工业废水中阿特拉津降解细菌的遗传和生理多样性

Genetic and physiological diversity among atrazine-degrading bacteria isolated from industrial wastewater

【目的】通过遗传学和生理学实验,揭示分离自工业废水的阿特拉津降解细菌具有遗传和生理多样性,为阐明阿特拉津生物降解的分子机理和阿特拉津降解细菌在污染环境生物修复中的应用提供新见解。【方法】用普通PCR方法检测菌株的阿特拉津降解基因,分析其降解基因组成;用基因组重复序列PCR技术(rep-PCR)分析降解菌株的基因组类型;用Western blot方法检测菌株阿特拉津降解途径的第一个酶三嗪水解酶(TrzN);用不同氮源(阿特拉津、莠灭净、扑草净、西玛津、氰草净、阿特拉通和氰尿酸)和碳源(蔗糖、葡萄糖、麦芽糖、乳糖、柠檬酸钠、乙酸钠和琥珀酸钠)培养降解菌株,通过检测培养液的OD600值,证明菌株能够利用的氮源和碳源种类。【结果】对分离自工业废水的27个阿特拉津降解菌株所进行的阿特拉津降解基因PCR检测表明,其降解基因组成分别为trzN-atzBC、trzN-atzABC和atzADEF;通过rep-PCR实验将27个阿特拉津降解菌株分为7个群;Western blot结果表明,27个菌株中有24个含有三嗪水解酶TrzN;氮源利用实验表明,2个菌株能够利用所有7种氮源生长,其余25个菌株只能利用其中的2-6种;碳源利用实验表明,10个菌株能够利用所有7种碳源生长,其余17个菌株只能利用其中的3-6种。【结论】分离自某工业废水的27株阿特拉津降解功能菌存在相当广泛的遗传和生理学上的多样性,trzN-atzABC降解基因组成为首次发现。

The purpose of this paper is to reveal the genetic and physiological diversityof atrazine-degrading bacteria isolated from industrial wastewater, to provide new insights for mo- lecular mechanism of atrazine biodegradation. [Methods] Atrazine-degrading genes of 27 atrazine-degrading strains were detected by the conventional PCR and their composition of atrazine-degrading genes was analyzed. The genome composition of these bacteria was analyzed by repetitive extragenic palindromic PCR （rep-PCR） genomic fingerprinting. Triazine hydrolase TrzN in bacteria, the first enzyme for atrazine-degrading pathway, was detected by Western blot analysis. Ni- trogen source （atrazine, ametryn, atratone, cyanazine, prometryn, simazine and cyanuric acid） and carbon source （glucose, sucrose, maltose, lactose, sodium citrate, sodium succinate and sodium ace- tate） used by these bacteria were analyzed by measuring absorbance of the culture at 600 nm. [Results] The conventional PCR experiments indicated that three main atrazine-degrading gene combinations （i） trzN-atzBC, （ii） trzN-atzABC, and （iii） atzADEF were observed respectively from either strain of 27 atrazine-degrading strains, rep-PCR analysis indicated that these bacteria could be differentiated into 7 clusters. Western blot analysis proved that 24 strains contained TrzN protein. Nitrogen source experiments showed that two strains could use all of the 7 compounds as nitrogen source and other 25 strains only could use 2-6 compounds as nitrogen source. Carbon source ex- periments showed that 10 strains could use all of the 7 compounds as carbon source and other 17 strains only could use 3-6 comlounds as nitrogen source. [Conclusion] These 27 atrazine-degrading strains isolated from industrial wastewater have their genetic and physiological diversity.