磷酸苯酯羧化酶同源基因在部分Thauera aromatica及Azoarcus evansii菌株中的多样性  

Diversity of Phenylphosphate Carboxylase Genes in Some Thauera aromatica and Azoarcus evansii Strains

作　　者：丁秦超 蒋福升[1] 钱朝东[1] 蒋莉娜 丁滨[1] 

机构地区：[1]浙江中医药大学生命科学学院,杭州310053

出　　处：《农业生物技术学报》2017年第3期470-476,共7页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学基金(No.LQ14C10004);浙江省"钱江人才计划"C;D类项目(No.QJD1302020)

摘　　要：苯酚是重要的化工原料,也是危害环境的重要酚类污染物之一。在反硝化细菌Thauera aromatica K172中,苯酚经过磷酸化和羧化可转化成毒性较低的4-羟基苯甲酸。磷酸苯酯羧化酶是这一过程的关键酶,由4个亚基α、δ、β和γ构成。本研究以编码δ和γ亚基的开放阅读框架(open reading frame,ORF)orf 5和orf 12为模板构建了DNA探针,通过Southern blot的方法,对部分T.aromatica及Azoarcus evansii菌株中相关基因的多样性进行了分析,发现这些菌株普遍含有多个orf 5同源序列,而杂交图谱显示该基因不具备属的特异性。相比之下,orf 12仅分布在T.aromatica K172和S100以及A.evansii T中,相关的基因簇为单一拷贝,且均位于一个Pst I消化的大小一致的片段中;在培养实验中也只有这3个菌株能够稳定地在反硝化条件下以苯酚为唯一碳源生长。因此推测,T.aromatica K172,S100和A.evansii T中降解代谢苯酚的途径是相同的,可能是在进化过程中获得了相同的苯酚代谢的基因或基因簇。本研究进一步证明了orf 12编码的γ蛋白是磷酸苯酯羧化酶的亚基之一,为深入探讨苯酚的无氧代谢提供了实验基础。Phenol is a key chemical engineering material, which is detrimental to the agroecological environment. In Thauera aromatica strain K172, a denitrifying bacterium, phenol undergoes phosphorylation and carboxylation and is converted into 4-hydroxybenzoic acid, which is less toxic. Phenylphosphate carboxylase, consists of α, δ, β and γ subunits, is a key enzyme of phenol degradation process. This study used the open reading frames, orf 5 and orf 12, which encode the δ and γ subunits, as the templates of DNA probes to analyze the diversity of related genes in strains of T. aromatica and Azoarcus evansii by Southern blot. The results indicated that these strains all contained several orf 5 homologs. The hybridization map showed there was no genus specificity of the gene. By contrast, orf 12 was only present in T. aromatica K172, S100 and A. evansii T and the gene cluster related to phenol degradation had a single copy in the genome. The gene was mapped to a PstⅠ digest fragment whose size was consistent among the bacterial strains. While in the bacterial culture experiments, only these 3 strains grew steadily on phenol as the only carbon source under the condition of denitrification. Therefore, our results suggested that T. aromatica K172, S100 and A. evansii T possessed the ability of phenol degradation through the same pathway. It is possible that the bacteria acquired the identical genes or gene cluster during evolution. This study provids new evidence of previous research that γ protein encoded by orf 12 is one subunit of phenylphosphate carboxylase. Meanwhile this work offers available scientific evidences for further studies of phenol degradation.

关 键 词：磷酸苯酯羧化酶 苯酚 降解代谢 硝酸呼吸 

分 类 号：S156[农业科学—土壤学] Q931[农业科学—农业基础科学]