深海多环芳烃降解菌新鞘氨醇杆菌H25的降解特性及降解基因

目的]为了从深海环境中筛选新的多环芳烃降解菌,了解其降解基因及降解特性.方法]以原油作为碳源从印度洋深海海水样品中富集筛选出降解能力较强的多环芳烃降解菌,并根据已报道的相关菌属的多环芳烃起始双加氧酶大亚基序列及侧翼序列设计兼并引物进行扩增.结果]获得了1株能够高效降解原油、柴油及多种多环芳烃的菌株H25.经16S rDNA序列系统发育分析表明它属于新鞘氨醇杆菌属(Novosphingobium)(96%).并从该菌株中扩增获得2条相似度为91.0%双加氧酶基因片段.2条序列在NCBI上Blastn分析表明均与菌株N.aromaticivorans DSM12444T的降解质粒pNL1上的双加氧酶大亚基具有最高相似度,分别为99.6%和91.0%.根据pNL1上的双加氧酶序列设计引物获得了包含H25双加氧酶大亚基及上下游序列的2个基因片段H25 Ⅰ(2.9kb)和H25Ⅱ(4.5kb).另外,单碳降解实验表明H25对联苯、2-甲基萘、2,6-二甲基萘、菲、二苯并噻吩、二苯并呋喃等均有较好的降解能力.结论]H25菌株是Novosphingobium属可能的新种.深海细菌在大洋环境多环芳烃污染的自然净化中起到一定作用,并在环境生物修复中有较大的应用前景.

Polycyclic aromatic hydrocarbon -degrading bacterium Novosphingobium sp. H25 isolated from deep sea and its degrading genes

Objective] The aim of this study is to isolate novel and efficient polycyclic aromatic hydrocarbon (PAH) degarading bacteria from deep sea. Methods] Bacteria in deep-sea water sample from Indian Ocean were enriched in the medium with crude oil as the carbon source. PAH-degrading bacteria were isolated and their degradation potential was tested by Gas Chromatography-Mass Spectrometry. PCR-primers were designed to detect the gene encoding the large subunit of aromatic-ring-hydroxylating dioxygenase. Results] A PAHs-degrading bacterium, named H25 was obtained. Several PAHs including 2-methynaphthalene, 2, 6-dimethynaphthalene, phenanthrene and dibenzothiopheneand dibenzofuran could be used as carbon sources for growth by strain H25. Analysis of 16S rDNA sequence showed that it belonged to genus Novosphingobium with highest similarity (96%) with previously described bacteria. Two fragments of the di-oxygenase gene were obtained by PCR with size of about 700bp, which were closest to the counterpart of N. aromati-civorans DSM12444 with 99.6% and 91.0% similarities. Furthermore, two fragments named H25I (2.9 kb) and H25II(4.5kb) containing the upstream and downstream sequences were obtained by another set of primers. Conclusion] Strain H25 was a novel PAH-degrading bacterium in deep sea environment, which might play a role in bioattenuation of PAH in oceanic environments and has potential in bioremediation of PAH contaminated environment.