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

在这些实验中，我们分析了多氯联苯降解基因的结构，并讨论了它们的进化起源。从几种细菌中筛选出2，3-二羟基联苯双加氧酶(BPHC)基因，并进行了克隆和分析。采用聚合酶链式反应的方法筛选联苯降解基因。然而，这种方法只适用于高度同源的基因。最近，我们从土壤中分离出一株不仅能降解多氯联苯，而且能降解苯和甲苯的细菌。这种细菌可以降解所有的多氯联苯同系物。该菌为革兰氏阳性菌，经鉴定为红球菌属。克隆了降解性基因，并对BPHC进行了测序。对BPHC相关基因进行计算机同源性分析，构建系统发育树。为了监测环境中的转基因微生物，我们设计了一种从土壤中提取细菌总DNA的方法。细菌组分经超速离心机浓缩后冷藏。总DNA是从这个细菌组分中提取出来的。以联苯降解基因为探针与土壤分离的DNA进行DNA杂交，但未观察到阳性信号。为了更好地理解，需要改变杂交条件，包括探针的设计。我们的实验得出结论，降解基因的进化受到环境的影响，如这些细菌的生存条件。

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.