Host-microbe systems in the ocean: identifying genetic determinants of Phaeobacter inhibens pathogenesis of the phytoplankton Emiliania huxleyi

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Microbes dominate marine habitats and these invisible life forms are responsible for driving the biogeochemical cycles that sustain life on our planet. Perhaps most important are phytoplankton, as these microalgae perform half of the photosynthesis on earth, removing carbon dioxide from the atmosphere and generating oxygen. One ubiquitous and abundant of these is Emiliania huxleyi, which can form blooms in the oceans as large as countries. Besides fixing carbon through photosynthesis, it can also mineralize it into calcite disks that cover its surface. These fall to the bottom of the ocean when the microalgae dies, where they can stay for over 50,000 years. E. huxleyi is also unique in that it uses biologically produced molecules to interact with higher (fish and birds) and lower (protist grazers and viruses) tropic levels within the marine food web. We have recently identified that E. huxleyi also closely interacts with bacteria, which can be pathogenic on their host and thereby accelerate release of calcite. One such bacterium, Phaeobacter inhibens, kills E. huxleyi by activating its programmed cell death pathway (PCD) through an effector molecule delivered through its type IV secretion system (T4SS). Viruses are also known to activate its PCD pathway and this cell lysis is a critical step in the microbial loop. We are the first group to show bacteria also occupy this niche. By sequencing all the RNA transcripts of P. inhibens genes (transcriptomics) when it is killing E. huxleyi, we aim to discover genes involved in bacterial pathogenesis of phytoplankton. We will then perform fitness experiments where a library of >200,000 mutants (RB-TnSeq) will be applied to their host, E. huxleyi, and those which are under and over represented during pathogenesis can be identified as fitness determinants. We will also use this mutant library to further characterize the genetic networks and pathways identified from the transcriptomics and RB-TnSeq in this pathogenic niche. This research proposal will elucidate how this novel pathogen targets E. huxleyi and will allow us to be the first to identify the genetic determinants of a phytoplankton pathogen.