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

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?

The deep biosphere is estimated to contain 10-33% of the Earth's biomass, but it is one of the most challenging and extreme environments on Earth. Despite these conditions, or perhaps because of them, these microorganisms are contributing to fundamental biogeochemical processes with planetary-scale impacts stimulating from mineral transformation, elemental cycles and oceanic crust weathering. Revealing the metabolic diversity, energy sources and biogeochemical transformations performed by candidate phylum JS1 bacteria, a key major group in the deep biosphere, promises to provide a wealth of novel information, much of which has significant scientific and potential biotechnological implications. Furthermore, the association of these bacteria with mesothermic petroleum reservoirs has links them to unique hydrocarbon degradation pathways.

However, given that this potential has not been demonstrated (unequivocally) for any cultured microbe from this environment suggests that the important metabolic pathways lie in the as yet uncharacterised and mainly uncultivated so called 'Dark Matter' bacteria. The current project will use modern molecular biological approaches linked to new cultivation method to uncover the metabolic and biogeochemical potential of bacteria in candidate phylum JS1. These resources available include fosmid clones, single sorted cells and enriched cultures of the JS1 phylum, and we will harness next generation sequencing approaches to derive the genomic architecture for this phylum. This provides significant potential to reveal genes and pathways of significant biotechnological importance. The team, which is internationally recognised in this area and has pioneered deep biosphere research, will ensure any potential biotechnological impact is realised through a combination of protection of Intellectual property and engagement with commercial partners (including the petroleum industries) as appropriate. Where it will not compromise possible exploitation the work will be disseminated widely through papers and conference attendance adding to the burgeoning international community engaged deep biosphere research. We will also hold a targeted workshop on exploitation of the JS1 genome information during the final phases of the project engaging both academic and commercial partners.

The public fascination with the idea of life buried in rocks deep below the ocean floor has been illustrated through the success of public engagement projects such as 'Adopt a Microbe from the Deep Bioshere' (http://sites.google.com/site/adoptamicrobe/). The team will tap into this interest through a suite of public engagement partnerships, National Museum of Wales and the Techniquest Science Discovery Centre, together with established forums, such events like this year's 'The Scott Lecture Series' (School of Earth and Ocean Sciences) and the Wales Gene Park Christmas lectures. These activities will be support through a web-based portal that will both inform the public as to the ongoing progress and allow them to meet and interact with the scientists performing the proposed research.