土壤氨氧化古菌适应酸性胁迫的ATP酶基因分子进化研究

Molecular Phylogeny Suggests a Key Role of ATPase Genes on Evolutionary Adaption of Soil Ammonia-oxidizing Archaea to Acid Stress

氨氧化古菌(Ammonia-oxidizingarchaea,AOA)被认为是酸性土壤硝化过程的主要微生物类群,但AOA如何适应酸性胁迫并发挥作用一直是研究难点,而ATP酶(ATPase)是能量代谢的关键,其编码基因可能在AOA适应酸性胁迫过程中发生了趋同性演化。据此,本研究针对5个不同种植年限的马尾松人工林酸性土壤(15 a、24 a、45 a、55 a、63 a),通过深度宏基因组测序获得7360亿碱基对,重构AOA氨单加氧酶amo A基因和ATP酶A亚基(ATPase subunit A)基因的系统发育进化谱系,研究AOA适酸的分子机制。结果表明:根据经典的amo A基因系统发育进化分类,所有5个森林土壤中优势AOA主要包括Nitrososphaerales和Ca.Nitrosotaleales两大类群,但Nitrososphaerales类群与中碱性土壤中的AOA古菌亲缘关系更近,与嗜酸的Ca.Nitrosotaleales类群亲缘关系较远,表明amo A基因的系统进化关系不能解释Nitrososphaerales在酸性土壤中的成功定殖。然而,基于ATPase subunit A基因的系统进化分析则发现,所有酸性森林土壤中嗜酸/耐酸氨氧化古菌均含有亲缘关系较近的V-ATPasesubunitA基因,表明氨氧化古菌可能通过基因水平转移获得V-ATPase基因适应酸性胁迫环境,较好地解释了氨氧化古菌适应酸性胁迫的生境扩展规律。随林龄的增加,Ca.Nitrosotaleales类群丰度先减少后增加,而Nitrososphaerales类群丰度先增加后减少,速效钾是显著影响AOA群落结构的重要环境因子。这些结果表明,不同种植年限下酸性人工林土壤中氨氧化古菌种群发生了明显的分化,V-ATPase基因水平转移可能是氨氧化古菌适应酸性胁迫的重要机制。

【Objective】Ammonia-oxidizing archaea(AOA)is considered the main microbial group catalyzing the nitrification process in acidic soils.However,how AOA adapts to acid stress remains largely uncertain,and we hypothesize that the gene encoding ATPase for microbial energy metabolism may likely play a key role in the adaption of AOA to acidic stress in these soils.【Method】In this study,736 billion base pairs were obtained by ultra-deep metagenomics sequencing of acidic soils of Masson pine plantations with five different planting years(15 a,24 a,45 a,55 a,63 a).The phylogenetic congruency between the AOA amoA gene and ATPase subunit A gene was reconstructed to study the molecular mechanism that may enable the acidophilic lifestyle of AOA.【Result】The habitat expansion of AOA to the acidic environment cannot be explained by the phylogenetic evolutionary trajectory of canonic amoA genes encoding ammonia monooxygenase.The dominant AOA taxa from all 5 forest soils were phylogenetically affiliated with Nitrososphaerales and Ca.Nitrosotaleales based on amoA gene phylogeny.Even though Nitrososphaerales is more distantly related to Ca.Nitrosotaleales,it is more closely related to neutral AOA in alkaline soils.Therefore,the phylogenetic law based on the amoA gene cannot explain the successful colonization of Nitrososphaerales in acidic soils.The phylogeny of ATPase subunit A genes indicated a single clade of AOA in all acidic forest soils.These results thus suggest that during the habitat expansion and evolution of AOA,the amoA and ATPase genes could have experienced different selection pressures to cope with acid stress,and the V-ATPase gene may be obtained through horizontal gene transfer to adapt to acid stress.With the increase in forest age,the abundance of Ca.Nitrosotaleales group first decreased and then increased,while the abundance of Nitrososphaerales group first increased and then decreased.Also,it was observed that soil bioavailable potassium is an important environmental factor that significantly affects the structure of the AOA community.【Conclusion】These results indicate that the AOA populations in acid plantation soils under different planting years were clearly differentiated,and the horizontal transfer of V-ATPase gene may be an important mechanism that enables AOA to survive acidic stress for the habitat expansion.