How are candidate phylum JS1 bacteria adapted for life in the deep sub-seafloor biosphere?

20世纪90年代中期，我们在日本海下约200米处采集的沉积物样本中发现了一组不寻常的细菌。我们不能在传统的实验室培养基上，在液体或培养皿中的琼脂上培养它们，但它们是通过直接从沉积物中测序16 S rRNA基因来鉴定的，这使我们能够将它们与其他已建立的细菌物种联系起来。我们和其他人进行的进一步研究表明，这些细菌在深海底生物圈的许多地方都非常丰富，它们构成了一个潜在的或候选的新部门或门，我们称之为JS 1。随着微生物学家开始更常规地使用DNA分析来研究细菌和微生物群落，很明显，绝大多数细菌物种不能在实验室中生长。例如，在海洋沉积物中，0.1%或更少的物种是未培养的;候选门JS 1细菌只是50个，可能是100个候选门中的一个，这些门没有培养的代表，只能根据它们的16 S rRNA基因进行鉴定。如果含有大肠杆菌等病原体的门（变形菌）和MRSA（厚壁菌门）或光合细菌（蓝细菌），或产生抗生素的放线菌，因为人们对它知之甚少，很难想象它对医学、工业和社会会产生什么样的后果。这项研究旨在组建一支强大的科学家合作团队，由卡迪夫小组领导，该小组在过去二十年中开创了深层生物圈研究的发展。这项合作提供了一个独特的机会，利用欧洲和美国最近的一些研究，开发使用荧光激活细胞分选和微流控激光捕获等技术分离的单个细胞进行基因组分析的方法。丹麦奥胡斯大学的合作者已经分离出了JS 1细菌的单细胞，该项目的首要目标之一是对其基因组进行测序。与此同时，我们在美国UNLV的合作者将致力于研究一个潜在相关的候选门（OP 9），他们从黄石公园的温泉中分离出单细胞。美国麻省理工学院的另一个项目合作伙伴已经从一个油藏中克隆了JS 1基因组DNA片段，并将与卡迪夫团队合作对这些片段进行测序。除了单细胞JS 1基因组测序外，我们还将尝试使用包括高压培养在内的各种系统，在实验室中改进这些细菌的培养。项目的这一部分还将涉及开发DNA标记，以进一步调查JS 1细菌在地球各地沉积物中的分布和多样性，其中许多样品已经储存在卡迪夫的-80 ° C档案中。从低浓度中扩增DNA的方法（QPCR）将用于定量地下沉积物中的JS 1细菌，并将与德国汉诺威的合作者合作使用一种称为CARD-FISH的方法来可视化和计数它们。因此，该项目的另一个目的是在实验室中获得高度富集JS 1细菌的培养物，并从这些培养物中提取DNA和RNA，这些培养物可以进行测序和分析，以与单细胞和克隆的基因组DNA进行比较。然而，JS 1细菌和其他微生物之间的相互作用可能在它们的活动和生物化学循环方面非常重要，因此我们也想了解与JS 1细菌相关的细菌和其他微生物的群落。尽管我们知道JS 1细菌在地球仪的深层生物圈沉积物中数量丰富且分布广泛我们对地球上最大的生物群之一（的确，它们可能构成地球上最丰富的生物群之一）的了解并不多。最终，该项目旨在回答这个问题：JS 1细菌如何适应深海海底生物圈中的生活？

In the mid-1990s we discovered an unusual group of bacteria in sediment samples collected from about 200 metres below the Japan Sea. We could not grow them on conventional laboratory media, in liquid or on agar in Petri dishes, but they were identified by sequencing 16S rRNA genes directly from the sediment that allowed us to relate them to other established bacterial species. Further studies that we and others carried out showed that these bacteria were highly abundant in many parts of the deep sub-seafloor biosphere, and that they constituted a potential or candidate new division or phylum, which we called JS1. As microbiologists started to use DNA analysis more routinely to investigate bacteria, and microbial communities generally, it became apparent that the vast majority of bacterial species could not be grown in the lab. For example, in marine sediment 0.1% or less of species were uncultivated; candidate phylum JS1 bacteria was just one of 50, possibly 100 candidate phyla which had no cultivated representative and could only be identified on the basis of their 16S rRNA genes. If the phyla containing pathogens like Escherichia coli (Proteobacteria) and MRSA (Firmicutes), or the photosynthetic bacteria (Cyanobacteria), or the antibiotic-producing Actinobacteria were as poorly understood it is difficult to imagine what the consequences would have been for medicine, industry and society.This research aims to assemble a strong team of collaborating scientists, led by the Cardiff group who have pioneered developments in deep biosphere research over the last two decades. This collaboration provides a unique opportunity to exploit some recent research in Europe and the USA, developing methods for genome analysis using individual cells isolated by techniques such as fluorescence-activated cell sorting and microfluidic laser capture. Single cells of JS1 bacteria have been isolated by collaborators at Aarhus University, Denmark, and one of the first aims of the proposed project is to sequence their genomes. In parallel, our collaborators at UNLV, USA, will be working on a potentially related candidate phylum (OP9), single cells of which they have isolated from hot springs in Yellowstone Park. Another project partner at MIT, USA, has cloned JS1 genomic DNA fragments from an oil reservoir, and will collaborate with the Cardiff team to sequence these fragments. In addition to the single cell JS1 genome sequencing, we will be trying to improve cultivation of these bacteria from sediments in the laboratory, using a variety of systems including high-pressure cultivation. This part of the project will also involve developing DNA markers to investigate further the distribution and diversity of JS1 bacteria in sediments from across the Earth, many samples of which are already stored in an archive at -80oC at Cardiff. Methods to amplify DNA from low concentrations (QPCR) will be used to quantify JS1 bacteria in sub-surface sediment, and a method called CARD-FISH to visualize and count them, will be used in partnership with a collaborator at Hannover, Germany. Another aim of the project, therefore, is to obtain cultures in the lab that are highly enriched in JS1 bacteria, and to extract DNA and RNA from these cultures that can be sequenced and analysed for comparison to the single cell and cloned genomic DNA. However, interactions between JS1 bacteria and other microbes are likely to be very important in terms of their activities and biogeochemical cycles, so we will also want to learn about the community of bacteria and other microorganisms with which JS1 bacteria are associated.Thus, although we know JS1 bacteria are abundant and widespread in deep biosphere sediments across the globe (indeed they may constitute one of the most abundant taxa on Earth), we don't know much else about them. Ultimately, this project aims to answer the question: how are JS1 bacteria are adapted for life in the deep sub-seafloor biosphere?