Elucidation of the homarine degradation pathway in the oceans
Context:
Primary biological production in the oceans exerts significant control over atmospheric CO2. Every day, phytoplankton transform 100 million tonnes of CO2 into thousands of different organic compounds (1). Most of these molecules (as metabolites) are biologically labile and converted back into CO2 within a few hours or days. The climate-carbon feedback loops mediated by this reservoir of labile dissolved organic carbon (DOC) depend on this network of microbes and metabolites. In other words, the resilience of the ocean to global changes(such as temperature rise and acidification) will depend on how this network responds to these perturbations.
Because of its short lifespan, this pool of labile DOC is difficult to observe. Yet these microbial metabolites are the most important carbon transport pathways in the ocean and are assimilated by marine bacteria as sources of carbon and energy. Knowledge of the main metabolic pathways (from genes to metabolites) is therefore essential for modelling carbon flows in the oceans. However, the diversity of these molecules remains largely unexplored and many of them have no annotated biosynthetic and/or catabolic pathways. This is the case for homarin (N-methylpicolinate), an abundant compound in the oceans. Homarine content can reach 400 mM in the marine cyanobacterium Synechococchus (2) and this ubiquitous organism contributes between 10 and 20% of global net primary production (3).Because of its abundance, homarine is probably an important metabolite in the carbon cycle.
Project:
In this thesis project, we aim to elucidate the homarine degradation pathway in the oceans.
Ruegeria pomeroyi DSS-3 is a Gram-negative aerobic bacterium and a member of the marine Roseobacter clade. Its close relatives account for around 10-20% of the bacterial plankton in the mixed coastal and oceanic layer (4). In the laboratory, DSS-3 can use homarine as its sole carbon source but to date, there is no information on the genes and catabolites involved in this process.
Comparative analysis of RNAseq experiments conducted on DSS-3 cultures grown with homarine or glucose (control) as a carbon source will enable us to identify the candidate genes involved in the degradation pathway. This pathway will also be studied using a metabolomic approach based on liquid chromatography coupled with very high resolution mass spectrometry. The difference in profile between DSS-3 metabolomes from cells grown on glucose as a carbon source and those from cells grown on homarine will help to detect catabolites in the pathway. Finally, the candidate genes will be cloned for recombinant expression in E. coli, the corresponding proteins purified and their activity characterized in order to reconstruct the entire homarine degradation pathway in vitro.
Analysis of the expression of these genes in data from the Tara Oceans project (5) will be the first step towards a better understanding of the role of homarine in the carbon cycle.
References :
(1) doi.org/10.1038/358741a0
(2) doi.org/10.1128/mSystems.01334-20
(3) doi.org/10.1073/pnas.1307701110
(4) doi.10.1038/nature03170
(5) https://fondationtaraocean.org/expedition/tara-oceans/
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