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Isolation and Gene Cloning of Metsulfuron-Methyl Degrading Strain and Resistant Strain

Author: HuangXing
Tutor: LiShunPeng
School: Nanjing Agricultural College
Course: Microbiology
Keywords: metsulfuron-methyl biodegradation and bioremediation herbicides resistance gene cloning
CLC: X172
Type: PhD thesis
Year: 2006
Downloads: 60
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Abstract


A bacterium S113 capable of degrading metsulfuron-methyl was isolated from metsulfuron-methyl contaminated soil. Based on the morphology, physiological and biochemical characteristics, and the homology analysis of its 16S rDNA sequence, S113 was identified preliminarily as Methylopila Sp.。S113 was capable of utilizing metsulfuron-methyl as the sole carbon source for its growth. This bacterium could degrade 98% of 50mg L-1 metsulfuron-methyl within 72h. Biological properties of S113 were studied. The optimal growth temperature and initial pH are 30℃, 7.0 respectively. The aeration had little effect on the growth of S113. The optimal medium for the growth of strain S113 was amylum as carbon source and organic nitrogen as nitrogen source.S113 could also degrade thifensulfuron-methy, bensulfuron, ethametsulfuron, but could not degrade chlorsulfuron and pyrazosulfuron. The optimal pH of S113 for degrading metsulfuron-methyl was 7.0. The degradation rate of metsulfuron-methyl by S113 was related positively to initial amount of inoculation. The aeration has little effect on degrading ability of S113。The speed of degradation of metsulfuron-methyl by S113 was related positively to initial amount of inoculum size. Peptone and yeast extract at certain concentration could promote the degradation of the strain.Metsulfuron-methyl hydrolase from metsulfuron-methyl degrading strain S113 was studied. The hydrolase in the bacterium was endoemzyme. The enzymatic reaction system was found as follows: Na2HPO4-NaH2PO4 buffer 2830μL, metsulfuron-methyl (1000 mg L-1) 150μL, crude enzyme 20μL (about 1.2μg crude protein), 30℃water bathing 30 min. The characteristics of metsulfuron-methyl hydrolase were determined. The pH suitable for keeping enzyme was 6-9, with the optimum pH 8.0. The activity of enzyme was down to 59.22% and zero when treated at 45℃for 30 min and 70℃for 30min respectively. Eight metal ions were chosen to study their effects on the metsulfuron-methyl hydrolase activity, and the results showed that 1 mmol L-1 Ca2+ could enhance the enzyme activity.A Strain of FLDA capable of degrading metsulfuron-methyl was isolated from sludge collected from a pesticide (metsulfuron-methyl) plant. Based on its morphology, physiological and biochemical characteristics, and the homology analysis of its 16S rDNA sequence, FLDA was identified preliminarily as Pseudomonas sp. FLDA could degrade 72.6% of 30 mg L-1 metsulfuron-methyl in liquid medium within 5 days. The optimal pH and temperature of FLDA for degrading metsulfuron-methyl was 7.0 and 30℃respectively. The degradation rate was related positively to initial inoculation rate. Enzyme distribution experiment showed that the metsulfuron-methyl degradeing enzyme in the bacterium was endoenzyme. The addition of strain FLDA could accelerate the degradation of metsulfuron-methyl in soil.A Strain of FLX capable of highly degrading thifensulfuron-methyl was isolated from the soil sample collected from a producing thifensulfuron-methyl pesticide plant after taming and enrichment. Based on analysis of phenotype, physiological and biochemical characteristics, and the homology analysis of its 16S rDNA sequence, FLX was identified preliminarily as Stenotrophomonas sp. FLX could degrade thifensulfuron-methyl in its 50 mg L-1 liquor medium, with 83.34% degrading rate in 48h. The optimal pH and temperature of FLX for degrading thifensulfuron-methyl were 7.0 and 35℃respectively. In the tested metal ions, Zn2+、Al3+、Cu2+、Ba2+、Fe3+ had little influence on FLX, while Hg2+、Co2+ inhibited its growth and degradation. The distribution experiment showed that the thifensulfuron-methyl hydrolysis enzyme in the bacterium was endoenzyme.The bioremediation of metsulfuron-methyl contaminated soil by inoculating S113 was studied under laboratory conditions. After addition of 108 cells g-1 dry soil into soil, 76.9% of metsulfuron-methyl at concentration of 10 mg kg-1 dry soil was degraded at 30d, whereas only 11.9% of metsulfuron-methyl was degraded in uninoculated soil. The degradation rate was related positively to the amount of inoculation. Only 39.6% of metsulfuron-methyl was degraded when the concentration of metsulfuron-methyl was 50 mg kg-1 dry soil. The optimal temperature for metsulfuron-methyl degradation by S113 in soil was 30℃. The addition of glucose and urea could accelerate the degradation of metsulfuron-methyl. Pour root and seed soaked with S113 could protect maize from the phytotoxicity of metsulfuron-methyl of 40, 80μg kg-1 in varying degrees. When the concentration of metsulfuron-methyl increased to 120μg kg-1, the effects were not distinct. It suggested that the metsulfuron-methyl in soil could be degraded effectively by inoculating S113.Two metsulfuron-methyl resistant strains, L6 and L36 were isolated from metsulfuron-methyl contaminated soil. Based on the homology analysis of its 16S rDNA sequence, morphology, physiological and biochemical characteristics, the L6, L36 were identified as Pseudomonas aeruginosa . Medium effective concentration (EC50) of metsulfuron-methyl against the growth of wild-type isolate PAO1 and resistant isolates L6, L36 were 0.36, 2.75, 2.89mM respectively; and the minimal inhibition concentrations (MICs) were 1.31mM, 6.03mM, 6.03mM respectively. The metsulfuron-methyl resistant strains showed no cross resistance with imazethapyr.Inhibition by metsulfuron-methyl of acetolactate synthase (ALS) activities of Pseudomonas aeruginosa wild-type isolate PAO1 and two resistant strains were assayed. The ALS activities of PAO1、L6、L36 were 391.4、122.8、120.4U respectively, the results showed ALS activities of resistant strains were significantly lower than that of PAO1. The activity of PAO1 ALS was inhibited 75.5% by 400nM SM, whereas the ALS produced by the resistant strains was not affected, which suggested that the occurrence of metsulfuron-methyl resistance were related to the decreased sensitivity of ALS to it. The ALS of L6 and L36 were not sensitive to metsulfuron-methyl. The ALS of L6 and L36 were sensitive to pH compared with that of PAO1.There was no difference among the sensitive of ALS produced by the SM sensitive or resistant strains to temperature. The ALS of PAO1, L6 and L36 were inhibited 68.6%, 45.3%, 46.2% by 500nM Val.The complete nucleotide sequences of two subunits (IlvI, IlvH) of acetolactate synthase (ALS) were cloned by PCR arnplification. The entire nucleotide sequences of ilvI, ilvH were 1725bp, 492bp in length, which encoded two polypeptides of 575, 164 amino acid residues respectively. The IlvI and IlvH subunits were highly conserved not only in Pseudomonas sp., but also in other organisms. By sequence blasting, an amino acid mutation Ala29(GCC)→Va129 (GTC) in IlvI subunit was found in L6 and L36, which might confer resistance of Pseudomonas aeruginosa to metsulfuron-methyl. The mutant ilvI gene from a resistant strain L36, containing the Ala29(GCC)→Va129 (GTC)mutation, was cloned by PCR amplification. The mutant ilvI gene was then ligated into vector pBBR1-MCS5, and shown to confer metsulfuron-methyl resistance in Pseudomonas aeruginosa when transfer into the wild-type sensitive strain PAO1. It was confirmed that the substitution of Ala to Val in the IlvI of ALS conferred metsulfuron-methyl resistance to Pseudomonas aeruginosa.

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