土壤中可编码乌头酸异构酶的芽胞杆菌菌株筛选及鉴定

Isolation and identification of soil Bacillus strains that encode aconitate isomerase

摘要:

[目的]以芽胞杆菌（Bacillus）为筛选对象，分离土壤中可编码乌头酸异构酶（aconitate isomerase，AI）的革兰氏阳性（Gram positive，G⁺）菌株，以丰富对AI分布的科学认识，为其生物学功能研究奠定理论与材料基础。[方法]采用土样高温预处理法、含反式乌头酸（trans-aconitic acid，TAA）唯一碳源的ACO固体平板培养法，结合16S rDNA基因序列同源性分析，筛选能够编码AI的芽胞杆菌目的菌株。[结果]共分离得到22株能够利用TAA碳源的细菌菌株，成功鉴定了其中的16株，分别为巨大芽胞杆菌（Bacillus megaterium）2株，阿氏芽胞杆菌（Bacillus aryabhattai）7株，短小芽胞杆菌（Bacillus pumilus）1株，未鉴定到种的芽胞杆菌（Bacillussp.）6株；且它们所含AI编码基因与已知AI基因在序列上存在差异。[结论]首次证明可编码AI的芽胞杆菌细菌种类具有多样性，暗示G⁺细菌广泛编码AI的可能性，更新了AI几乎只在G^-细菌中分布的观点，为后续深入挖掘AI基因及其生物学功能研究提供更多可用微生物资源。

关键词:乌头酸异构酶;芽胞杆菌;分离鉴定;乌头酸顺反异构体

Abstract:

[Objective] Purpose of this work was to specifically screen the Gram positive (G⁺) bacteria of Bacillus that could encode the aconitate isomerase (AI) enzyme, to enrich our understanding of the distribution of AI and to provide theoretical and material basis for further research. [Methods] Through heat pretreatment of soil sample, plate cultivation by using ACO solid medium containing trans-aconitic acid (TAA) as the sole carbon source and the 16S rDNA sequences homologous analysis, the Bacillus target strains that encode AI can be isolated. [Results] We totally isolated 22 bacterial strains that could utilize TAA carbon from the ACO plate, and 16 of which were successfully classified as:2 strains of Bacillus megaterium, 7 strains of Bacillus aryabhattai, one Bacillus pumilus strain, and 6 undetermined Bacillus sp. strains; besides, we found that the AI-coding genes of these 16 Bacillus isolates were different from the already cloned ones in DNA sequence. [Conclusion] The species diversity of Bacillus bacteria encoding AI enzyme is rich, indicating the encoding ability in more G⁺ bacteria and updating the old opinion that considered AI distribution mainly in G^- hosts. Our research provided available microbial resource for further studies on the identification of AI gene as well as its biological significance.

Key words:aconitate isomerase;Bacillus;isolation and identification;cis-trans aconitate isomers