邻氯硝基苯降解菌Pseudomonas putida OCNB-1的分离、鉴定与降解质粒研究

摘要：以邻氯硝基苯为唯一碳源、氮源和能源从某化工厂废水处理站分离出一株细菌, 经微生物自动测定仪WSVTK-R07.02和16SrRNA扩增测序鉴定为Pseudomonas putida；在 培养基pH为8.0，培养温度32 ℃，摇床转速120 rpm 条件下考察了该菌株降解邻氯硝基苯的能力, 菌株42 h内将起始浓度为1.07 mM的对邻氯硝基苯降解近80%；经质粒检测,在菌株OCNB-1中发现一条质粒条带,选用7种内切酶对质粒pOCNB-1进行单酶切,得出质粒大小约为32 Kb；抗生素抗性实验表明菌株对红霉素、氨苄青霉素、青霉素钠的抗性与质粒无关，对利福平、氯霉素的抗性与质粒有关；通过细菌接合，质粒转移到无降解邻氯硝基苯能力的Pseudomonas.stutzeri受体菌中，接合子Pseudomonas.stutzeri能利用邻氯硝基苯为唯一碳源、氮源和能源，证明质粒为降解质粒。

Studies on Isolation, Characterization and Degradative Plasmid of o-Chloronitrobenzene-degrading Strain Pseudomonas putida OCNB-1

Abstract：A bacterial strain that took o-chloronitrobenzene as sole carbon, nitrogen and energy source was isolated from the activated sludge of waste treatment plant of a chemical factory. The strain was identified as Pseudomonas putida based on its morphology, physiological, biochemical properties, automatic biometrical system BioMerieux Vitek and 16SrRNA sequence analysis. The strain degraded o-chloronitrobenzene of 1.07 mM original concentration about 85% in 42 h when grown in pH8.0, temperature 32 ℃ and rotation speed 120 rpm. A plasmid strip was found from strain OCNB-1 through plasmid detection，The plasmid size was determined finally to be 32 kb approximately using 7 kinds of restriction enzyme for single cutting the plasmid pOCNB-1 respectively. The strain has resistance to erythromycin, ampicillin, benzylpenicillin sodium, rifampicin, chloramphenicol, only the resistance to rifampicin and chloramphenicol was related to the plasmid pOCNB-1. The plasmid obtained was transferred to Pseudomonas.stutzeri through bacterial conjugation and the conjugant could grow using o-chloronitrobenzene as sole carbon, nitrogen and energy source. Therefore, it could be deemed that the plasmid carried the gene for o-chloronitrobenzene degradation.