Research Projects
The section below defines in more detail the roles and projects of the individual consortium members. These interact to form the integrated research programme described in the project background page.
Plymouth Marine Laboratory
Marine microbial communities - seasonal and inter-annual variability (contributing to Hypotheses 1 and 2).
The aim is to design oligonucleotide arrays that can be used to characterise the diversity of the microbial assemblage at the English Channel sampling site L4 (50.25 N, 4.216 W) and on the Atlantic Meridional Transect (AMT). We plan to base the array on published phylogenetic gene sequences (RDP database and MegX) of marine bacteria, on PML sequence data from the English Channel, on bacterial artificial chromosome (BAC) and fosmid library clones that we will produce for L4 in this project and on metagenomic and genomic sequences that are in the public domain. We will screen metagenomic libraries for phylogenetic markers and genes associated with biogeochemical cycling.
Metagenomic analyses of virus-driven nutrient regeneration on microbial growth in marine ecosystems - (contributing to Hypothesis 1).
The main non-predatory loss route for primary production by phytoplankton in virus-induced cell lysis and as much as one-quarter of the photosynthetically-fixed organic carbon is recycled back to the dissolved fraction by viral lysis. Virus infection of phytoplankton will lead to cell lysis and release of labelled cell contents (including labelled progeny viruses) that will become available to a microbial community. Downstream analysis will include spotting metagenomic libraries of key gene clusters (from both virus and bacteria libraries) onto microarray slides as part of the consortium chip.
Aberdeen, Newcastle, Warwick Universities and Centre for Ecology & Hydrology
The response of ammonia-oxidising bacteria (AOB) to environmental change caused by increased atmospheric carbon dioxide using transcriptomic and proteomic approaches - (contributing to Hypothesis 2).
The team will use a combination of cultured and naturally occurring AOB communities to identify physiological responses to perturbations in carbon dioxide, pH and ammonia and to relate those to changes in natural communities and to nitrification rates. Pure cultures of AOB isolated from marine and freshwater environments will be incubated at a range of pH values and ammonia concentrations and the differential responses will be determined using microarray analysis and proteomic analysis, with simultaneous assessment of growth rate and activity.
Transcriptomic and proteomic responses will be characterised in enrichment cultures and compared with those of pure cultures. In addition, SIP analysis following incubation of enrichment cultures and environmental samples with 13C-labelled CO2 will be used to identify which strains are active, and which genes are expressed, at different ammonia concentrations and pH values.
Cardiff University
Diversity and function in the marine sediments - (contributing to Hypotheses 1 and 3).
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The aim will be to combine molecular biological, geomicrobiological and ecological approaches to compare microbial population diversity and function in marine sediments. Focusing on the coupling between degradation/re-mineralisation processes and the microbial populations in sediments, so as to correlate process rates with the size and productivity, structural and functional diversity of the sediment prokaryotic community. |
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A key aim of the project is to use the comparative analysis of metagenomes to improve our understanding as to how biogeochemical processes are integrated between microbial communities in the sediment/benthic and pelagic compartments of marine ecosystems. The oceanic (pelagic-dominated) AMT site to be investigated by other partners in the consortium will provide a comparison where processes are not influenced directly by sediments. |
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Centre for Ecology & Hydrology
Analyses and curation of data - (underpinning Hypotheses 1, 2 and 3).Overseeing the consortium activities for data analysis and bioinformatics, to ensure timely exchange and publication of data as well as to co-ordinate the evolution of the consortia website. Responsibility for the overall co-ordination, curation and integration of the consortium's resulting data sets, accessing Genespring through the EGTDC, and ensuring all microarray data is submitted in MIAME compliant format to the central EGTDC server.
Function specific genomics using RNA-Stable Isotope Probing - (contributing to Hypotheses 1, 2 and 3).
The first role will involve underpinning of the consortium's use of SIP (for 16SrRNA or mRNA work) by liaising with Warwick University group for technical developments, and to supply expertise to consortium members specifically applying RNA-SIP in their laboratories.
The focus of experimental data generation for period 12 - 36 months will be through analyses of L4 samples and as part of the Bergen mesocosm experiments. Specifically, tracing 13C-carbon fixation and subsequent transfer within the microbial fraction of marine communities. (Back to top of page)
Sheffield University and Essex University
Metagenomics of biogeochemical cycling in sediments - (contributing to Hypotheses 1, 2 and 3).
This research will investigate the activity and genetic potential of key bacterial functional groups involved in biogeochemical cycling in estuarine sediments in the Colne estuary. Metagenomic libraries will be constructed from sediments to permit characterisation of functional genes and operons involved in nitrogen cycling, in particular nitrate reduction.
Together with other environmentally-relevant nitrogen cycling gene sequences this will allow development of a suite of probes for use in microarray experiments to determine spatial genetic variation in response to nutrient/salinity gradients, and to investigate how indigenous bacterial communities respond to changes in nutrients, salinity and temperature.
The research will be conducted in collaboration with other members of the consortium to investigate sulphate reduction and methanogenesis (Cardiff), polymeric carbon degradation (Liverpool) and correlation to taxonomic markers (Newcastle), and the application of SIP approaches (in collaboration with Warwick) to identify active components of the sediment bacterial community.
Liverpool University
Degradation of polymeric carbon by marine microorganisms - (contributing to Hypotheses 1 and 3).
A combination of techniques will be used to analyse the microbial population that colonises polymeric carbon in situ using a combination of genomic and proteomic techniques. Particularly interested in actual polysaccharide degraders and will be seeking to identify both community markers and functional genes, and determine their abundance.
SIP analysis of nucleic acids with 13C cellulose as source will be used to ground observations and further refine the selection of probes for the consortium's microarray.
Polysaccharide hydrolases have been extensively classified into a large number of families on the basis of their domain structures at the amino acid sequence level. The team will perform proteomic analysis of colonised cellulose baits in parallel to identify polysaccharide hydrolases expressed in situ. Sequence information obtained by MALDI-ToF and MS-MS QToF will add a further dimension to the identification of partial gene sequences for the most ecologically active and relevant marine polysaccharide degraders.
Newcastle University
Linking function and phylogeny: the distribution of functional genes across divergent marine taxa using an integrated metagenomic approach - (contributing to Hypothesis 1).
This project links specifically with nitrogen-fixation at Stirling and the degradation of polymeric carbon at Liverpool. Newcastle will analyse metagenomic DNA libraries from L4 sediment samples and prepare 15N-enriched (DNA-SIP and RNA-SIP) fosmid libraries from environmental and experimental samples. High molecular weight BAC libraries will be constructed from representative environmental metagenome libraries using the Nycodenz method and cells isolated from the environmental samples used to construct metagenomic libraries and analysed for function (niffH) and phylogenetic affiliation (16S) using a combination of FISH and in situ PCR.
The response of natural communities of ammonia-oxidising bacteria to changes in pH, DIC and ammonia availability using SIP-enabled metagenomics - (contributing to Hypotheses 1 and 2).
The aim of the project is to test if changes in ammonia and DIC concentration, and pH engendered by increasing atmospheric carbon dioxide affect the composition and gene expression in naturally occurring aquatic ammonia-oxidising bacteria. 13C-based stable isotope probing will be used in a hierarchical approach whereby 16S rRNA surveys of 13C-enriched DNA and RNA will provide a framework within which to interpret data from fosmid and cDNA libraries from the same samples.
National Oceanography Centre & Warwick University
To link biodiversity and function of natural populations of Prochlorococcus and Synechococcus using isotopic tracer, molecular and flow cytometric sorting techniques - (contributing to Hypotheses 1 and 2).
This project will make specific use of flow cytometric sorting technology to analyse natural picocyanobactererial populations. This methodology will complement the use of SIP being used elsewhere within the consortium. We propose to use tandem MS with synthetic labelled peptides as internal standards to quantify changes in cyanobacterial protein components that are likely to be of critical importance in amino acid uptake and processing.
In order to assess whether specific cyanobacterial populations have the 'genetic' capacity to utilise only certain N and P substrates, we will use environmental DNA derived from flow sorted Synechococcus and Prochlorococcus populations to perform PCR and construct BAC or fosmid libraries targeted to N utilisation genes and P regulatory genes.
Stirling University
Metagenomic analysis of marine carbon and nitrogen fixers - (contributing to Hypothesis 2).
This team will examine the diversity and expression of the key genes involved in C and N fixation in marine communities. The aim is to identify the most significant organisms carrying out these processes with a particular focus on those microbes that contribute to new production by either assimilating nitrate/nitrite or by fixing N2.
The metagenomic transcriptome will be amplified by RT-PCR using degenerate primers incorporating recognition sites for class IIS endonucleases targeting for I and form II rbcL (encoding the large subunit of RubisCO) and nifH.
Metagenomic libraries will be constructed in amplifiable fosmid vectors from each study site and screened with the functional (nifH and rbcL) and 16S rRNA genes for common phenotypes identified above.
Warwick University - Nicholas Mann
Gene expression in virus-infected phytoplankton cells under nutrient-replete and deplete conditions - (contributing to Hypotheses 1 and 2).
Microarray studies (MIAME compliant) will be carried out to monitor expression of phage S-PM2 genes during the course of infection of P-replete and P-deplete Synechococcus cells. This transcriptional analysis will be coupled to proteomic analysis. Synechococcus proteins characteristic of the P-deplete state will be identified by 2D-PAGE, tryptic digestion and MALDI-TOF. Proteins characteristic of S-PM2 P-deplete pseudolysogenic cells will be identified.
These studies should permit the sequential pattern of phage gene expression in infected cells, and also in cells undergoing pseudolysogeny. These results taken together with those from the proteomic studies will yield a clear picture of the subtle phage-host interactions taking place in the oligotrophic marine environment. (Back to top of page)
Warwick University - Colin Murrel and Centre for Ecology & Hydrology
The role of methylotrophic bacteria in the biogeochemical cycling of one-carbon compounds including biogenic gases - (contributing to Hypotheses 1 and 3).
The team will investigate the cycling of atmospheric trace gases such as CH4 dimehtylsulphide (DMS), CH3C1 and CH3Br in the marine environment and we now have functional gene probes and excellent databases of sequences for methanotrophs (pmoA, nmoX, mxaF) and the methyl halide degrading methylotrophs (cmuA). We will use DNA-SIP techniques to determine the distribution, diversity and activity of these methylotrophs in the environment and to asses their role in biogeochemical cycling of biogenic gases.
The team will use a recently developed microarray for the detection of pmoA and amoA genes (encoding the active site polypeptides of methane and ammonia monooxygenases respectively) in environmental samples.
Time-course studies will be carried out during SIP experiments in order to determine the distribution of pulses of 13C-labelled carbon through different populations of bacteria through analysis of 16S rRNA gene libraries constructed from heavy 13C-DNA generated in SIP experiments.