极端酸性矿山环境微生物基于CRISPR系统的适应性免疫机制

Adaptive immunity mechanisms of microorganisms in extreme acid mine environment based on CRISPR system

摘要:

[目的] 通过对酸性矿山环境中嗜酸硫杆菌属（Acidithiobacillus）、脱硫弧菌属（Desulfovibrio）、钩端螺旋菌属（Leptospirillum）、硫化杆菌属（Sulfobacillus）、酸原体属（Acidiplasma）和铁质菌属（Ferroplasma）的100株冶金微生物基因组中CRISPR-Cas系统的结构特征和同源关系进行生物信息学分析，在基因组水平上解析冶金微生物基于CRISPR系统对极端环境的适应性免疫机制。[方法] 从NCBI网站下载基因组序列，采用CRISPR Finder定位基因组中潜在的CRISPR簇。分析CRISPR系统的组成结构与功能：利用Clustal Omega对重复序列（repeat）分类；将间隔序列（spacer）分别与nr数据库、质粒数据库和病毒数据库比对，获得注释信息；根据Cas蛋白的种类和同源性对酸性矿山环境微生物的CRISPR-Cas系统分型。[结果] 在100株冶金微生物基因组中共鉴定出415个CRISPR簇，在176个cCRISPR簇中共有80种不同的重复序列和4147条间隔序列。对重复序列分类，发现12类重复序列均能形成典型的RNA二级结构，Cluster 10中的重复序列在冶金微生物中最具有代表性。间隔序列注释结果表明，这些微生物曾遭受来自细菌质粒与病毒的攻击，并通过不同的防御机制抵抗外源核酸序列的入侵。冶金微生物细菌的大部分CRISPR-Cas系统属于I-C和I-E亚类型，而古菌的CRISPR-Cas系统多为I-D亚类型，两者基于CRISPR-Cas系统的进化过程中存在显著差异。[结论] 酸性矿山环境微生物的CRISPR结构可能采用不同免疫机制介导外源核酸序列与Cas蛋白的相互作用，为进一步揭示极端环境微生物的适应性进化机理奠定了基础。

关键词:酸性矿山环境;CRISPR-Cas系统;重复序列;间隔序列;适应机制

Abstract:

[Objective] To investigate the adaptive mechanism of bioleaching microorganisms to extreme environment based on CRISPR system at the genomic level, bioinformatic analyses were performed on the structural characteristics and homology of CRISPR-Cas system in genomes of 100 species from genera (Acidithiobacillus, Desulfovibrio, Leptospirillum, Sulfobacillus, Acidiplasma and Ferroplasma) in acid mine environment. [Methods] We downloaded genome sequences from NCBI website and identified potential CRISPR arrays by using CRISPR Finder. The composition, structure and function of each CRISPR system were analyzed, where repeats were classified by Clustal Omega and spacers were aligned and annotated according to nr database, plasmid database and viral database, respectively. The CRISPR-Cas system of microorganisms in acid mine environment was classified based on the types and homology of Cas proteins. [Results] Among the genomes of 100 bioleaching microorganisms, we found 415 CRISPR arrays. There were 80 different repeats and 4147 spacers in 176 confirmed CRISPR arrays. All the 12 types of repeat sequences in each cluster could form typical RNA secondary structure and the sequence of cluster 10 was the most representative one among all the bioleaching microorganisms. The annotation results showed that these microorganisms have been attacked by bacterial plasmids and virus, and have resisted the invasion of foreign nucleic acid sequences through different defense mechanisms. Most of the CRISPR-Cas systems of bioleaching bacteria belong to I-C and I-E subtypes, while most of the CRISPR-Cas systems of archaea belong to I-D subtype. There are significant differences between them in the evolution process based on the CRISPR-Cas system. [Conclusion] The CRISPR structure of acid mine environment microorganisms may mediate the interaction between foreign nucleic acid sequence and Cas protein based on different immune mechanisms, which provides a foundation for further revealing the adaptive evolution mechanism of extreme environment microorganisms.

Key words:acid mine environment;CRISPR-Cas system;repeat;spacer;adaptive mechanism