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

光合微生物席是浅水海洋环境中重要的生态系统，因为它们为这些通常氮有限的环境提供生物可利用的氮，然而，人们对固定氮的种群（重氮营养体）知之甚少。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席中主要的重氮营养菌。然而，由于转录和翻译后调控，该基因的存在和/或转录并不一定表明这些生物在该系统中主要负责N2固定。在提出的研究中，光合微生物垫的重氮营养群落将使用组合技术进行研究。首先，nifH基因和转录本测序将确定潜在的重氮营养物，并为下一个任务设计引物和探针。其次，通过nifH定量PCR和RT定量PCR确定潜在重氮营养体的丰度和季节演替。第三，将结合15N2孵育实验、原位杂交和纳米级二次离子质谱（nanoSIMS）研究。使用卤素标记的酰胺和纳米sims（称为EL-FISH或HISH-SIMS）进行原位杂交将评估富含15N的微生物群落，从而可以鉴定固定N2的微生物群落并在单细胞水平上量化其代谢活动。

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.