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

智力上的功绩。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，对Keystone海洋蓝藻Trichodesum和Crocosphaera响应高二氧化碳选择的适应性变化产生独特的见解几项研究表明，在未来的高CO2海洋中，生物地球化学关键蓝藻Trichodesum和Crocosphaera的固氮率可能会大幅增加，但这些都使用了相同有限的培养分离株，并认为细胞只短暂地适应了高CO2。然而，这位研究人员的新结果表明，在每个重氮菌属中都存在着大量不同的适应高和低二氧化碳的生态型。此外，在用毛霉进行的为期四年的初步实验进化研究中，PI在500-700代高二氧化碳选择后观察到了巨大的适应反应--但以一种完全意想不到的方式。所有6个重复的高CO2适应细胞系都显示出对氮气固定率的强烈结构性上调。这些非常高的氮气固定率继续存在，尽管培养物已经切换回低二氧化碳条件很多个月了。在这些细胞系中，nif操纵子和N同化基因的表达也上调，基因组的许多基因间隔区的表达也是如此。研究人员推测，细胞内氮气固定系统的结构性上调可能是毛霉和冠藻在高二氧化碳长期选择下的一种共同的适应性反应。该项目将在一项为期四年的实验性进化研究中检验这一假说，以确定这两种重氮菌的高二氧化碳和低二氧化碳专化生态型对二氧化碳增加的适应反应。研究人员将在380ppm和750ppm二氧化碳条件下从每个属的复制良好的细胞系中培养具有代表性的适应高和低二氧化碳的生态型，最多培养1000代。他们将定期进行“切换”实验，以测量在低二氧化碳条件下短暂(一周)生长的高二氧化碳精选细胞系的氮气和二氧化碳固定率和生长率，反之亦然。这些转换实验将允许筛选在表型表达速率参数上表现出适应性变化的细胞系，例如在毛霉菌初步研究中观察到的那些参数。CO2选择的细胞系的进化机制将通过比较它们的基因组、转录体和蛋白质组随时间的变化相对于参考基因组的变化来检验，使用在前一选择期间每月存档的冷冻样本。对这些分子和生化变化的检查将与一系列深入的生理和生物地球化学分析相协调。这种结合的方法将允许评估重氮营养蓝藻的潜在适应机制，从插入、复制、单核苷酸多态和转位突变到假定的非编码RNA表达、蛋白质表达和翻译后蛋白质修饰的改变，然后允许研究人员将这些机制与它们对生态系统水平的生物地球化学过程的潜在影响直接联系起来，如氮气和二氧化碳的固定。最后，研究小组将确定高二氧化碳的长期选择对毛藻和冠藻对铁和磷的需求有多大影响，因为对氮气固定的结构性上调也将对未来高二氧化碳海洋中这两种关键营养物质对重氮菌的限制产生重大影响。该项目将支持南加州大学和世界卫生组织的研究生论文工作，以及未被充分代表的本科生生物学专业的研究活动。由南加州大学资助的旨在整合科学和社会应对气候变化的“2020”计划每年举办一系列公众宣传和专业讨论会，将加强该项目的公共教育和宣传工作。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，以获得对固氮蓝藻的适应反应的机械理解，以及它们控制的关键生物地球化学循环对海洋化学和气候变化的影响。

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