Exploring B vitamin biosynthesis and exchange in understudied marine stramenopiles and their communities
Microbes are the dominant form of life on Earth. Environmental sequencing has revealed the extent of the diversity of microbial life in all environments, not least the ocean. In this vast aquatic environment microbes, both prokaryotic and eukaryotic, play essential roles in the cycling of nutrients, primary productivity and form the basis of all food webs. These single-celled organisms do not live in isolation, rather they form complex communities, a dominant feature of which is the exchange of nutrients. In many areas of the ocean the concentration of some macro and micronutrients are limiting to the growth of some organisms. In addition, many organisms appear to have dispensed with the ability to biosynthesise various micronutrients, for example, B vitamins. Instead, they rely on an external source to satisfy their requirement. Here I explore the role of B vitamin exchange in microbial communities, with a particular focus on the marine protists of the stramenopile group of eukaryotes. An initial investigation took environmental sea water samples and applied different B vitamin and macronutrient amendments to assess if any of these compounds were limiting for the growth of the microbial community as a whole in this area of the ocean (English Channel), or if growth was co-limited by both B vitamin and macronutrient concentration. The results suggested that none of the B vitamins tested were a limiting factor for growth of the majority of species, either prokaryotic or eukaryotic. However, some changes to the microbial assemblage were observed when assessing the relative transcriptional activities of the organisms in the samples. For example, the bacterial genus Litorivivens was more transcriptionally active (high ratio of ribosomal RNA reads to ribosomal DNA reads) under the addition of vitamins B2, B3, B5, B6 and B9. The eukaryotic genera Ostreococcus and Picochlorum were also more active in the same condition. The stramenopiles display considerable variety in morphology and lifestyle, from parasitic soil-based oomycetes to photosynthetic planktonic species such as the diatoms. A comparative genomics approach was employed to investigate the B vitamin biosynthetic capabilities of this diverse group and therefore deduce any likely auxotrophies for these nutrients. Identifying auxotrophic species hints at potential requirements for nutrient exchange with an external partner. The results of this study showed that the metabolism of stramenopile species is as diverse as the physical attributes of the members of the group. Of the species analysed in this experiment 64% were predicted to be auxotrophic for B12 and this trait was spread across the group in a mosaic fashion. For the vitamin B3 the analysis shows a clear dichotomy in the utilisation of one of two alternative pathway branches. This indicates multiple gene transfer events have occurred across the group to allow for this change in metabolic routes. Biosynthesis for some of the B vitamins, namely B5 and B6 appears to be a universal trait in the stramenopiles. The results also demonstrate that B vitamin auxotrophy, in general, is more common in the heterotrophic species of the group than in the phototrophs. Finally, metagenomics was employed to probe the community surrounding an enigmatic marine stramenopile, Incisomonas marina, one of only a few MAST (MArine STramenopile) species that have been cultured. MASTs represent the hitherto understudied and unknown heterotrophic cohort of the stramenopiles. This is in contrast to the comparatively well studied and understood ochrophytes, the photosynthetic members of the stramenopiles, who have thousands of representatives in culture. I. marina was originally isolated from Nova Scotia, Canada with a cohort of bacterial species, with which it has been maintained in culture. This provides a closed community to interrogate for nutrient exchange and interaction. Analysis of pathways for B vitamin and amino acid biosynthesis revealed that no one single species of the one eukaryote and 23 bacterial species in the community encodes a complete biosynthesis pathway for the full suite of either the B vitamins or the amino acids. This suggests some level of exchange for these essential nutrients indicating a community-based approach to metabolism. Overall, the work outlined in this thesis suggests that B vitamins play an essential role in shaping the communities within which stramenopiles reside. Given the ecological prevalence of this group, these types of interactions between members of the community to share nutrients and exchange metabolites are essential to the health and function of the environments in which they live. Furthering our understanding of these processes will aid our understanding of microbial communities as a whole and the biogeochemical processes that they dictate.
B vitamins, Metabolic pathways, Metagenomics, Microbial communities, Protists, Stramenopiles
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