Cloning of the organophosphorus pesticide hydrolase gene clusters of seven degradative bacteria isolated from a methyl parathion contaminated site and evidence of their horizontal gene transfer Ruifu Zhang 1 , Zhongli Cui 1 , Xiaozhou Zhang 1 , Jiandong Jiang 1 , Ji-Dong Gu 2 & Shunpeng Li 1, * 1 Department of Microbiology, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, 6 Tongwei Road, 210095, Nanjing, Jiangsu, P.R. China; 2 Laboratory of Environmental Microbiology and Toxicology, Department of Ecology & Biodiversity, The University of Hong Kong, Kadoorie Biological Sciences Building, Pokfulam Road, Hong Kong SAR, P.R. China (*author for correspondence: e-mail: lsp@njau.edu.cn) Accepted 2 September 2005 Key words: horizontal gene transfer, organophosphorus hydrolase gene cluster, signal peptide, degradation Abstract Seven organophosphorus pesticide-degrading bacteria harboring the methyl parathion degrading (mpd) gene were isolated from a methyl parathion contaminated site. In this study, the 4.7 kb mpd gene cluster, conserved in all seven bacteria capable of degrading methyl parathion, was cloned and further analysis revealed that this cluster contained five ORFs and the mpd gene was associated with a mobile element, IS6100. In addition to mpd gene ORF and tnpA ORF, three other ORFs showed high homology to the permease component of ABC-type transport system, the general secretion pathway protein B, and the RNA polymerase sigma 70 factor, respectively. The mpd genes of these 7 strains were subcloned and expressed in E. coli, SDS-PAGE and zymogram analysis showed that two expression products of mpd genes in E. coli were found, but the one without signal peptide showed the hydrolytic activities. Our evidences collectively suggest that mpd gene cluster may be disseminated through horizontal gene transfer based on phylogenetic analysis of the cluster and their host bacterial strains, and comparisons of GC content of the cluster and respective host’s chromosome. Introduction Organophosphorus pesticides such as parathion and methyl parathion are used worldwide to con- trol major insect pests. These insecticides are potent acetylcholinesterase (AchE) inhibitors. Since AchE is also present in all vertebrates, the potential damage by this class of insecticides to non-target organisms is extremely high, and such pesticides are now being banned in developed countries. However, they remain the major insec- ticides in agricultural pest management of the developing countries. Bacterial enzymatic detoxification of organo- phosphorus pesticide has become the focus of many studies, because it is economical and effective. A number of soil bacteria, belong to the genera of Flavobacterium (Sethunathan & Yoshida 1973), Pseudomonas (Serdar et al. 1982), Agrobacterium (Horne et al. 2002), have been found capable of degrading parathion, methyl parathion and related organophosphorus pesticides. These bacteria were isolated from diverse geographical locations, but all of them synthesized organophosphorus pesticide hydrolases (OPH), which were encoded by the organophosphorus pesticide degrading (opd) gene responsible for the first step reaction in the degra- dation of parathion or methyl parathion prior to the formation of p-nitrophenol (McDaniel et al. 1988). The opd genes from P. diminuta GM and Biodegradation (2006) 17: 465–472 Ó Springer 2006 DOI 10.1007/s10532-005-9018-6