帕米尔高原可培养需氧冷适应细菌及古菌多样性

Diversity of aerobic cold-adapted bacteria and archaea isolated from the Pamir Plateau

摘要:

【目的】帕米尔高原是贫营养、高辐射、干燥的冷环境，可能蕴藏丰富的冷适应微生物资源。本研究基于分离培养技术，探究帕米尔高原不同海拔梯度及不同培养条件下的冷适应微生物多样性。【方法】针对采集自帕米尔高原1 000-2 000 m、2 000-3 000 m、3 000-4 000 m和4 000-5 000 m四个海拔梯度的土壤样品，选用TSA和R2A两种培养基于4℃进行冷适应细菌的分离培养，NOM和F6两种培养基于4℃和15℃进行冷适应古菌的分离培养。根据16S rRNA基因序列同源性对分离菌株进行鉴定，分析不同海拔梯度和不同培养条件的物种多样性及之间的差异性。【结果】本研究从帕米尔高原共分离得到419株需氧原核微生物，16S rRNA基因测序鉴定结果表明，分离菌株隶属于2个域、5个门、8个纲、18个目、28个科、49个属及118个种，为115种冷适应细菌及3种古菌。冷适应细菌中，γ-变形菌纲(Gammaproteobacteria)为第一优势纲，放线菌纲(Actinobacteria)中物种多样性最为丰富。高海拔地区的冷适应细菌物种多样性存在差异，海拔1 000-4 000 m的优势菌群均为γ-变形菌纲，而4 000-5 000 m高海拔地区放线菌纲占比最高。古菌域微生物隶属于钠线菌属(Natrinema)和盐陆生菌属(Haloterrigena)，且仅在1 000–2 000 m低海拔地区，培养温度为15℃时分离得到。4种培养基分离菌株中，γ-变形菌纲在TSA和R2A培养基分离的冷适应细菌中占优越地位，而NOM和F6培养基仅分离得到盐杆菌纲(Halobacteria)的古菌。此外，本研究获得23株潜在新物种，隶属于放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和广古菌门(Euryarchaeota)5个门。【结论】本研究从帕米尔高原分离获得了大量冷适应细菌以及古菌资源，揭示了帕米尔高原不同海拔的冷适应细菌及古菌分布多样性，以及不同培养条件下的分离效果，为冷适应细菌及古菌资源的开发与利用及其生态学功能研究提供了菌株材料和科学理论支持。

Abstract:

[Objective] The Pamir Plateau is characterized by a cold and oligotrophic environment exposed to strong radiation, in which cold-adapted microorganisms are abundant. Using a cultivation-dependent approach, we aimed to reveal the diversity of cold-adapted bacteria and archaea present in the Pamir Plateau. [Methods] Soil samples were collected from a gradient of altitudes in the Pamir Plateau, from which aerobic bacteria and archaea were isolated by different cultivation methods. For the isolation of cold-adapted bacteria, two bacterial culture media were used and the incubation was only conducted at 4℃. For cultivation of archaeal ones, two archaeal culture media were employed, and cultivation was at two temperatures, i.e., 4℃ and 15℃. Sampling was conducted at four altitudes:1 000-2 000 m, 2 000-3 000 m, 3 000-4 000 m and 4 000-5 000 m. All microbial isolates were classified based on their 16S rRNA gene sequences. [Results] A total of 419 strains of aerobic prokaryotes were isolated from the soil samples, which belonged to 118 different species, including 115 bacterial species and 3 archaeal species. These microorganisms fell into 49 genera, 28 families, 18 orders, 8 classes, 5 phyla, and 2 domains. Gammaproteobacteria was the predominant order of cold-adapted bacteria and Actinomycetes constituted the most diverse one. Gammaproteobacteria dominated at altitude of 1 000-4 000 m and Actinobacteria prevailed at altitude of 4 000-5 000 m (the highest altitude). The archaeal isolates belonged to haloarchaea of Natrinema and Haloterrigena, and they were obtained from the samples at altitude of 1 000-2 000 m and at the cultivation temperature of 15℃. Furthermore, among the four culture media used in this work, Gammaproteobacteria isolates were mainly obtained with the two bacterial media whereas the haloarchaea only grew on the archaeal media. Additionally, 23 strains represented new taxa among all isolates, and they belonged to Actinobacteria, Proteobacteria, Bacteroidetes, Firmicutes and Euryarchaeota.[Conclusion] A number of cold-adapted bacteria and three archaeal species were isolated as pure cultures from the Pamir Plateau soil samples. These microorganisms exhibited an altitude-dependent distribution. Furthermore, we found that the isolation experiment was benefitted from utilization of different culture conditions, which was a strategy to be exploited in future work. Together, the present study has yielded the first insights into the composition and diversity of cold-adapted bacteria and archaea in the Pamir Plateau. The obtained microorganisms provide unique resources for exploration of their ecological functions and biotechnological applications.