Identifying N2-fixing microorganisms in photosynthetic microbial mats by Stable Isotope Probing (SIP) and nanometer-scale Secondary Ion Mass Spectrometry (nanoSIMS)

光合成微生物垫在浅水海洋环境中是重要的生态系统，因为它们为这些通常氮有限的环境提供生物有效氮，但固定氮的种群(重氮菌)却知之甚少。对nifH基因和转录本的测序表明，单细胞和丝状蓝藻以及异养细菌可能是微生物垫中的优势重氮菌。然而，该基因的存在和/或转录并不一定表明这些生物由于转录和翻译后调节而主要负责该系统中的氮气固定。在这项拟议的研究中，将使用多种技术组合来研究光合作用微生物垫的重氮营养群落。首先，nifH基因和转录本测序将识别潜在的重氮菌，并为下一步任务设计引物和探针。其次，通过nifH定量聚合酶链式反应和定量逆转录聚合酶链式反应来确定潜在的重氮菌的丰度和季节演替。第三，这将与15N2孵化实验、原位杂交和纳米级二次离子质谱仪(NanSIMS)的研究相结合。使用卤素标记的酪胺和纳米SIMS(所谓的EL-FISH或HISH-SIMS)的原位杂交将评估富含15N的微生物群落，从而能够识别固定氮气的微生物群落，并在单细胞水平上量化它们的代谢活动。

Phototsynthetic microbial mats are important ecosystems in shallow-water marine environments since they supply these often nitrogen-limited environments with biologically available nitrogen, however the populations that fix nitrogen (diazotrophs) are poorly understood. Sequencing of the nifH genes and transcripts indicated that unicellular and filamentous cyanobacteria as well as heterotrophic bacteria could represent the dominant diazotrophs in microbial mats. However, the presence and/or transcription of this gene do not necessarily indicate that those organisms are primarily responsible for N2 fixation in this system due to transcriptional and posttranslational regulation. In the proposed research, the diazotrophic community of photosynthetic microbial mats will be studied using a combination of techniques. First, nifH gene and transcript sequencing will identify the potential diazotrophs and allows the design of primers and probes for the next task. Second, the abundance and seasonal succession of potential diazotrophs will be determined by nifH quantitative PCR and quantitative RT PCR. Third, this will be combined with 15N2 incubation experiments, in situ hybridization and investigations with nanometer-scale Secondary Ion Mass Spectrometry (nanoSIMS). In situ hybridization using halogen labeled tyramides and nanoSIMS (so called EL-FISH or HISH-SIMS) will assess the microbial community enriched in 15N, thereby allowing the identification of the microbial community that fixed N2 and the quantification of their metabolic activities at the single cell level.