The Genetic Characteristics And Degradation Properties Of A Thiodiglycol-degrading Bacterium

Thiodiglycol (TDG or TG) is dual-use chemical that can be used both in chemical weapon production and chemical industries. It is both the precursor for the synthesis of sulfur mustard and the main hydrolyzing product of it, which is one of the most toxic Chemical Warfare Agents (CWAs) to date. TDG is relatively persistent in the environment. It may remain several years or even decates in soil, posing a high risk of conteminating groudwater as it's fully miscible with water. It may also react with products of mustard degradation and other chemicals to produce more toxic and persistent chemicals in the environment. TDG is listed in the chemical weapons convention treaty and must be degraded in a mineralization process.Biological degradation is considered to be one of the most desirable methods to treat persistent and harmful organic pollutants because it is economic, safe, and efficient. Biodegradation has been playing a important role in the destroying of sulfur mustard stockpile. A combination of increasing academic and industrial attentions has been put on bioremediation technology. The pure culture capable of completely catabolizing TDG is the key factors in biological degradation. What's more, characterizing microbial genetic and degradation properties of TDG are more important step not only will help to understand the biodegradation process but open new door towards discovering gene evolution process under environmental presure. Microorganisms capable of degrading TDG used in the study were originaly isolated from sulfer mustard contaminated soil by Professor Li Hong. This work further purify and investigate the molecular genetics and degradation properties of the microorganisms. The mian results are listed as followings:1. The purification and identification of degrading strains. Two cultures based on colony morphology were isolated using selective medium from TDG-degrading freeze dried bacterial culture provided by Professor Li Hong, named01S and01B. The two strains were both confirmed to blong to Burkholderia sp. by16SrDNA sequeacing. Based on16SrDNA gene sequences analysis and construction of phylogenetic tree, the two strains had a high homology to Burkholderia xenovorans LB400(>97%).2. Generation properties of TDG-degrading strain Burkholderia sp.01S. Based on orthogonal experiment, the optimum growth conditions of the strain are:pH7.0, temperature30℃, and concentration of TDG500ppm. LC-MS analysis indicated that TDG was degradation by strain and not by non-biological degradation, In domestication experiment, TDG were added into basal salt medium at increased concentration levels as sole carbon sources. The result showed that the maximum tolerance concentration was1100ppm. A broad spectrum of substrate degradation experiments indicated that Burkholderia sp.01S was able to grow on butanol, TDG, glycerine and succinate as the sole carbon source. This is the first time to report that Burkholderia sp. could degrade organic sulfur compound (TDG). However, B. xenovorans LB400has no ability to degrade TDG3. Genome sequencing and basic analysis of Burkholderia sp.01S. The genome size of Burkholderia sp.01S is9.12Mb. The GC content is approximately63.07%. A total of8632genes have been predicted, which covered85.36%of the genome. The average gene length is880bp. The intergenic region length is1,303,046bp, which covered14.63%of the genome. Comparative genomic analysis showed that B. xenovorans LB400and Burkholderia sp.01S shared5812orthologous genes. The phylogenetic tree constructed based on orthologous genes suggested Burkholderia sp.01S is close to B. xenovorans LB400. Comparativ analysis also discovered a good correspondence between the core chromosomes of B. xenovorans LB400and genome of Burkholderia sp.01S, while the mega plasmid of B. xenovorans LB400was not found in Burkholderia sp.01S. The similarity search for degradation genes of the sulfur-containing organic compounds showed that they were present in both strains. Combined with Burkholderia sp.01S's proteomics data, the homologous gene of LysR-type transcriptional regulator BdhR may play a role in the TDG degradation.4. Preliminary exploration of horizontal gene transfer. The possibility of horizontal gene transfer from Burkholderia sp.01S into soil microflora and B. xenovorans LB400was simulated under laboratory conditions. TDG was used as a selective pressure. The experimental results are not conclusive to date. Without TDG selection pressure, Burkholderia sp.01S lost TDG degrading-ability. The growth of B. xenovorans LB400was severely suppressed when TDG presented as co-carbon source with tryptone as co-nutrients.In summary, this work was innovative as follows:1. This is the first time to report that Burkholderia sp. can degrade thiodiglycol.2. This is the first time to report the genome sequence of Burkholderia sp.01S of thiodiglycol degradation starin.