Collaborative Research: Gene content, gene expression, and physiology in mesopelagic ammonia-oxidizing archaea

智力上的功绩。生物如何对其物理、化学和环境做出反应是海洋生态学的中心问题。对于生活在中层海洋--海洋的“黄昏地带”--的微生物来说，有效的应对措施对于利用从表层海洋下沉的间歇性有机和无机化合物尤为重要。这些生物体必须有一套新陈代谢和调节策略来应对环境的变异性，但这些策略在很大程度上是未知的。了解代谢基因表达的时间和原因对于我们理解海洋中的营养物质再矿化至关重要。海洋第一类(MG1)古生菌是中、深海生物中普遍存在的、数量丰富的微生物，是研究这些问题的有前途的模式生物。MG1古生菌是一种化学自养细菌，它将氨氧化为能量，并为生物量固定碳，因此，在海洋耦合的碳和氮循环中发挥着核心作用。尽管MG1在历史上一直难以培养，但最近的努力已经成功地将具有代表性的MG1古生菌从开阔的海洋引入养殖，并证明了它们在温室气体一氧化二氮生产中的重要性。该项目利用独特的MG1培养和最近测序的其中一种生物-菌株CN25的基因组草稿来研究MG1古生菌对其化学环境变化的生理和转录反应，特别是：1.在一系列能量可获得性和亚硝酸盐胁迫下生长的CN25细胞的比较转录组学将识别可用于诊断自然种群生理状态的选定基因2。MG1古菌基因组和转录组学知识的改进将有助于彻底重新解释现有的元基因组和元转录数据集，并在未来的研究中提供更好的上下文理解。研究人员将对指数生长期中期和稳定期收获的CN25细胞与饥饿细胞进行比较转录组学研究。在高硝酸盐浓度和低PO2条件下生长的细胞与在标准条件下生长的细胞的转录本将被描述。一种链特异的高密度RNAseq方法将用于检查假定的ORF、多顺反子操纵子和小RNA的表达，除了基因表达谱外，还具有改善基因组注释的辅助好处。最后，研究人员将对另外两个从公海分离的MG1菌株以及来自环境调查的单个细胞的基因组进行测序，并利用与CN25基因组的组合来重新分析现有的元基因组和元翻译数据集。这些结果将定义模型中层微生物对一系列化学环境的转录反应，并展示物理化学环境如何诱导基因表达和基因内容的变化，从而导致温室气体产生。这项工作将迅速产生一些新的知识，了解一些最普遍但迄今难以捉摸的微生物如何对地球化学变化做出反应，并塑造我们对海洋氮循环的不断演变的理解。该项目的科学和社会影响将是阐明生物和化学海洋学家广泛感兴趣的一种模式海洋生物产生温室气体的机制。转录组测序将改进CN25基因组的组装，CN25基因组是来自公海的MG1考古子的第一个基因组。基因组和转录组都将是研究人员在海洋中使用元基因组学、元转录组学和元蛋白质组学的重要参考，因为这些技术依赖于由DNA序列和生理学组成的知识库。因此，这些结果对现有和未来的研究都有价值。这项拟议的研究将通过为两名职业生涯早期的研究人员、一名博士后研究人员和一名中学教师提供支持，促进对下一代海洋科学家的教育、教学和培训。

Intellectual Merit. How organisms respond to their physical and chemical and environment is a central question in marine ecology. For microbes living in the mesopelagic - the ocean's "twilight zone" - an efficient response is particularly important to capitalize on the intermittent delivery of organic and inorganic compounds sinking from the surface ocean. These organisms must have a suite of metabolic and regulatory strategies used to cope with environmental variability, but these strategies are largely unknown. Understanding when and why metabolic genes are expressed is critical to our understanding of nutrient remineralization in the ocean. Marine group 1 (MG1) archaea are ubiquitous, abundant microbes in the meso- and bathypelagic and promising model organisms for investigating these questions. MG1 archaea are chemolithoautotrophs that oxidize ammonia for energy and fix carbon for biomass, and as such, play a central role in the ocean's coupled carbon and nitrogen cycles. Though MG1 have historically eluded cultivation, recent efforts have been successful at bringing representative MG1 archaea from the open ocean into culture and demonstrating their importance in the production of the greenhouse gas nitrous oxide. This project takes advantage of unique MG1 cultures and the recently sequenced draft genome of one of the organisms - strain CN25 - to investigate the physiological and transcriptional responses of MG1 archaea to variations in their chemical environment, specifically:1. Comparative transcriptomics of CN25 cells grown under a range of energy availability and nitrosative stress will identify select genes that can be used to diagnose the physiological state of natural populations2. Improvements in the genomic and transcriptomic knowledge of MG1 archaea will facilitate a thorough reinterpretation of existing metagenomic and metatranscriptomic datasets, as well as provide a better contextual understanding in future studiesThe investigators will conduct comparative transcriptomics of CN25 cells harvested in mid-exponential growth and stationary phase versus starved cells. Transcriptomes of cells grown at high nitrate concentrations and low pO2 with those grown in standard conditions will be characterized. A strand-specific, high-density RNAseq approach will be used to examine the expression of putative ORFs, polycistronic operons, and small RNAs, which, in addition to gene expression profiling, has the ancillary benefit of improving genome annotation. Finally, the investigators will sequence the genomes of two additional MG1 strains isolated from the open ocean, as well as single cells from environmental surveys, and leverage the combination with the CN25 genome to reanalyze available metagenomic and metatranscriptomic datasets. The results will define the transcriptional response of a model mesopelagic microbe to a range of chemical environments, and show how the physicochemical environment induces changes in gene expression and gene content that result in greenhouse gas production. This work will rapidly generate new knowledge of how some of the most ubiquitous, yet heretofore elusive, microorganisms respond to geochemical variability and shape our evolving understanding of the marine nitrogen cycle.Broader Impacts. The scientific and societal impact of the project will be to elucidate the mechanisms of greenhouse gas production in a model marine organism that is of broad interest to biological and chemical oceanographers. Transcriptome sequencing will improve the assembly of the CN25 genome, the first genome of an MG1 archaeon from the open ocean. Both the genome and transcriptomes will be important references for researchers using metagenomics, metatranscriptomics, and metaproteomics in the ocean, as these techniques are reliant on a knowledgebase composed of both DNA sequence and physiology. Thus, the results add value to both existing and future studies. The proposed research will advance education, teaching, and training for the next generation of marine scientists by providing support for two early-career investigators, one postdoctoral researcher, and a secondary school teacher.