Abstract
The genes encoding enzymes involved in the initial reactions during degradation of 4-chloronitrobenzene (4CNB) were characterized from the 4CNB utilizer Pseudomonas putida ZWL73, in which a partial reductive pathway was adopted. A DNA fragment containing genes coding for chloronitrobenzene nitroreductase (CnbA) and hydroxylaminobenzene mutase (CnbB) were PCR-amplified and subsequently sequenced. These two genes were actively expressed in Escherichia coli, and recombinant E. coli cells catalyzed the conversion of 4CNB to 2-amino-5-chlorophenol, which is the ring-cleavage substrate in the degradation of 4CNB. Phylogenetic analyses on sequences of chloronitrobenzene nitroreductase and hydroxylaminobenzene mutase revealed that these two enzymes are closely related to the functionally identified nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas strains JS45 and HS12. The nitroreductase from strain ZWL73 showed a higher specific activity toward 4CNB than nitrobenzene (approximately at a ratio of 1.6:1 for the recombinant or 2:1 for the wild type), which is in contrast to the case where the nitroreductase from nitrobenzene utilizers Pseudomonas pseudoalcaligenes JS45 with an apparently lower specific activity against 4CNB than nitrobenzene (0.16:1) [Kadiyala et al. Appl Environ Microbiol 69:6520–6526, 2003]. This suggests that the nitroreductase from 4-chloronitrobenzene utilizer P. putida ZWL73 may have evolved to prefer chloronitrobenzene to nitrobenzene as its substrate.
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References
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 72:248–254
Bryant C, DeLuca M (1991) Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae. J Biol Chem 266:4119–4125
Davis JK, He Z, Somerville CC, Spain JC (1999) Genetic and biochemical comparison of 2-aminophenol 1,6-dioxygenase of Pseudomonas pseudoalcaligenes JS45 to meta-cleavage dioxygenases: divergent evolution of 2-aminophenol meta-cleavage pathway. Arch Microbiol 172:330–339
Davis JK, Paoli GC, He Z, Nadeau LJ, Somerville CC, Spain JC (2000) Sequence analysis and initial characterization of two isozymes of hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45. Appl Environ Microbiol 66:2965–2971
Groenewegen PE, Breeuwer P, van Helvoort JM, Langenhoff AA, de Vries FP, de Bont JA (1992) Novel degradative pathway of 4-nitrobenzoate in Comamonas acidovorans NBA-10. J Gen Microbiol 138:1599–1605
Haggblom MM (1992) Microbial breakdown of halogenated aromatic pesticides and related compounds. FEMS Microbiol Rev 9:29–71
Johnson GR, Spain JC (2003) Evolution of catabolic pathways for synthetic compounds: bacterial pathways for degradation of 2,4-dinitrotoluene and nitrobenzene. Appl Microbiol Biotechnol 62:110–123
Kadiyala V, Nadeau LJ, Spain JC (2003) Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols. Appl Environ Microbiol 69:6520–6526
Katsivela E, Wray V, Pieper DH, Wittich RM (1999) Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium, strain LW1. Appl Environ Microbiol 65:1405–1412
Lendenmann U, Spain JC (1996) 2-Aminophenol 1,6-dioxygenase: a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS45. J Bacteriol 178:6227–6232
Lessner DJ, Johnson GR, Parales RE, Spain JC, Gibson DT (2002) Molecular characterization and substrate specificity of nitrobenzene dioxygenase from Comamonas sp. strain JS765. Appl Environ Microbiol 68:634–641
Linch AL (1974) Biological monitoring for industrial exposure to cyanogenic aromatic nitro and amino compounds. Am Ind Hyg Assoc J 35:426–432
Liu SJ, Steinbüchel A (2000) Exploitation of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid). Appl Microbiol Biotechnol 53:545–552
Liu H, Wang SJ, Zhou NY (2005) A new isolate of Pseudomonas stutzeri that degrades 2-chloronitrobenzene. Biotechnol Lett 27:275–278
Nishino SF, Spain JC (1993) Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes. Appl Environ Microbiol 59:2520–2525
Park HS, Kim HS (2000) Identification and characterization of the nitrobenzene catabolic plasmids pNB1 and pNB2 in Pseudomonas putida HS12. J Bacteriol 182:573–580
Park HS, Lim SJ, Chang YK, Livingston AG, Kim HS (1999) Degradation of chloronitrobenzenes by a coculture of Pseudomonas putida and a Rhodococcus sp. Appl Environ Microbiol 65:1083–1091
Peterson FJ, Mason RP, Hovsepian J, Holtzman JL (1979) Oxygen-sensitive and -insensitive nitroreduction by Escherichia coli and rat hepatic microsomes. J Biol Chem 254:4009–4014
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Shimizu M, Yasui Y, Matsumoto N (1983) Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium—a series of chloro- or fluoro-nitrobenzene derivatives. Mutat Res 116:217–238
Somerville CC, Nishino SF, Spain JC (1995) Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45. J Bacteriol 177:3837–3842
Takenaka S, Murakami S, Shinke R, Hatakeyama K, Yukawa H, Aoki K (1997) Novel genes encoding 2-aminophenol 1,6-dioxygenase from Pseudomonas species AP-3 growing on 2-aminophenol and catalytic properties of the purified enzyme. J Biol Chem 272:14727–14732
Takenaka S, Murakami S, Kim YJ, Aoki K (2000) Complete nucleotide sequence and functional analysis of the genes for 2-aminophenol metabolism from Pseudomonas sp. AP-3. Arch Microbiol 174:265–272
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882
Wu JF, Shen XH, Zhou YG, Liu SJ (2004) Characterization of p-chloronitrobenzene-degrading Comamonas sp. CNB-1 and its degradation of p-chloronitrobenzene. Acta Microbiol Sin 44:8–12
Wu JF, Sun CW, Jiang CY, Liu ZP, Liu SJ (2005) A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli. Arch Microbiol 183:1–8
Wu JF, Jiang CY, Wang BJ, Ma YF, Liu ZP, Liu SJ (2006) Novel partial reductive pathway for 4-chloronitrobenzene and nitrobenzene degradation in Comamonas sp. strain CNB-1. Appl Environ Microbiol 72(3):1759–1765
Zhen D, Liu H, Wang SJ, Zhang JJ, Zhao F, Zhou NY (2006) Plasmid-mediated degradation of 4-chloronitrobenzene by newly isolated Pseudomonas putida ZWL73. Appl Microbiol Biotechnol (in press). DOI 10.1007/s00253-006-0345-2
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This research was supported by a grant from National Natural Science Foundation of China (NSFC30230010).
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Y.X. and J.-F.W. equally contributed to this work.
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Xiao, Y., Wu, JF., Liu, H. et al. Characterization of genes involved in the initial reactions of 4-chloronitrobenzene degradation in Pseudomonasputida ZWL73. Appl Microbiol Biotechnol 73, 166–171 (2006). https://doi.org/10.1007/s00253-006-0441-3
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DOI: https://doi.org/10.1007/s00253-006-0441-3