| Synthetical pyrethroids (SPs), which are synthetic version of an extract from the chrysanthemum, are now the major class of insecticides used for insect control in agriculture and households as a replacement for more toxic and environmentally persistent organochlorine and organophosphorus pesticides. Currently, SPs annual sales are estimated to reach above 1.5 billion dollars and rank the second in global insecticide sales. In China, SPs account for 1/3 of total pesticides application area.Although pyrethroid pesticides generally have lower acute oral mammalian toxicity than organophosphate and organochlorine insecticides, exposure to pyrethroid pesticides might cause damage to reproductive, immune, endocrine and cardiovascular systems. Especially, most pyrethroid pesticides possess acute toxicity to some nontarget organisms, such as bees, fish, and aquatic invertebrates, with LC50 values less than 0.5μg/L and factor of safety lower than 0.063 to fish.Great concerns have been raised about the persistence and degradation of pyrethroid pesticides in the environment. Bioremediation based on the degradation ability of microorganisms was an environment friendly method in the treatment of pollutants.The aim of this study was to isolate pyrethroid-degrading strains, elucidate the degradation pathway, clone the degradation-related genes, which would be helpful to research the gene function and establish foundation of construct the genetic engineered strain by enhanced degrading capacity.Three novel bacteria strains designated LQY-7T, LQY-18Tand BA-3Twere isolated from activated sludge in a synthetic pyrethroid-manufacturing wastewater treatment facility in Yangnong Chemical Group Co., LTD., Jiangsu Province, China by using a classic enrichment method. Strains LQY-7T, LQY-18Tand BA-3T showed obviously distincted morphological, chemotaconomic and phylogentic characteristics from their cloest neighbors. Based on the polyphasic taxonomic results, the isolated strains LQY-7T, LQY-18T and BA-3Twere assigned to the members of the genus Flavobacterium, Sphingobacterium and Sphingobium.LQY-7T cells are Gram-negative rods,0.3-0.7μm in width and 1.3-2.0μm in length, non-spore-forming, and motile by gliding. Congo red is not absorbed and flexirubin-type pigments are not produced. Colonies on TSA are yellow, glistening, sticky, convex and circular with entire margins. The major fatty acids (>5%) are iso-C15:0(39.5%), iso-C15:1 G (26.3%), iso-C17:03-OH (7.9%), anteiso-C15:0(6.3%) and iso-Ci1:0 3-OH (5.6%). The major isoprenoid quinone is MK-7 (87%) and MK-6 (13%), the DNA G+C content is 34 mol%. on the basis of the phylogenetic evidence and fatty acids together with the phenotypic characteristics, strain LQY-7T should be classified as representing a novel species of the genus Flavobacterium, for which the name Flavobacterium haoranii sp. nov. is proposed.LQY-18T cells are Gram-negative, non-motile, non-spore-forming, non-flagellated, strictly aerobic rods, approximately 0.6-1.4μm in length and 0.3-0.6μm in diameter. After 3 days of incubation on TSA, colonies are 1.0-2.0 mm in diameter, yellowish, convex, circular and smooth with entire margins. The almost complete 16S rRNA gene sequence of strain LQY-18T indicated that strain LQY-18T belongs to the genus Sphingobacterium of the phylum Bacteroidetes and showed sequence similarities of 89.1-92.9% with recognized species of the genus Sphingobacterium. Phylogenetic analysis demonstrates strain LQY-18T belongs to the genus Sphingobacterium and forms a subclade with Sphingobacterium shayense CCTCC AB 209006T. The major respiratory quinone is MK-7. The major polar lipid is phosphatidylethanolamine and an unknown aminolipid and several unknown polar lipids are also detected. Sphingolipid is present. The major cellular fatty acids are summed feature 3 (C16:1ω6c and/or C16:1ω7c), iso-C15:0 and iso-C17:0 3-OH. The DNA G+C content of the type strain is 40.3 mol%. On the basis of phenotypic, genotypic and phylogenetic properties. Strain LQY-18T should be classified as a novel species of the genus of Sphingobacterium, for which the name Sphingobacterium wenxiniae sp. nov. is proposed.BA-3T cells are aerobic, Gram-negative, catalase-and oxidase-positive, non-motile, non-sporulating rods with rounded ends, approximately 0.6-0.8μm wide and 1.0-1.2μm long. Colonies on 10-fold-diluted LB agar are circular (0.2-0.4 cm in diameter), convex and cream-white. The major quinone is ubiquinone Q-10. The Major fatty acids(>5%) are summed feature 8 (C18:1ω6c and/or C18：1ω7c), C16:0, summed feature 3 (C16:1ω6c and/or C16:1ω7c) and C14:0 2-OH. The polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), sphingoglycolipids (SGL1-SGL2), glycolipid (GL) and phosphatidylcholine (PC). The major cellular polyamine is spermidine. DNA G+C content is 63.8 mol%. Strain BA-3T should be classified as a novel species of the genus of Sphingobium, for which the name Sphingobium jiangsuense sp. nov.BA-3Tis proposed.The primers were constructed based on the published sequence of the pyrethroid-hydrolying gene. The pyrethroid-hydrolying gene(pytH) was cloned by PCR from JZ-2. The analysis and alignment of the pyrethroid-hydrolying gene revealed that this gene has 840 bases, which coding for the pyrethroid hydrolase contained 280 amino acids. The pytH gene from the strain JZ-2 was linked to the expression plasmid vector pET29a, the expression strain of the gene was gained by transfer of the expression plasmid into strain BL21 (DE3).Error Prone PCR was used to generate PytH variants improvement in hydrolysis of substrates and a mutation was obtained. The variant, LB37 which had two site mutations (Y19F, H127R), exhibited increase energy for bifenthrin hydrolysis.According to the results we obtained two site-mutation variants by site-directed mutagenesis. Study of their kinetic parameters for hydrolysis of bifenthrin was performed. Results showed that M19 exhibited a 5.5-fold increase in kcat/Km values and H127R exhibited a 14-fold increase in kcat/Km values. The best variant Mn7 displayed a 6-fold increase in kcat and 70-fold increase in kcat/Km values, this is synthetical effect of two mutants.The proposal metabolic pathway of 3-Phenoxybenzoic acid was deduced as follows: strain JZ-2 metabolized 3-Phenoxybenzoic acid to m-hydroxybenzoic acid and catechol; catechol then metabolized by ortho cleavage pathway, m-hydroxybenzoic acid could not be degraded.The reported catechol 1,2-dioxygenasegenes were aligned using Clustalx software and degenerated primers were designed according to the conserved sequence. A 400bp length segment was amplified from JZ-2 genomic DNA, and sequence analysis showed a significant level of homology (98%) to catechol 1,2-dioxygenase of Sphingomonas sp. KA1 plasmid pCAR3 (GeneBank Aeeession No.AB270530). Gene catA was cloned into the suicide vector PJQ200SK for knocking out of the catA in JZ-2 and the resultant strain JZ-2-dcatA lost the ability to degrade catechol and the feedback control of hydroquinone bring on the consequence that JZ-2-AcatA can just degrade 3-PBA slightly, about 30%in 24 hours. When used strain JZ-2 to degrade the SPs residue in Tea in Lishui, Zhejiang Province, we found that the degradation rate reached 55-75%, showed that strain JZ-2 has a strong prospect.A genetically engineered microorganism(GEM) designated as JZ-2-mbhDHIM, capable of mineraling 3-PBA was successfully constructed by homologous recombination. The 3-HBA-degrading genes mbhDHIM was inserted into the chromosome of JZ-2 by using a homologous recombination vector with sacB gene as counter selectable marker. The GEM was relatively stable and without bringing any antibiotic marker. The homologous recombination events were confirmed by PCR detection. |
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