Isolation And Identification Of Chlororthalonil-Degrading Strains, The Degrading Characteristics And Cloning Of The Degrading Gene

Chlorothalonil (CTN) as an agricultural tetrachlorinated benzonitrile fungicide has been widely used and caused great pollution. As the toxicity of CTN in the environment, exploration of various CTN-degrading bacteria to clean-up the pollutant is of immense importance.Three strains of bacteria (designated as CTN-11, CTN-2, CTN-4 respectively) capable of degrading CTN efficiently, were isolated from a long-term CTN contaminated soil. Based on the comparative analysis of the 16S rRNA gene and morphological as well as biochemical characterization, these strains were identified as Ochrobactrum sp.CTN-11, Pseudomonas sp. CTN-2 and Rhizobiumsp. CTN-4, respectively. It was the first time for us to report a strain capable of degrading CTN belong to the genera Rhizobium. Strain CTN-11, which was selected for further study of its growth and degrading characteristics. The optimum growth is observed at 30℃,1-3% NaCl and pH 7.0. The optimal carbon source for the growth of CTN-11 was glucose, D-fructose and sucrose but it failed to utilize D-lactose. The optimal nitrogen source for the growth of CTN-11 was organic nitrogen source tryptone and yeast extract and ammonium sulfate was the best inorganic nitrogen. Strain CTN-11 did not used sodium nitrate as sole nitrogen source for growth. Strain CTN-11 showed resistant to ampicillin, penicillin, gentamicin, streptomycin, kanamycin, chloramphenicol, spectinomycin.Strain CTN-11 and CTN-4 showed little faster degradation rate than strain CTN-2 and 50 mg l-1 CTN was degraded to an undetectable level in 48 h. All the three degraders were markedly different from the previously reported CTN co-metabolic degraders, and they could efficiently degrade CTN when CTN was used as the sole carbon source. Strain CTN-11 could degrade CTN efficiently in a relatively broad range of temperatures from 20 to 40℃and the optimal temperature was at 35℃. Strain CTN-11 showed the highest degrading activity at pH values between 6.0 and 7.0, and lost its degrading ability when the pH value was below 5.0, it could aslo degrade CTN better when pH value between 8.0-9.0. The effect of alternative carbon sources on CTN biodegradation by strain CTN-11 were tested. It was found that alternative carbon sources could enhance the CTN degradation but not necessary for CTN biodegradation. It was found that Fe3+, Zn2+, Mn2+, Ca2+and Cu2+(1 mM) could enhance the CTN degradation, while, Ni2+, Co2+or Mg2+have no significant effect on the CTN degradation. The metabolite from CTN degradation by strain CTN-11 was identified as hydroxy-trichloroisophthalonitrile(CTN-OH) by second-order MS analysis. Comparison of the completely degradation product in water phase in the three degraders, CTN-OH was also the end product in the strain CTN-2 and CTN-4. Dechlorination of CTN under anaerobic conditions indicated that hydrolytic dechlorination reaction was involved in the degradation of CTN, rather than the oxidative dechlorination reaction. It was found that 50 mg l-1 CTN were degraded to a non-detectable level in both sterile soil and non-sterile soil, suggesting that strain CTN-11 could be successfully used for the removal of CTN from contaminated soils.Comparison the dechlorination mechanism of the reported CTN degraing strain Ochrobactrum anthropi SH35B and Ochrobactrum. sp. CTN-11, as a result, a GST-like gene was cloned by PCR method from Ochrobactrum sp. CTN-llwhich had 88% similarities with that from Ochrobactrum anthropi SH35B, however, it was not functionally expressed at the presence of glutathione. These data indicated that CTN was not reductively dechlorinated by thiolytic substitution catalyzed by GST in Ochrobactrum. sp. CTN-11. The different degrading mechanisms of CTN between Ochrobactrum sp. CTN-11 and Ochrobactrum anthropi SH35B indicated the dechlorination metabolic diversity of CTN in the same genera. Chlorothalonil hydrolytic dehalogenase (Chd) gene (chd) were cloned from three degraders by the PCR or shotgun method, which had highly conservatism with similarity ranged from 99.8 to 100%, even though three CTN degraders were placed in the different phylogenesis genera. The analysis of the gene and promotor sequence of insertion fragment (3991bp) from strain CTN-11 revealed that this gene fragment had three complete ORF, which were identified as potential transposase gene, ATP binding protein gene and chd gene and placed in the same transcriptional orientation, a putative promotor in front of the potential transposase gene could start transcription and a independent promotor were discovered ahead of chd gene. This study found the logistical proof and the molecular basis of the horizontal transfer of the chd gene, suggesting the horizontal gene transfer is the pathway for the microbial community to adapting CTN contaminated ecology environment and help them obtain the CTN-degrading ability.