采用功能性单细胞方法重新审视格雷罗内格罗潮间带微生物垫中的固氮作用。
Revisiting N₂ fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach.
作者信息
Woebken Dagmar, Burow Luke C, Behnam Faris, Mayali Xavier, Schintlmeister Arno, Fleming Erich D, Prufert-Bebout Leslie, Singer Steven W, Cortés Alejandro López, Hoehler Tori M, Pett-Ridge Jennifer, Spormann Alfred M, Wagner Michael, Weber Peter K, Bebout Brad M
机构信息
1] Departments of Chemical Engineering, and of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA [2] Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA [3] Department of Microbiology and Ecosystem Science, Division of Microbial Ecology, University of Vienna, Vienna, Austria.
1] Departments of Chemical Engineering, and of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA [2] Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA.
出版信息
ISME J. 2015 Feb;9(2):485-96. doi: 10.1038/ismej.2014.144. Epub 2014 Oct 10.
Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N₂ fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N₂ fixation, whereas (15)N₂ tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of (15)N₂-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in (15)N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% (15)N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N₂ fixation in the intertidal mats, whereas support for significant N₂ fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.
光合微生物席是复杂的分层生态系统，其中高初级生产力产生了对氮的需求，部分需求通过固氮作用来满足。在这些微生物席中检测到了蓝细菌和异养细菌（如δ-变形菌）的固氮酶还原酶（nifH）基因及转录本，但其对固氮作用的贡献却知之甚少。我们采用了抑制剂操纵实验、nifH测序和单细胞同位素分析相结合的方法，来研究墨西哥格雷罗内格罗附近的奥霍德列夫雷湖潮间带微生物席中的活跃固氮群落。使用特定代谢抑制剂的乙炔还原试验表明，硫酸盐还原菌和蓝细菌成员都对固氮作用有贡献，而整体水平的（15）N₂示踪实验仅支持蓝细菌的贡献。蓝细菌和nifH聚类III（包括δ-变形菌硫酸盐还原菌）序列在nifH基因库中占主导地位，而nifH转录本库则由与鞘丝藻属相关的序列主导。通过高分辨率二次离子质谱（NanoSIMS）对（15）N₂培养的微生物席样品进行单细胞同位素分析发现，蓝细菌富含（15）N，在鞘丝藻属丝状体中检测到的富集程度最高（平均为4.4原子百分比（15）N），而通过催化报告沉积荧光原位杂交（CARD-FISH）鉴定的δ-变形菌没有明显富集。我们研究了CARD-FISH对同位素组成的潜在稀释效应，并得出结论，稀释偏差不足以影响我们的结论。我们的综合数据提供了证据，表明蓝细菌成员，特别是鞘丝藻属，对潮间带微生物席中的固氮作用有积极贡献，而未发现靶向的δ-变形菌硫酸盐还原菌有显著固氮活性的支持证据。
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