极端嗜酸硫杆菌Acidithiobacillus spp.种群的遗传多态性及分子地理学研究

Microorganisms are the most abundant and potentially most diverse organisms in the biosphere. However, to date, knowledge of the origin, maintenance and distribution of diversity on terrestrial microorganisms remains limited. Biogeography constitutes a cornerstone approach for studying biodiversity patterns at different taxonomic levels in the prokaryotic world. In this regard, the importance of geographic barriers influencing microbial speciation is subjected to great debate. .A DNA sequence-based approach can help overcome these challenges, because the bacteria falling into sequence clusters can correspond to ecologically distinct populations. The ecotype model is highly attractive as it describes evolutionary and ecological principles that might account for microbial population structures and speciation. To date, research on bacterial population genetics has been restricted almost entirely to pathogenic and relevant symbiotic bacteria，knowledge about the relevance of pedigree geography and ecological strategies in natural populations of free-living bacteria is extremely limited..We recently showed that the extremely acidophilic bacterium Acidithiobacillus ferrooxidans can be isolated from different parts of the world in sites as diverse as mining area, hot sulfur spring. Moreover, since their natural habitats are ecologically extremely diverse, different Acidithiobacillus ferrooxidans strains exhibit a high degree of interstrain genetic variability. The extreme conditions and geographical isolation of its environments are optimal circumstances for observing allopatric speciation..In this work, we are going to evaluate the importance of different evolutionary forces in shaping the genetic structure of Acidithiobacillus ferrooxidans populations distributed in six acidic mining areas, by using phylogenetic and population genetics approaches based on multilocus sequence analyses coupled to genomic fingerprintings (PFGE, rep-PCR, AFLP)..To conclude, our specific objectives are: (ⅰ)to identifying the smallest sequence clusters corresponding to ecologically meaningful putative ecotypes, and test whether putative ecotypes exhibit specie-like properties.(ⅱ) to clarify whether putative ecotypes of Acidithiobacillus populations coexist in a particular environment, indicating the occurrence of sympatric differentiation among closely related iron-oxidizing Acidithiobacillus populations.(ⅲ)to examine to which degree geographic distance has an influence on genitic structure in Acidithiobacillus spp. and test whether barriers to dispersal or ecological selection are primarily responsible for its differentiation.(ⅳ)To test whether there has been genetic divergence and local adaptation of Acidithiobacillus populations to prevailing environmental conditions.

目前国内外对陆生环境微生物种群内微多样性及谱系地理分化的研究报道较少。由于栖息环境的隔离和极端的生存条件，极端微生物成为研究材料的首选。我们前期研究发现，地理分布较广的极端嗜酸细菌Acidithiobacillus ferrooxidans存在显著的种群遗传多态性，但不清楚地理隔离和生态位差异对其系统发育和种群分化的影响。本项目根据种群遗传学、生态学原理，采用持家基因多序列分析、分子指纹分型、宏基因组文库构建及生理生态参数的测定，通过生态型模拟(ES)和AdaptML软件分析亚种群生态生理参数与遗传多态性参数的相关性，在不同空间尺度范围，阐明异质环境的生态位和地理隔离对亲缘关系非常相近亚种群的产生和分化的作用及成种模式；同时采用q-PCR、RT-PCR分析不同选择环境对核心基因和适应性基因在转录水平上的差异，揭示环境选择对种群分化的影响。为了解微生物谱系地理和多样性维持机制提供理论依据。

项目在不同空间尺度范围，阐明异质环境的生态位和地理隔离对亲缘关系非常相近亚种群的产生和分化的作用及成种模式；同时采用q-PCR、RT-PCR分析不同选择环境对核心基因和适应性基因在转录水平上的差异，揭示环境选择对种群分化的影响。为了解微生物谱系地理和多样性维持机制提供理论依据。.结果显示，不同地理来源极端嗜酸硫杆菌Acidithiobacillus spp.的分离，采用3种培养基，从六个样地，包括新疆浮云、湖北大冶、云南腾冲、广东云浮、江西德兴和江西永平分离了330多株菌株，所有分离的菌株在16S-23SrRNA间隔区序列和16S rRNA序列系统发育树中可被划分为5大类群，表明Acidithiobacillus spp.存在较大的遗传多样性。通过对30株具有代表性菌株的RubisCO 一型cbbL基因和二型cbbM基因分析显示，发现21株菌的cbbL基因是双拷贝，分别为cbbL1和cbbL2基因；9株菌只检测到了cbbL1，没有检测到cbbL2。在系统发育树上，基于cbbL1基因序列将它们划分为3个序列型。同样基于cbbL2基因也被划分为3个序列型，因此cbbL1和cbbL2基因序列型的划分基本一致。此外，MLST分型技术被用于42株嗜酸硫杆菌序列分型，所有菌株共产生8种ST型，ST1是出现频率最高的ST型，其中有5种ST型由单一的分离物形成；10株A. ferridurans只有两种ST型，分别是ST7和ST8。其中分离自湖北大冶的5株菌同属于一个ST型。.此外，高通量测序显示，酸性矿山环境中细菌具有较高的多样性，在高层次分类单元（taxon）门（phylum）一级水平共有31个系统发育类群，其中测序reads丰度超过5%的包括proteobacteria（31.7%）和Chloroflexi（5.0%）；序列超过1%的系统发育类群有Bacteroidetes（4.0%）、Firmicutes（2.4%）、Acidobacteria（2.3 %）、Actinobacteria（1.5%）、Nitrospira（1.4%）和Deltaproteobacteria（1.0%）。RDA分析表明，Fe2+、SO42-、Co、Zn 和 EC等环境因子对沉积层环境中群落结构的组成起决定性作用。