Transcriptional regulation of bacterial nitrous oxide emissions in complex ecosystems

项目目的和目的：如果我们要减少细菌N2O的排放，那么了解它们是如何在环境和转录水平上受到调控的是一个必不可少的先决条件。本研究将：1.确定细菌sRNA在控制反硝化作用中的作用。我们已经绘制了脱氮副球藻(Paracoccus denitriicans，PD)产N2O和消耗N2O培养物的初级转录组，并鉴定了160个小RNA(SRNA)，其中40%在不同条件下差异表达。细菌sRNA是一系列生理过程的重要调节者，但在反硝化作用中却是前所未有的。我们已经在实验上证实了2/5测试的sRNA，它们会影响PD N2O的排放。我们需要了解SRNA对反硝化的影响。学生将在PD的反硝化培养物中脉冲表达这些sRNA，提取细胞总RNA，并使用RNA-seq测量转录组。一旦确定了这些靶点，就会通过转录/翻译融合和生理学实验进一步验证它们的有效性。学生有机会在此基础上从分支I(N2O生产者和消费者)和II(仅N2O消费者)细菌中识别具有代表性的物种的N2O诱导的sRNA。这一目标将确定一个在反硝化细菌和环境中保守的核心sRNA组，它可能作为全球缓解N2O的目标，并为调节这一重要的生物地球化学循环建立一个新的教条。比较环境变量对模拟土壤(PD)、发酵双杆菌(DF)和海洋(Ruegeria Pomeroyi)反硝化细菌N2O产生的影响。在厌氧、富含N3-、铜限制的条件下，Pd(分支I)释放的N2O是富铜培养的~4000倍，这是由于铜限制的细胞中NOSZ的下调。在其他反硝化菌中是否存在这种情况，将通过用RP(海洋)和DF(土壤，分支II)重复这些实验并测量对反硝化装置转录的影响来进行测试。这将证明铜对N2O排放的监管是否跨越了来自其他环境的反硝化物种。我们假设碳源和NO3-的比例也影响nosZ的转录，我们将在一定的电子供体/受体比范围内，研究铜充足和铜缺乏条件下野生型PD、DF和RP的生理和转录组。这一目标将决定控制这一转变的监管网络以及碳、硝酸盐、铜在肥料制度中的混合和地表径流的影响。3.确定在复合生态系统中用于N2O生产/消费的生化和基因调控。土壤和水环境中的N2O是由系统发育上不同的细菌和古细菌氨氧化菌和反硝化细菌产生的。群落组成和活性成分是由环境条件驱动的，并决定了哪些生物主导一氧化二氮的产生。来自格伦索(与爱丁堡大学合作)和水生(Wensum和Yare)具有不同排放特性的旱地草原泥炭灰壤样本的土壤的比较化验记学将被用来识别与这些环境变化相关的功能和调节基因。主要的活性菌株将被分离和鉴定。这一目标将增加对产生N2O的生物体调控网络的多样性和共性的了解，而不是传统的“实验室大鼠”

Project Aims and Objectives: If we are to mitigate bacterial N2O emissions then understanding how they are environmentally and transcriptionally regulated is an essential pre-requisite. This studentship will:1. Establish the role of bacterial sRNA in controlling denitrification.We have mapped the primary transcriptome of N2O producing versus consuming cultures of Paracoccus denitrificans (Pd) and identified 160 small RNAs (sRNA), 40% of which are differentially expressed between the conditions. Bacterial sRNA are important regulators of a range of physiological processes, but never before in denitrification. We have experimentally confirmed 2/5 tested sRNAs, which impact on Pd N2O emissions. We need to understand how sRNA impact on denitrification. The student will pulse express these sRNA in denitrifying cultures of Pd, extract total cellular RNA and measure the transcriptome using RNA-seq. Once these targets have been identified they are further validated by transcriptional /translational fusions and physiology experiments. The student has the scope to build on this to identify N2O inducible sRNA of representative species from clade I (N2O producers and consumers) and II (N2O consumers only) bacteria. This objective will identify a core sRNAome - conserved across denitrifying bacteria and environments, that may act as global targets for N2O mitigation, and establish a new dogma for regulation of this important biogeochemical cycle.2. Compare how environmental variables impact on N2O production in model soil ((Pd), Dyadobacter fermentans (Df)) and marine (Ruegeria pomeroyi (Rp)) denitrifiers.Under anaerobic, NO3--rich, Cu-limited conditions, N2O release by Pd (clade I) is ~4000 times higher than in Cu-replete cultures; this is due to down-regulation of nosZ in Cu-limited cells. Whether this is the case in other denitrifiers, will be tested here by repeating these experiments with Rp (marine) and Df (soil, clade II) and measuring the effect on transcription of the denitrification apparatus. This will demonstrate if the copper regulation of N2O emissions spans denitrifying species from other environments. We hypothesise that the ratio of carbon source to NO3- also impacts on nosZ transcription and we will examine the physiology and transcriptome of wild-type Pd, Df and Rp under Cu-replete and Cu-deplete conditions at a range of electron donor:acceptor ratios. This objective will determine the regulatory network which controls this switch and the impact of carbon:nitrate:copper mixes in fertilizer regimes and surface run off. 3. Determine the biochemistry and gene regulation used for N2O production /consumption in complex ecosytems. N2O in soil and aquatic environments is generated by phylogenetically diverse bacterial and archaeal ammonia oxidisers and denitrifiers. Community composition and active components are driven by environmental conditions and determine which organisms dominate nitrous oxide production. Comparative metatranscriptomics of soil from an upland grassland peaty podzol soil at Glensaugh (in collaboration with University of Edinburgh) and aquatic (Wensum and Yare) samples, with different emission characteristics, will be used to identify functional and regulatory genes associated with these environmental variations. Dominant active strains will be isolated and characterised. This objective will increase knowledge of the diversity, and commonality, of regulatory networks of N2O producing organisms beyond traditional 'laboratory-rats'