Genetic analysis of bacteria which are capable of degrading halogenated aromatic compounds and molecular evolution of that ability

能够降解卤代芳香族化合物的细菌的遗传分析以及该能力的分子进化

• 批准号: 03454067
• 负责人: YANO Keiji
• 金额: $ 4.22万
• 依托单位: Nagaoka University of technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454067/
• 关键词: PCB; Degradation Genes; Cloning; PCR; Molecular Evolution; クロ-ニング

In these experiment, we analyzed the structure of PCB degradation genes and discussed about their evolutionary origin. The gene responsible for 2,3-dihydroxybiphenyl dioxygenase (bphc) was screened from several bacteria and were cloned for further analysis. A PCR method was used for screening of biphenyl degradation genes. However this method was suitable only for highly homologous genes.Recently we have isolated a bacterium from soil which can degrade not only PCB but also benzene and toluene. This bacterium can degrade all of the PCB congeners. It is a gram positive bacterium which was identified as a species of Rhodococcus. Degradative genes were cloned and the sequencing of bphC was done. A computer analysis of homology of bphC related genes was done and a phylogenic tree was constructed. These results suggested that amino acid differences effect the differences of the activity.To monitor genetically manipulated microorganism in the environment, we have devised a method for isolating total bacterial DNA from soil. Bacterial fraction was concentrated by ultracentrifuge and stored in deep freezer. Total DNA was isolated from this bacterial fraction. DNA hybridization was done with the soil isolated DNA using biphenyl degradation genes as probes, but a positive signal was not observed. The hybridization conditions including probe designing should be changed for a better understanding.We conclude from our experiment that the evolution of degradative genes are effected by the environment such as the living condition of these bacteria.

本实验分析了多氯联苯降解基因的结构，并对其进化起源进行了探讨。从几种细菌中筛选出2，3-二羟基联苯双加氧酶（bphc）的基因，并进行克隆以作进一步分析。采用PCR方法筛选联苯降解基因。最近我们从土壤中分离到一株能降解多氯联苯、苯和甲苯的细菌。这种细菌可以降解所有的PCB同系物。它是一种革兰氏阳性细菌，被鉴定为红球菌属的一种。克隆了降解基因，并对bphC基因进行了序列测定。对bphC相关基因进行计算机同源性分析，构建了bphC基因的进化树。这些结果表明，氨基酸的差异影响活性的差异。为了监测环境中的基因操纵微生物，我们设计了一种从土壤中提取细菌总DNA的方法。通过超离心浓缩细菌级分并储存在深冻器中。从该细菌级分中分离总DNA。以联苯降解基因为探针，与土壤分离的DNA进行杂交，未观察到阳性信号。为了更好的理解，杂交条件包括探针设计都需要改变。我们从实验中得出结论，降解基因的进化受到环境的影响，如细菌的生活条件。