Collaborative research: Adaptation of key N2-fixing cyanobacteria to changing CO2

智力优势。这项研究将采用实验进化技术和最先进的分子方法的新组合，以产生独特的见解，在关键的海洋蓝藻Trichodesmium和Crocosphaera的适应性变化，以应对高CO2的选择。几项研究表明，N2固定率的生态化学关键蓝藻Trichodesmium和Crocosphaera可能会在未来的高CO2海洋显着增加，但是这些都使用了相同的有限的培养分离物组，并且认为细胞仅仅短暂地适应了升高的CO2。然而，研究人员的新结果表明，在每个固氮菌属中存在着广泛的高CO2和低CO2适应生态型。此外，在对束毛藻进行的为期四年的初步实验进化研究中，PI观察到高CO2选择500-700代后的大适应性反应-但以完全出乎意料的方式。所有六个重复的高CO2适应细胞系表现出强烈的组成性上调N2固定率。这些非常高的N2固定率持续存在，即使培养物已被切换回低CO2条件许多个月。在这些细胞系中，nif操纵子和N同化基因的表达也被上调，基因组的许多基因间区域的表达也是如此。研究人员假设，细胞固氮系统的组成性上调可能是在CO2浓度升高的扩展选择下，束毛藻和Crocosphaera的共同适应性反应。本项目将在一项为期四年的实验性进化研究中检验这一假设，以确定这两种固氮生物的高CO2和低CO2专门生态型对CO2增加的适应性反应。研究人员将在380 ppm和750 ppm CO2的条件下，在复制良好的细胞系中，从每个属中培养具有代表性的高和低CO2适应生态型，最多可达1000代。他们将定期进行“切换”实验，以测量N2和CO2固定率以及在低CO2下短暂生长（一周）的高CO2选择细胞系的生长率，反之亦然。这些转换实验将允许筛选在表型表达速率参数中表现出适应性变化的细胞系，例如在初步束毛藻研究中观察到的那些。通过比较其基因组、转录组和蛋白质组随时间相对于参考基因组的变化，使用在前一个选择期每月存档的冷冻样品，检查CO2选择细胞系的进化机制。这些分子和生物化学变化的检查将与一系列深入的生理和地球化学分析相协调。这种结合的方法将允许评估固氮蓝藻中的潜在适应机制，范围从插入缺失、复制、单核苷酸多态性和转座突变到改变的推定非编码RNA表达、蛋白质表达和翻译后蛋白质修饰，然后让研究人员将这些机制与它们对生态系统的潜在影响直接联系起来-如N2和CO2固定。最后，研究小组将确定高CO2的长期选择如何影响Trichodesmium和Crocosphaera的铁和磷需求，因为N2固定的组成性上调也将对未来高CO2海洋中这两种关键营养物质对固氮生物的限制产生重大影响。该项目将支持南加州大学和WHOI的研究生论文工作，以及代表性不足的本科生物学专业的研究活动。该项目的公众教育和宣传工作将通过由南加州大学资助的“2020”倡议赞助的年度系列公众宣传和专业研讨会得到加强，该倡议旨在整合科学和社会对气候变化的反应。该项目最大的科学影响很可能是开发了一种结合海洋科学和进化生物学的开创性新方法，以便从机理上了解固氮蓝藻的适应性反应及其控制的关键海洋地球化学循环，以改变海洋化学和气候

Intellectual Merit. This study will employ a novel combination of experimental evolution techniques and state-of-the-art molecular methods to yield unique insights into adaptive changes in the keystone marine cyanobacteria Trichodesmium and Crocosphaera in response to selection by high CO2 Several studies have suggested that N2-fixation rates of the biogeochemically-critical cyanobacteria Trichodesmium and Crocosphaera may increase dramatically in the future high CO2 ocean, but these have all used the same limited set of cultured isolates and considered cells only briefly acclimated to elevated CO2. The investigator's new results, however, demonstrate that a broad diversity of high- and low-CO2 adapted ecotypes exists within each diazotroph genus. Furthermore, in a preliminary four year experimental evolution study with Trichodesmium, the PIs observed large adaptive responses following 500-700 generations of selection by high CO2- but in a completely unexpected way. All of the six replicate high CO2-adapted cell lines exhibited strong constitutive up-regulation of N2 fixation rates. These very elevated N2 fixation rates continued, even though the cultures have were switched back to low-CO2 conditions for many months. Expression of the nif operon and N assimilatory genes was also up-regulated in these cell lines, as is expression of many intergenic regions of the genome. The investigators hypothesize that constitutive up-regulation of cellular N2 fixation systems may be a common adaptive response of both Trichodesmium and Crocosphaera under extended selection by elevated CO2. This project will test this hypothesis in a four-year experimental evolution study to determine the adaptive responses of both high- and low-CO2 specialized ecotypes of these two diazotrophs to increased CO2. The investigators will grow representative high- and low-CO2 adapted ecotypes from each genus in well-replicated cell lines at 380 ppm and 750 ppm CO2 for up to 1000 generations. Periodically, they will perform "switch" experiments to measure N2 and CO2 fixation rates and growth rates of high CO2-selected cell lines grown briefly (one week) at low CO2, and vice versa. These switch experiments will allow screening for cell lines which exhibit adaptive changes in phenotypically-expressed rate parameters, such as those observed in the preliminary Trichodesmium study. Evolutionary mechanisms in the CO2-selected cell lines will be examined by comparison of changes in their genomes, transcriptomes, and proteomes over time relative to reference genomes, using frozen samples archived monthly during the preceding selection period. Examination of these molecular and biochemical changes will be coordinated with an in-depth array of physiological and biogeochemical analyses. This combined approach will allow an evaluation of potential adaptive mechanisms in diazotrophic cyanobacteria ranging from indel, duplication, single nucleotide polymorphism, and transposition mutations to altered putative non-coding RNA expression, protein expression, and post-translational protein modifications, and then allow the investigators to link these mechanisms directly with their potential impacts on ecosystem-level biogeochemical processes like N2 and CO2 fixation. Finally, the research team will determine how long term selection by high CO2 affects the iron and phosphorus requirements of Trichodesmium and Crocosphaera, since constitutive up-regulation of N2 fixation would also have major implications for limitation of diazotrophs by these two critical nutrients in the future high CO2 ocean.Broader Impacts. This project will support graduate student dissertation work at USC and WHOI, as well as research activities by under-represented undergraduate biology majors. Public education and communication efforts for this project will be enhanced by an annual series of public outreach and professional colloquia sponsored by our USC-funded "2020" initiative intended to integrate scientific and societal responses to climate change. The biggest scientific impact of this project may well be the development of a pioneering new approach combining marine science and evolutionary biology, in order to obtain a mechanistic understanding of the adaptive responses of N2-fixing cyanobacteria and the key biogeochemical cycles that they control to changing ocean chemistry and climate