RESUMO
A bacterium(designated as LW-3), capable of degrading Chlorimuron-ethyl, was isolated from the long-term contaminated soil by Chlorimuron-ethyl. Based on physiological and biochemical analyses and sequences comparison of 16S rDNA, strain LW-3 was identified as Pseudomonas sp. LW-3 could use Chlorimuron-ethyl as sole nitrogen source for growth. The optimum pH and temperature for degradation of Chlorimuron-ethyl were pH 6.5~7.2 and 30?C~35?C, at same temperature the pH change to the Chlorimuron -ethyl degrading influence is large. When the pH and temperature were pH 6.5 and 32?C, 50 mg/L Chlori- muron-ethyl could be degraded to 70%~80% level within 7 days.
RESUMO
Construction of multifunctional pesticides-degrading genetically engineered microorganisms (GEMs) is increasing important in the bioremediation of various pesticides contaminants in environment. However, construction of genetically stable GEMs without any exogenous antibiotic resistance is thought to be one of the bottlenecks in GEMs construction. In this article, homologous recombination vectors with the recipient's 16S rDNA as homologous recombination directing sequence (HRDS) and sacB gene as double crossover recombinants positive selective marker were firstly constructed. The methyl parathion hydroalse gene (mpd) was inserted into the 16S rDNA site of the carbofuran degrading strain Sphingomonas sp. CDS-1 by homologous recombination single crossover in the level of about 3.7 x 10-(7) - 6.8 x 10(-7). Multifunctional pesticides-degrading GEMs with one or two mpd genes inserted into the chromosome without any antibiotic marker were successfully constructed. The homologous recombination events were confirmed by PCR and southern blot methods. The obtained GEMs were genetically stable and could degrade methyl parathion and carbofuran simultaneously. The insertion of mpd gene into rrn site did not have any significant effect on recipient' s physiological and original degrading characteristics. The methyl parathion hydrolase (MPH) was expressed at a relatively high level in the recombinants and the recombinant MPH specific activity in cell lysate was higher than that of original bacterium (DLL-1) in every growth phase tested. The highest recombinant MPH specific activity was 6.22 mu/tg. In this article, we describe a first attempt to use rRNA-encoding regions of Sphingomonas strains as target site for expression of exogenous MPH, and constructed multifunctional pesticides degrading GEMs, which are genetically stable and promising for developing bioremediation strategies for the decontamination of pesticides polluted soils.