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

光合微生物垫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，但是对固氮菌（固氮菌）的种群知之甚少。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以代表微生物垫中的优势固氮菌。然而，该基因的存在和/或转录并不一定表明这些生物体主要负责该系统中的N2固定，由于转录和翻译后调节。在拟议的研究中，光合微生物垫的固氮菌群将使用技术的组合进行研究。首先，nifH基因和转录本测序将确定潜在的固氮生物，并允许为下一个任务设计引物和探针。第二，潜在的固氮生物的丰度和季节性演替将通过nifH定量PCR和定量RT PCR来确定。第三，这将与15 N2孵育实验，原位杂交和纳米级二次离子质谱（nanoSIMS）的调查相结合。使用卤素标记的酪酰胺和nanoSIMS（所谓的EL-FISH或HISH-SIMS）的原位杂交将评估富含15 N的微生物群落，从而允许鉴定固定N2的微生物群落并在单细胞水平上定量其代谢活性。

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.