RUI: High throughput culturing and ecophysiological analyses of low light adapted Prochlorococcus ecotypes

海洋蓝细菌原绿球藻是地球上已知的最小但最丰富的光养生物，对世界开阔海洋的初级生产和生物地球化学循环做出了重大贡献。自20年前首次描述以来，这种生物已成为一种得到充分研究的海洋微生物，正在被开发为跨尺度系统生物学的模型系统。自1988年以来，已获得至少26个菌株，根据其生理生化特性，将其分为两个不同的生态类型：强光适应型（HL）和弱光适应型（LL）。超过80%的菌株属于HL适应型，这是由至少两个不同的系统发育分支。其他分离株（6）被认为是LL适应的，分布在几个系统发育分支中，其中两个分支由单一培养分离株代表。有证据表明，这两个HL适应分支可能演变的最适温度的差异，导致其人口的不同水平分布。目前尚不清楚的是不同的LL适应分支的表型和/或生态基础以及这些分支在空间和时间上的分布，在很大程度上是因为代表每个分支的分离株很少。因此，尽管相对大量的菌株和数据的原绿球藻，只有一半的故事，更占主导地位的HL适应生态型，是了解的系统发育关系和分布。本计画将研究组成适应LL的原绿球藻生态型的不同系统发育分支的生理特性，这些生理特性对生态学和进化有贡献。这种表征将通过高通量培养和生理评估实验来实现，这些实验利用多孔板来纯化新的LL适应性分离株、生理测定和连接到流式细胞仪的96孔采样系统。该项目将分离新的无菌培养物，并附带生态生理学和系统发育信息，可供生物海洋学家，海洋地球化学建模者，微生物学家和进化生物学家的自然种群分布，生理机制，基因组学，转录组学和蛋白质组学的世界上最丰富的光养生物的进一步研究。例如，16 S-23 S rRNA序列可用于重新设计或开发用于LL适应原绿球藻种群和特定生态型的环境检测的新引物，这反过来将使研究人员开始了解它们在更深的真光层中的生态作用。新的原绿球藻分离株将提供给其他研究人员，并存放在一个更大的培养收藏，如海洋浮游植物培养中心。基因序列将保存在公共数据库GenBank中，本研究的结果将为选择特定菌株进行进一步的基因组序列分析提供信息。此外，该项目还将为几名本科生提供研究机会和财政支持，以开展研究项目，一名生物学硕士学生，以及USM第一位缅因州系统合作微生物学博士生。

The marine cyanobacterium, Prochlorococcus, is the smallest yet most abundant known phototroph on the planet and contributes significantly to primary production and biogeochemical cycling in the world's open oceans. Since its first description 20 years ago, this organism has become a well-studied marine microbe that is being developed as a model system for cross-scale systems biology. At least 26 isolates have been obtained since 1988, from which two distinct ecotypes were identified based on the relative photophysiological characteristics: high-light (HL) adapted and low-light (LL) adapted. More than 80% of the isolates belong to the HL adapted ecotype, which is composed of at least two distinct phylogenetic clades. The other isolates (6) are considered LL adapted and are spread among several phylogenetic clades, two of which are represented by single cultured isolates. Evidence indicates that the two HL adapted clades likely evolved from differences in temperature optima resulting in distinct horizontal distributions of their populations. What is not clear are the phenotypic and/or ecological underpinnings of the different LL adapted clades and the distributions of these clades in space and time, in large part because there are so few isolates representing each clade. Thus, despite the relatively large number of isolates and data on Prochlorococcus, only half the story, that of the more dominant HL adapted ecotypes, is understood in terms of the phylogenetic relationships and distributions. This project will study the physiological characteristics that contribute to the ecology and evolution of different phylogenetic clades that make up the LL adapted Prochlorococcus ecotype. This characterization will be achieved through high throughput culturing and physiological assessment experiments that utilize multi-well plates for purifying new LL adapted isolates, physiological determinations, and a 96-well sampling system attached to a flow cytometer.Broader Impacts: This project will isolate new, axenic cultures with accompanying ecophysiological and phylogenetic information that could be used by biological oceanographers, biogeochemical modelers, microbiologists and evolutionary biologists for further studies of natural population distributions, physiological mechanisms, genomics, transcriptomics, and proteomics of the world's most abundant phototroph. For instance, the 16S-23S rRNA sequences can be used to redesign or develop new primers for environmental detection of LL adapted Prochlorococcus populations and specific ecotypes, which in turn will enable researchers to begin to understand their ecological role in the deeper euphotic zone. New Prochlorococcus isolates will be made available to other researchers as well as deposited in a larger culture collection such as the Center for the Culture of Marine Phytoplankton. Gene sequences will be deposited in the public database, GenBank, and the results from this study will provide information to choose specific strains for further genome sequence analysis. In addition, this project will also provide research opportunities and financial support for several undergraduates to carry out research projects, a MS student in Biology, and the first University of Maine System Cooperative PhD student in Microbiology at USM.