Applied Microbiology and Biotechnology

, Volume 73, Issue 6, pp 1452–1462 | Cite as

Biodegradation of methyl parathion and p-nitrophenol: evidence for the presence of a p-nitrophenol 2-hydroxylase in a Gram-negative Serratia sp. strain DS001

  • Suresh B. Pakala
  • Purushotham Gorla
  • Aleem Basha Pinjari
  • Ravi Kumar Krovidi
  • Rajasekhar Baru
  • Mahesh Yanamandra
  • Mike Merrick
  • Dayananda Siddavattam
Environmental Biotechnology


A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component “A” were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component “A”, typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia.


Parathion hydrolase p-Nitrophenol hydroxylase component A Serratia sp Biodegradation Catabolomics 



We thank Dr. Reddanna for providing GCMS facility and the University Grants Commission, New Delhi and the Department of Science and Technology, New Delhi for providing financial support to carry out the research work. ABP is a recipient of Junior Research Fellowship from UGC, New Delhi. DS is also a recipient of financial support from Institute of Life Sciences. MM is supported by the Biotechnology and Biological Sciences Research Council, UK.

Supplementary material

253_2006_595_MOESM1_ESM.doc (48 kb)
Fig. 1Mass spectrum identifying p-nitrophenol, a degradation product of methyl parathion (DOC 48 640 KB)
253_2006_595_MOESM2_ESM.doc (42 kb)
Fig. 2Mass spectrum identifying dimethyl thiophosphoric acid, a degradation product of methyl parathion (DOC 43 520 KB)
253_2006_595_MOESM3_ESM.doc (60 kb)
Fig. 3Mass spectrum identifying 4-nitrocatechol, a key intermediate in degradation of PNP (DOC 60 928 KB)
253_2006_595_MOESM4_ESM.doc (95 kb)
Fig. 4Mass spectrum identifying 1,2,4-benzenetriol, a key intermediate in degradation of PNP (DOC 97 280 KB)
253_2006_595_MOESM5_ESM.doc (862 kb)
Fig. 52D analysis of the proteome of Serratia sp. strain DS001 cells grown in a glucose, b methylparathion, and cp-nitrophenol (DOC 882 688 KB)


  1. Barnes H, Folkard AR (1951) The determination of nitrites. Analyst 76:599–603CrossRefGoogle Scholar
  2. Benning MM, Kuo JM, Raushel FM, Holden HM (1994) Three-dimensional structure of phosphotriesterase: an enzyme capable of detoxifying organophosphate nerve agents. Biochemistry 33:15001–15007CrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  4. Brown KA (1980) Phosphotriesterases of Flavobacterium sp. Soil Biol Biochem 12:105–112CrossRefGoogle Scholar
  5. Cassidy MB, Lee H, Trevors JT, Zablotowicz RB (1999) Chlorophenol and nitrophenol metabolism by Sphingomonas sp. UG 30. J Ind Microbiol Biotechnol 23:232–241CrossRefGoogle Scholar
  6. Chaudhry GR, Ali AN, Wheeler WB (1988) Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp. Appl Environ Microbiol 54:288–293Google Scholar
  7. Chauhan A, Samanta SK, Jain RK (2000) Degradation of 4-nitrocatechol by Burkholderia cepacia: a plasmid-encoded novel pathway. J Appl Microbiol 88:764–772CrossRefGoogle Scholar
  8. Chen WX, Tan ZY, Gao JL, Li Y, Wang ET (1997) Rhizobium hainanense sp. nov., isolated from tropical legumes. Int J Syst Bacteriol 47:870–873Google Scholar
  9. Cho CM, Mulchandani A, Chen W (2004) Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos. Appl Environ Microbiol 70:4681–4685CrossRefGoogle Scholar
  10. Daubaras DL, Saido K, Chakrabarty AM (1996) Purification of Hydroxyquinol 1,2-dioxygenase and Maleylacetate reductase: the lower pathway of 2,4,5-trichlorophenoxyacetic acid metabolism by Burkholderia cepacia AC1100. Appl Environ Microbiol 62:4276–4279Google Scholar
  11. Duffes F, Jenoe P, Boyaval P (2000) Use of two-dimensional electrophoresis to study differential protein expression in divercin V41-resistant and wild-type strains of Listeria monocytogenes. Appl Environ Microbiol 66:4318–4324CrossRefGoogle Scholar
  12. Errampalli D, Tresse O, Lee H, Trevors JT (1999) Bacterial survival and mineralization of p-nitrophenol in soil by green fluorescent protein-marked Moraxella sp. G21 encapsulated cells. FEMS Microbiol Ecol 30:229–236CrossRefGoogle Scholar
  13. Harper LL, Mcdaniel CS, Miller CE, Wild JR (1988) Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes. Appl Environ Microbiol 54:2586–2589Google Scholar
  14. Hayatsu M, Hirano M, Tokuda S (2000) Involvement of two plasmids in Fenitrothion degradation by Burkholderia sp. strain NF100. Appl Environ Microbiol 66:1737–1740CrossRefGoogle Scholar
  15. Jain RK, Dreisbach JH, Spain JC (1994) Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp. Appl Environ Microbiol 60:3030–3032Google Scholar
  16. Kadiyala V, Spain JC (1998) A two-component monooxygenase catalyzes both the hydroxylation of p-nitrophenol and the oxidative release of nitrite from 4-nitrocatechol in Bacillus sphaericus JS905. Appl Environ Microbiol 64:2479–2484Google Scholar
  17. Kitagawa W, Kimura N, Kamagata Y (2004) A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101. J Bacteriol 186:4894–4902CrossRefGoogle Scholar
  18. Leung KT, Tresse O, Errampalli D, Lee H, Trevors JT (1997) Mineralization of p-nitrophenol by pentachlorophenol-degrading Sphingomonas spp. FEMS Microbiol Lett 155:107–114CrossRefGoogle Scholar
  19. Leung KT, Campbell S, Gan Y, White DC, Lee H, Trevors JT (1999) The role of the Sphingomonas species UG 30 pentachlorophenol-4-monooxygenase in p-nitrophenol degradation. FEMS Microbiol Lett 173:247–253CrossRefGoogle Scholar
  20. Liu H, Zhang J, Wang S, Zhang X, Zhou N (2005) Plasmid-borne catabolism of methylparathion and p-nitrophenol in Pseudomonas sp. strain WBC-3. Biochem Biophys Res Commun 334:1107–1114CrossRefGoogle Scholar
  21. Loper JC (1968) Histidinol dehydrogenase from Salmonella typhimurium. Crystallization and composition studies. J Biol Chem 243:3264–3272Google Scholar
  22. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  23. Mcdaniel CS, Harper LL, Wild JR (1988) Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase. J Bacteriol 170:2306–2311Google Scholar
  24. Mitra D, Vaidyanathan CS (1984) A new 4-nitrophenol 2-hydroxylase from a Nocardia sp. Biochem Int 8:609–615Google Scholar
  25. Mulbry WW, Karns JS (1989) Purification of three parathion hydrolases from gram-negative bacterial strains. Appl Environ Microbiol 55:289–293Google Scholar
  26. Mulbry WW, Karns JS, Kearney PC, Nelson JO, Mcdaniel CS, Wild JR (1986) Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta. Appl Environ Microbiol 51:926–930Google Scholar
  27. Muller DM, Schlomann M, Reineke W (1996) Maleylacetate reductases in chloroaromatic-degrading bacteria using the modified ortho pathway: comparison of catalytic properties. J Bacteriol 178:298–300Google Scholar
  28. Munnecke DM, Hsieh DP (1976) Pathways of microbial metabolism of parathion. Appl Environ Microbiol 31:63–69Google Scholar
  29. Murray KC, Duggleby J, Sala-Trepat JM, Williams PA (1972) The metabolism of benzoate and methylbenzoates via the meta-cleavage pathway by Pseudomonas arvilla mt-2. Eur J Biochem 28:301–310CrossRefGoogle Scholar
  30. Prakash D, Chauhan A, Jain RK (1996) Plasmid-encoded degradation of p-nitrophenol by Pseudomonas cepacia. Biochem Biophys Res Commun 224:375–381CrossRefGoogle Scholar
  31. Ramanathan MP, Lalithakumari D (1999) Complete mineralization of methylparathion by Pseudomonas sp. A3. Appl Biochem Biotechnol 80:1–12CrossRefGoogle Scholar
  32. Rani NL, Lalithakumari D (1994) Degradation of methyl parathion by Pseudomonas putida. Can J Microbiol 40:1000–1006CrossRefGoogle Scholar
  33. Roldan MD, Blasco R, Caballero FJ, Castillo F (1998) Degradation of p-nitrophenol by the phototrophic bacterium Rhodobacter capsulatus. Arch Microbiol 169:36–42Google Scholar
  34. Serdar CM, Murdock DC, Rohde MF (1989) Parathion hydrolase gene from Pseudomonas diminuta MG: subcloning, complete nucleotide sequence, and expression of the mature portion of the enzyme in Escherichia coli. Biotechnology 7:1151–1155Google Scholar
  35. Siddaramappa R, Rajaram KP, Sethunathan N (1973) Degradation of parathion by bacteria isolated from flooded soil. Appl Microbiol 26:846–849Google Scholar
  36. Singh BK, Walker A (2006) Microbial degradation of organophosphorus compounds. FEMS Microbiol Rev 30:428–471CrossRefGoogle Scholar
  37. Somara S, Siddavattam D (1995) Plasmid mediated organophosphate pesticide degradation by Flavobacterium balustinum. Biochem Mol Biol Int 36:627–631Google Scholar
  38. Spain JC, Gibson DT (1991) Pathway for biodegradation of p-nitrophenol in a Moraxella sp. Appl Environ Microbiol 57:812–819Google Scholar
  39. Takeo M, Yasukawa T, Abe Y, Nihara S, Marda Y, Negoro S (2003) Cloning and characterization of a 4-nitrophenol hydroxylase gene cluster from Rhodococcus sp. PN1. J Biosci Bioeng 95:139–145Google Scholar
  40. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882CrossRefGoogle Scholar
  41. Weigent DA, Nester EW (1976) Purification and properties of two aromatic aminotransferases in Bacillus subtilis. J Biol Chem 251:6974–6980Google Scholar
  42. Zhang R, Cui Z, Jaindong J, Jain H, Xiangyang G, Sphunpeng L (2005) Diversity of organophosphorus pesticide degrading bacteria in a polluted soil and conservation of their organophosphorus hydrolase genes. Can J Microbiol 51:337–343CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Suresh B. Pakala
    • 1
  • Purushotham Gorla
    • 1
  • Aleem Basha Pinjari
    • 1
  • Ravi Kumar Krovidi
    • 2
  • Rajasekhar Baru
    • 2
  • Mahesh Yanamandra
    • 2
  • Mike Merrick
    • 3
  • Dayananda Siddavattam
    • 1
  1. 1.Department of Animal SciencesUniversity of HyderabadHyderabadIndia
  2. 2.Discovery Research, Dr. Reddy’s Laboratories Ltd.HyderabadIndia
  3. 3.Department of Molecular MicrobiologyJohn Innes CentreNorwichUK

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