西南印度洋中脊深海沉积物砷抗性菌的富集分离与多样性分析

Enrichment and diversity analyis of arsenite-resistant bacteria in deep sea sediment samples from southwest Indian Ocean ridge

摘要:

【目的】为了筛选深海砷抗性菌，了解印度洋中脊深海沉积物砷抗性菌的多样性情况。【方法】通过砷富集培养，筛选出砷抗性菌；通过变性梯度凝胶电泳（DGGE）分析与构建16S rDNA 克隆文库法两种手段分析了富集物中砷抗性菌的多样性。利用兼并引物PCR，从抗性菌株中克隆与砷抗性相关的基因。【结果】共筛选到8株砷抗性菌，分别属于γ-proteobacteria的5个不同的属。其中，菌株AS-I1-3的抗性最高，可以在26×10-3 mol/L三价砷存在的情况下生长，该菌株的16S rDNA 序列与菌株Pseudoalteromonas tetraodoni IAM同源性最高 (100%)。DGGE分析显示，该菌是富集物中的最优势菌，其次是盐单胞菌（Halomonas）和海杆菌（Marinobacter）。16S rDNA 克隆文库分析结果进一步证明，与菌株AS-I1-3序列相同的克隆子占整个克隆文库的72.5%；而与菌株AS-I1-5 (Halomonas meridiana, 100%) 和菌株Mn-I1-6（Marinobacter vinifirmus，99%）序相同的克隆子分别占10%和7.5%。然而，利用兼并引物PCR，仅能从2株非优势菌中克隆到了与砷抗性相关的基因，表明菌群中的优势抗性菌可能有其他的抗性机制。【结论】在深海环境中存在着多种砷抗性菌，其中Pseudoalteromonas属的菌株是该富集条件下的砷抗性优势菌。这些砷抗性菌在大洋环境砷元素的生物地球化学循环中的作用有待进一步研究。

Abstract:

[Objective] The aim of this study was to assess the diversity of arsenite-resistant bacteria in deep sea sediment of the southwest Indian Ocean Ridge. [Method] Arsenite-resistant bacteria in the deep-sea sediment were enriched with 2×10-3 mol/L NaAsO2 and isolated on plates. Their diversity was further examined by Denaturing gradient gel electrophoresis (DGGE) and 16S rDNA library analysis. Furthermore, the gene encoding putative arsenite efflux pump was detected with degenerate primers among these isolates. [Result] Eight arsenite-resistant isolates were obtained; they belonged to five different genera of γ-proteobacteria. One isolate named As-I1-3 can grow in presence of 26×10-3 mol/L NaAsO2, showed highest similarity to Pseudoalteromonas tetraodonis IAM (100%). DGGE result showed that As-I1-3 was dominant in arsenite enriched community, followed by isolate As-I1-5 (Halomonas meridiana,100%) and Mn-I1-6 (Marinobacter vinifirmus, 99%). 16S rDNA library analysis reconfirmed the result of DGGE, which showed that the three bacteria occupied 72.5%, 10% and 7.5% of the total OTUs (operational taxonomic units), respectively. However, only from other two isolates which were not dominant, the gene encoding a putative arsenite efflux pump was obtained. [Conclusion] Different bacteria of arsenite-resistance inhabited in deep sea environment, among which P. tetraodonis (100%) showed highest resistance; they might play a certain role in the geobiocycling of arsenic element in marine environments.