在好氧条件下多数细菌利用一套相似酶系统（pahABCDEF）完成各类PAHs的上游降解过程，目前对编码这些酶的基因的多样性缺少系统的研究。本文对功能明确的PAHs羟基化双加氧酶（pahA）与芳烃羟基化双加氧酶（ArhA）在进化上的关系进行了研究，在此基础上收集了数据库中所有的pahA与相关的ArhA的全长序列并基于加氧酶α亚基（A3）构建了系统发育树，界定了pahA在ArhA的系统发育树上所处的范围，研究了pahA的多样性。研究表明pahA3在系统发育树上可以分为两类，分别为来自变形菌门与放线菌门的PAHs降解菌。在pahA3的系统发育树的大部分分支存在功能明确的pahA，处于同一分支的pahA通常来自亲缘关系密切的PAHs降解菌且具有相同的底物范围，少数未经功能验证的pahA分支代表了新类型的pahA。pahA中电子传递蛋白存在多个起源，其中在pahA3系统发育树的多个分支中存在的Rubredoxin类型的氧化还原蛋白在已有的研究中被忽略了。本研究中还发现pahA中同一类型的电子传递蛋白也可能具有不同的起源，一些pahA的电子传递蛋白来源于其它ArhA。本研究利用功能明确的pahBCDEF检索并收集了数据库中相关的基因，利用pahA作为PAHs降解菌的标记基因对这些pahBCDEF进行了注释，在此基础上构建系统发育树以研究它们的多样性。结果表明，pahBCDEF的系统发育树及pahA3系统发育树的各个分支一一对应。pahBCF在系统发育树上存在多个起源且与一般芳烃降解过程中的相应基因有关。pahDE与其他芳烃降解过程基因无关，在系统发育树上有着单一起源。pahD只出现在变形菌门的PAHs降解菌中，而所有PAHs降解菌中都有共同起源的pahE，这表明pahE可以作为PAHs降解菌的标记基因。本研究对各类PAHs降解基因的组织方式进行了比较，研究发现，PAHs降解基因在基因组上有着高度多样的组织形式，在系统发育树上处于同一分支的pahABCDEF基因在基因组上通常形成相同的基因簇，反之亦然。通过全基因组比较，本研究清晰的呈现了许多细菌中PAHs降解基因簇整体的水平转移。利用pahE作为标记基因并结合基因组比较，本研究预测了两类新的pahA。其中一类pahA来自于α变形菌纲，在进化上与典型的联苯羟基化双加氧酶基因关系密切，这类pahA中有两个酶在曾被证实能够催化PAHs反应。另一类pahA来自Burkholderiales，在这些菌株中pahBCDEF均与已知变形菌门的PAHs降解菌中相应基因有着共同的起源，但pahA却与放线菌门的pahA更加接近。
Most bacteria degrade PAHs through a similar upper pathway under aerobic condition. A similar set of enzymes (pahABCDEF) responsible for catalizing the reactions in the pathway is present in all the well-studied PAHs degraders. Until now, the diversity of genes encoding these enzymes lacks comprehensive and in-depth investigation. In this work, all available pahABCDEF sequences as well as the corresponding genomes in Genbank are collected, the diversity of individual gene is studied and compared, and the way in which these genes are organised in the genome are compared, several new type of genes involved in PAHs degradation are predicted.Aromatics ring hydroxylating dioxygense (ArhA) can be classified into four major class, PAHs ring hydroxylating dioxygense (pahA) belongs to the IVth class of ArhA, which containing one oxygenase component and two electron transfer components (ETCs): reductase and redoxin. Based on the phylogenetic relationship of the oxygenase component, pahA can be classified into two major groups, each from PAHs degrading Proteobacteria or Actinobacteria, separately. Highly similar oxygenase sequences generally come from bacteria with close relationship. Various types of ETCs are present in pahA, however pahA with similar oxygenase component usually comprise same type of ETCs. There is a tight relationship between the oxygenase and ETCs during the evolution process of pahA.The diversity of pahBCDEF genes vary with one another. There are several different origins for pahBCF genes, these genes are closely related to the counterpart genes involved in the degradation of other aromatic hydrocarbons in their phylogenetic trees. Both pahD and pahE genes have a single origin and their phylogenetic trees contain no counterpart genes in other aromatics degradation process. pahD gene is presented in PAHs degrading Proteobacteria but not Actinobacteria, pahE genes is presented in all PAHs degraders, which suggests pahE gene can serve as a promising biomarker in PAHs degradation process. If two PAHs degraders contains highly similar pahX gene, they generally contains highly similar pahY genes (X, Y can be ABCDEF), however two pahX genes with same origin can not promise pahY genes with same origin.pahABCDEF genes are generally distributed in a region of 10s kb in the genomes of PAHs degraders, these genes can be organised into various types of gene clusters related to PAHs degradation. PAHs degraders with highly similar pahX genes generally contain the same gene clusters and vice versa. Clear signals of horizontal gene transfer are revealed through whole genome comparison and the comparion of PAHs degrading gene clusters, in these cases all pahABCDEF genes are transferred as a whole.Two new groups of pahA genes are predicted by ultilizing pahE genes as biomarker gene. The first group is from PAHs degraders belonging to α-Proteobacteria, these pahA are tightly related to ArhA typically involved in biphenyl and toluene degradation, two members in this group have been demonstrated to be involvled in PAHs degradation. Another new type of pahA is from Burkholderiales, the pahBCDE genes in these strains are tightly related to the typical PAHs degrading Burkholderiales in the phylogenetic trees, however, pahA is more closely related to pahA belonging to PAHs degrading Actinobacteria.
