotics makes it useful for the multi-purpose bioremediation of contaminated sites in both agricultural and industrial environmental settings.


Cite this paper: Iyer, R. , Stepanov, V. and Iken, B. (2013) Isolation and molecular characterization of a novel pseudomonas putida strain capable of degrading organophosphate and aromatic compounds. Advances in Biological Chemistry, 3, 564-578. doi: 10.4236/abc.2013.36065.

[1]   Grillo, G., Karalliedde, L. and Senanayake, N. (1989) Organophosphorus insecticide poisoning. British Journal of Anaesthesia, 63, 736-750.

[2]   van der Hoek, W. and Konradsen, F. (2006) Analysis of 8000 hospital admissions for acute poisoning in a rural area of Sri Lanka. Clinical Toxicology (Philadelphia), 44, 225-231.

[3]   van der Hoek, W., Konradsen, F., Athukorala, K. and Wanigadewa, T. (1998) Pesticide poisoning: A major health problem in Sri Lanka. Social Science & Medicine, 46, 495-504.

[4]   Eddleston, M., Eyer, P., Worek, F., Mohamed, F., Senarathna, L., von Meyer, L., Juszczak, E., Hittarage, A., Azhar, S., Dissanayake, W., Sheriff, M.H., Szinicz, L., Dawson, A.H. and Buckley, N.A. (2005) Differences between organophosphorus insecticides in human self-poisoning: A prospective cohort study. Lancet, 366, 1452-1459.

[5]   Jeyaratnam, J., Lun, K.C. and Phoon, W.O. (1987) Survey of acute pesticide poisoning among agricultural workers in four Asian countries. Bulletin of the World Health Organization, 65, 521-527.

[6]   Calvert, G.M., Karnik, J., Mehler, L., Beckman, J., Morrissey, B., Sievert, J., Barrett, R., Lackovic, M., Mabee, L., Schwartz, A., Mitchell, Y. and Moraga-McHaley, S. (2008) Acute pesticide poisoning among agricultural workers in the United States, 1998-2005. American Journal of Industrial Medicine, 51, 883-898.

[7]   US Congress, Office of Technology Assessment (1992) Disposal of chemical weapons: Alternative technologiesBackground Paper. US Government Printing Office.

[8]   Eddleston, M., Karalliedde, L., Buckley, N., Fernando, R., Hutchinson, G., Isbister, G., Konradsen, F., Murray, D., Piola, J.C., Senanayake, N., Sheriff, R., Singh, S., Siwach, S.B. and Smit, L. (2002) Pesticide poisoning in the developing world: A minimum pesticides list. Lancet, 360, 1163-1167.

[9]   Buckley, N.A., Karalliedde, L., Dawson, A., Senanayake, N. and Eddleston, M. (2004) Where is the evidence for treatments used in pesticide poisoning? Is clinical toxicology fiddling while the developing world burns? Journal of Toxicology-Clinical Toxicology, 42, 113-116.

[10]   Jeyaratnam, J. (1990) Acute pesticide poisoning: A major global health problem. World Health Statistics Quarterly, 43, 139-144.

[11]   World Health Organization (1990) The public health impact of pesticides use in agriculture. WHO, Geneva, 128. Accessed 1 July 2012

[12]   Kesavachandran, C.N., Fareed, M., Pathak, M.K., Bihari, V., Mathur, N. and Srivastava, A.K. (2009) Adverse health effects of pesticides in agrarian populations of developing countries. In: Whitacre, D.M., Ed., Reviews of Environmental Contamination and Toxicology, 200, Springer Science + Business Media, New York, 33-52.

[13]   Coupe, R.H., Manning, M.A., Foreman, W.T., Goolsby, D.A. and Majewski, M.S. (2000) Occurrence of pesticides in rain and air in urban and agricultural areas of Mississippi, April-September 1995. Science of the Total Environment, 248, 227-240.

[14]   Schipper, P.N., Vissers, M.J. and van der Linden, A.M. (2008) Pesticides in groundwater and drinking water wells: Overview of the situation in the Netherlands. Water Science and Technology, 57, 1277-1286.

[15]   Sethunathan, N. and Yoshida, T. (1973) A Flavobacterium sp. that degrades diazinon and parathion. Canadian Journal of Microbiology, 19, 873-875.

[16]   Kawahara, K., Tanaka, A., Yoon, J. and Yokota, A. (2010) Reclassification of a parathione-degrading Flavobacterium sp. ATCC 27551 as Sphingobium fuliginis. Journal of General and Applied Microbiology, 56, 249-255.

[17]   Serdar, C.M., Gibson, D.T., Munnecke, D.M. and Lancaster, J.H. (1982) Plasmid involvement in parathion hydrolysis by Pseudomonas diminuta. Applied and Environmental Microbiology, 44, 246-249.

[18]   Segers, P., Vancanneyt, M., Pot, B., Torck, U., Hoste, B., Dewettinck, D., Falsen, E., Kersters, K. and De Vos, P. (1994) Classification of Pseudomonas diminuta Leifson and Hugh 1954 and Pseudomonas vesicularis Busing, Doll, and Freytag 1953 in Brevundimonas gen. nov. as Brevundimonas diminuta comb. nov. and Brevundimonas vesicularis comb. nov., respectively. International Journal of Systematic Bacteriology, 44, 499-510.

[19]   Caldwell, S.R., Newcomb, J.R., Schlecht, K.A. and Raushel, F.M. (1991) Limits of diffusion in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta. Biochemistry, 30, 7438-7444.

[20]   Dumas, D.P., Caldwell, S.R., Wild, J.R. and Raushel, F.M. (1989) Purification and properties of the phosphotriesterase from Pseudomonas diminuta. Journal of Biological Chemistry, 264, 19659-19665.

[21]   Dumas, D.P., Wild, J.R. and Raushel, F.M. (1989) Diisopropylfluorophosphate hydrolysis by a phosphotriesterase from Pseudomonas diminuta. Biotechnology and Applied Biochemistry, 11, 235-243.

[22]   Siddavattam, D., Khajamohiddin, S., Manavathi, B., Pakala, S. and Merrick, M. (2003) Transposon-like organization of the plasmid-borne organophosphate degradation (opd) gene cluster found in Flavobacterium sp. Applied and Environmental Microbiology, 69, 2533-2539.

[23]   Somara, S., Manavathi, B., Tebbe, C. and Siddavattam, D. (2002) Localisation of identical organophosphorus pesticide degrading (opd) genes on genetically dissimilar indigenous plasmids of soil bacteria: PCR amplification, cloning and sequencing of the opd gene from Flavobacterium balustinum. Indian Journal of Experimental Biology, 40, 774-779.

[24]   Horne, I., Qiu, X., Russell, R.J. and Oakeshott, J.G. (2003) The phosphotriesterase gene opdA in Agrobacterium radiobacter P230 is transposable. FEMS Microbiology Letters, 222, 1-8.

[25]   Horne, I., Sutherland, T.D., Harcourt, R.L., Russell, R.J. and Oakeshott, J.G. (2002) Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate. Applied and Environmental Microbiology, 68, 3371-3376.

[26]   Chaudhry, G.R., Ali, A.N. and Wheeler, W.B. (1988) Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp. Applied and Environmental Microbiology, 54, 288-293.

[27]   Harper, L.L., McDaniel, C.S., Miller, C.E. and Wild, J.R. (1988) Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes. Applied and Environmental Microbiology, 54, 2586-2589.

[28]   Pandeeti, E.V., Chakka, D., Pandey, J.P. and Siddavattam, D. (2011) Indigenous organophosphate-degrading (opd) plasmid pCMS1 of Brevundimonas diminuta is selftransmissible and plays a key role in horizontal mobility of the opd gene. Plasmid, 65, 226-231.

[29]   Wilson, K. (1990) Preparation of genomic DNA from bacteria. In: Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. Eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York.

[30]   Frank, J.A., Reich, C.I., Sharma, S., Weisbaum, J.S., Wilson, B.A. and Olsen, G.J. (2008) Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and Environmental Microbiology, 74, 2461-2470.

[31]   Baker, G.C., Smith, J.J. and Cowan, D.A. (2003) Review and re-analysis of domain-specific 16S primers. Journal of Microbiological Methods, 55, 541-555.

[32]   Sprenger, W.W., Dijkstra, A., Zwart, G.J., Agterveld, M.P., Noort, P.C. and Parsons, J.R. (2003) Competition of a parathion-hydrolyzing Flavobacterium with bacteria from ditch water in carbon-, nitrateand phosphate-limited continuous cultures. FEMS Microbiology Ecology, 43, 45-53.

[33]   Borodina, T.A., Lehrach, H. and Soldatov, A.V. (2003) DNA purification on homemade silica spin-columns. Analytical Biochemistry, 321, 135-137.

[34]   Hannon, G.J. (2010) FASTX-Toolkit.

[35]   Zerbino, D.R. and Birney, E. (2008) Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome Research, 18, 821-829.

[36]   Hernandez, D., Francois, P., Farinelli, L., Osteras, M. and Schrenzel, J. (2008) De novo bacterial genome sequencing: Millions of very short reads assembled on a desktop computer. Genome Research, 18, 802-809.

[37]   Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. Journal of Molecular Biology, 215, 403-410.

[38]   Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K. and Madden, T.L. (2009) BLAST+: Architecture and applications. BMC Bioinformatics, 10, 421.

[39]   Smith, D.R., Quinlan, A.R., Peckham, H.E., Makowsky, K., Tao, W., Woolf, B., Shen, L., Donahue, W.F., Tusneem, N., Stromberg, M.P., Stewart, D.A., Zhang, L., Ranade, S.S., Warner, J.B., Lee, C.C., Coleman, B.E., Zhang, Z., McLaughlin, S.F., Malek, J.A., Sorenson, J.M., Blanchard, A.P., Chapman, J., Hillman, D., Chen, F., Rokhsar, D.S., McKernan, K.J., Jeffries, T.W., Marth, G.T. and Richardson, P.M. (2008) Rapid whole-genome mutational profiling using next-generation sequencing technologies. Genome Research, 18, 1638-1642.

[40]   Li, H. and Durbin, R. (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics, 25, 1754-1760.

[41]   Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J. and Higgins, D.G. (2007) Clustal W and Clustal X version 2. Bioinformatics, 23, 2947-2948.

[42]   Felsenstein, J. (1989) PHYLIP-Phylogeny inference package. Cladistics, 5, 164-166.

[43]   Felsenstein, J. and Churchill, G.A. (1996) A Hidden Markov Model approach to variation among sites in rate of evolution. Molecular Biology and Evolution, 13, 93-104.

[44]   Stover, B.C. and Müller, K.F. (2010) TreeGraph 2: Combining and visualizing evidence from different phylogenetic analyses. BMC Bioinformatics, 11, 7.

[45]   Iyer, R., Iken, B. and Tamez, T. (2012) Isolation, molecular and biochemical identification of Paraoxon-metabolizing Pseudomonas species. Journal of Bioremediation and Biodegradation, 2, 2011, 5.

[46]   Anzai, Y., Kim, H., Park, J.Y., Wakabayashi, H. and Oyaizu, H. (2000) Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. International Journal of Systematic and Evolutionary Microbiology, 50, 1563-1568.

[47]   Moore, E.R.B., Mau, M., Arnscheidt, A., Bottger, E.C., Hutson, R.A., Collins, M.D., VandePeer, Y., DeWachter, Y. and Timmis, K.N. (1996) The determination and comparison of the 16S rRNA gene sequences of species of the genus Pseudomonas (sensu stricto) and estimation of the natural intrageneric relationships. Systematic and Applied Microbiology, 19, 478-492.

[48]   Mulet, M., Lalucat, J. and García-Valdés, E. (2010) DNA sequence-based analysis of the Pseudomonas species. Environmental Microbiology, 12, 1513-1530.

[49]   Ait Tayeb, L., Ageron, E., Grimont, F. and Grimont, P.A.D. (2005) Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates. Research in Microbiology, 156, 763-773.

[50]   Hilario, E., Buckley, T.R. and Young, J.M. (2004) Improved resolution on the phylogenetic relationships among Pseudomonas by the combined analysis of atpD, carA, recA and 16S rDNA. Antonie van Leeuwenhoek, 86, 51-64.

[51]   Peix, A., Ramírez-Bahena, M.H. and Velázquez, E. (2009) Historical evolution and current status of the taxonomy of genus Pseudomonas. Infection, Genetics and Evolution, 9, 1132-1147.

[52]   Yamamoto, S. and Harayama, S. (1998) Phylogenetic relationships of Pseudomonas putida strains deduced from the nucleotide sequences of gyrB, rpoD and 16S rRNA genes. International Journal of Systematic and Evolutionary Microbiology, 48, 813-819.

[53]   Yamamoto, S., Kasai, H., Arnold, D.L., Jackson, R.W., Vivian, A. and Harayama, S. (2000) Phylogeny of the genus Pseudomonas: Intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD genes. Microbiology, 146, 2385-2394.

[54]   Wang, S., Liu, Z., Tang, H., Meng, J. and Xu, P. (2007) Characterization of environmentally friendly nicotine degradation by Pseudomonas putida biotype A strain S16. Microbiology, 153, 1556-1565.

[55]   Tang, H., Yao, Y., Wang, L., Yu, H., Ren, Y., Wu, G. and Xu, P. (2012) Genomic analysis of Pseudomonas putida: Genes in a genome island are crucial for nicotine degradation. Scientific Reports, 2, 377.

[56]   Fujita, M., Ike, M. and Kamiya, T. (1993) Accelerated phenol removal by amplifying the gene expression with a recombinant plasmid encoding catechol-2,3-oxygenase. Water Research, 27, 9-13.

[57]   Takeo, M., Maeda, Y., Okada, H., Miyama, K., Mori, K., Ike, M. and Fujita, M. (1995) Molecular cloning and sequencing of the phenol hydroxylase gene from Pseudomonas putida BH. Journal of Fermentation and Bioengineering, 79, 485-488.

[58]   Shimao, M., Nakamura, T., Okuda, A. and Harayama, S. (1996) Characteristics of transposon insertion mutants of mandelic acid-utilizing Pseudomonas putida strain A10L. Bioscience, Biotechnology and Biochemistry, 60, 1051-1055.

[59]   Mulbry, W.W. and Karns, J.S. (1989) Parathion hydrolase specified by the Flavobacterium opd gene: Relationship between the gene and protein. Journal of Bacteriology, 171, 6740-6746.

[60]   Rani, N.L. and Lalithakumari, D. (1994) Degradation of methyl parathion by Pseudomonas putida. Canadian Journal of Microbiology, 40, 1000-1006.

[61]   Santos, P.M. and Sa-Correia, I. (2007) Characterization of the unique organization and co-regulation of a gene cluster required for phenol and benzene catabolism in Pseudomonas sp. M1. Journal of Biotechnology, 131, 371-378.

[62]   Cowles, C.E., Nichols, N.N. and Harwood, C.S. (2000) BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida. Journal of Bacteriology, 182, 6339-6346.

[63]   Cafaro, V., Notomista, E., Capasso, P. and Di Donato, A. (2005) Regiospecificity of two multicomponent monooxygenases from Pseudomonas stutzeri OX1: Molecular basis for catabolic adaptation of this microorganism to methylated aromatic compounds. Applied and Environmental Microbiology, 71, 4736-4743.

[64]   Ramos, J.L., Duque, E., Godoy, P. and Segura, A. (1998) Efflux pumps involved in toluene tolerance in Pseudomonas putida DOT-T1E. Journal of Bacteriology, 180, 3323-3329.

[65]   Ramos, J.L., Duque, E., Gallegos, M.T., Godoy, P., RamosGonzalez, M.I., Rojas, A., Teran, W. and Segura, A. (2002) Mechanisms of solvent tolerance in gram-negative bacteria. Annual Review of Microbiology, 56, 743-768.

[66]   Kim, K., Lee, S., Lee, K. and Lim, D. (1998) Isolation and characterization of toluene-sensitive mutants from the toluene-resistant bacterium Pseudomonas putida GM73. Journal of Bacteriology, 180, 3692-3696.

[67]   Hearn, E.M., Patel, D.R. and van den Berg, B. (2008) Outer-membrane transport of aromatic hydrocarbons as a first step in biodegradation. Proceedings of the National Academy of Sciences of the United States of America, 105, 8601-8606.