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PCB降解基因的结构，并讨论了它们的进化起源。从几种细菌中筛选了2,3-二羟基联苯双加氧酶（bphc）基因，并进行了克隆，以供进一步分析。采用PCR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种细菌，它不仅能降解多氯联苯，还能降解苯和甲苯。这种细菌可以降解所有的多氯联苯同源物。它是一种革兰氏阳性细菌，被鉴定为红球菌的一种。克隆了bphC的降解基因，并对其进行了测序。对bphC相关基因进行了计算机同源性分析，并构建了系统发育树。这些结果表明，氨基酸的差异影响了活性的差异。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分离细菌总DNA的方法。细菌馏分采用超离心浓缩，冷冻保存。从细菌部分中分离出总DNA。以联苯降解基因为探针，对土壤分离的DNA进行了DNA杂交，但未观察到阳性信号。为了更好的理解，杂交条件包括探针的设计都需要改变。我们从实验中得出结论，降解基因的进化受到环境的影响，例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.