Collaborative Research: Next generation physiology: a systems-level understanding of microbes driving carbon cycling in marine sediments

目前，在实验室条件下培养绝大多数(99%)深海微生物是不可能的。由于大多数微生物对培养的顽固性，将特定的基因类型与其原位表型联系起来一直是几十年来微生物学中的一个问题--由于这些群落的复杂性，一直很难弄清楚微生物群落的哪些成员在做什么。这些研究中使用的新技术允许对在不同条件下活跃的微生物细胞进行相对直接的标记，从而允许使用这些“下一代生理学”技术来识别活跃地消耗不同化合物的单个细胞。在这项技术中，代谢活跃的细胞用AA化合物标记，并通过荧光染色检测这些细胞。这使得数百种分类群的新陈代谢可以在几十种不同的条件下并行进行。这些研究将重点放在加利福尼亚州海湾瓜伊马斯盆地的热液活动沉积物上。在那里，一系列广泛的未培养的古菌和细菌的门级别的谱系被假设参与包括碳氢化合物在内的复杂有机分子的降解。吸收和分解这些分子的微生物将被识别出来，他们的新陈代谢将与它们的基因组相关，这将使我们能够理解这些微生物如何能够靠碳氢化合物为生，并帮助我们进一步了解这些化合物是如何在环境中分解的。研究人员利用热门博客、公开演讲和杂志文章来教育和告知公众微生物在创造我们周围世界中的重要性，他们的实验室将为本科生研究人员提供令人兴奋的研究机会。该项目的目的是研究海洋沉积物中微生物碳循环的生理控制，并将特定的基因类型与其原位表型联系起来。研究人员最近开发了一种新的、高度可并行性和适应性的方法，允许以无与伦比的吞吐量研究未培养微生物的微生物功能和代谢相互作用。为了实现这一目标，他们结合了(1)通过生物正交非规范氨基酸标签(BONCAT)标记翻译活性细胞，(2)通过荧光激活细胞分类对单个活性细胞进行分类，以及(3)通过大规模平行基因测序研究活性细胞的身份和基因组成。这导致并行读出数百个未培养的分类群的翻译活性，从而允许确定数百个分类群在数十种不同条件下的功能活性。之所以选择瓜伊马斯盆地沉积物进行这些研究，是因为它们的温度梯度很大，电子供体的身份和可用性各不相同，而且存在为系统提供脂肪烃和芳香烃的碳氢化合物渗漏。这意味着这些沉积物可能具有高度多样化的代谢潜力，他们的研究将使人们能够更深入地了解海洋沉积物中的碳循环和其他地球化学过程。研究人员将在环境样本中识别那些涉及碳氢化合物、复杂底物和异养/自养活动的微生物，并在此之后进行有针对性的全基因组鸟枪测序，以直接识别活跃地利用这些生活方式的微生物，并提供能够利用特定底物的细胞的基因构成。这项工作将为发展中的深海沉积物微生物学领域提供基准研究，并为未来环境微生物学的生理学研究奠定基础。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

It is currently impossible to grow the vast majority (99%) of deep-sea microbes under laboratory conditions. Because of this recalcitrancy of most microbes to cultivation, linking specific genotypes with their in situ phenotypes has been a problem in microbiology for decades - it has been very difficult to figure out which members of microbial communities are doing what, due to the complexity of these communities. The new techniques used in these studies allow for a relatively straightforward labeling of the microbial cells that are active under different conditions, allowing for these "next generation physiology" techniques to be used to identify the individual cells actively consuming different compounds. In this technique metabolically active cells are labeled with aa compounds with the detection of these cells by fluorescence staining. This allows for the metabolism of hundreds of taxa to be followed under dozens of different conditions in parallel. These studies will focus on the hydrothermally active sediments of Guaymas Basin in the Gulf of California. There, a broad range of phylum-level lineages of uncultured archaea and bacteria are hypothesized to engage in the degradation of complex organic molecules, including hydrocarbons. The microbes that assimilate and break down these molecules will be identified, and their metabolism will be correlated with their genomes, allowing for an understanding of how these microbes are able to live on a diet of hydrocarbons, and helping us to further understand how these compounds break down in the environment. The investigators make use of topical blogs, public talks and magazine articles to educate and inform the general public on the importance of microbes in creating the world around us, and their laboratories will provide undergraduate researchers with exciting research opportunities. The aim of this project is to investigate physiological controls of microbial carbon cycling in marine sediments and to link specific genotypes with their in situ phenotype. The investigators recently developed a new, highly parallelizable, and adaptable approach that allows for the studying microbial function and metabolic interactions in uncultured microbes at unrivaled throughput. To achieve this goal they combine the (1) labeling of translationally active cells via bioorthogonal non-canonical amino acid tagging (BONCAT), (2) sorting of individual, active cells by fluorescence activated cell sorting, and (3) study of the identity and genetic makeup of active cells by massive parallel gene sequencing. This results in a readout of the translational activity of hundreds of uncultured taxa in parallel, which allows for the functional activity of hundreds of taxa under dozens of different conditions to be determined. Guaymas Basin sediments were chosen for these studies due to their steep gradients in temperature, variety of electron donor identity and availability, and the presence of hydrocarbon seeps supplying aliphatic and aromatic hydrocarbons to the system. This means that these sediments are likely to have highly diverse metabolic potential, and their study will allow for a deeper understanding of carbon-cycling and other geochemical processes in marine sediments. The researchers will identify those microbes in environmental samples that are involved in hydrocarbon, complex substrate and heterotrophic/autotrophic activity, and follow this with targeted whole genome shotgun sequencing to directly identify the microbes actively utilizing those lifestyles, and provide the genetic makeup of cells that are able to exploit specific substrates. This work will provide a benchmark study for the developing field of deep-sea sediment microbiology and lay the foundation for future physiological studies in environmental microbiology.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.