Nutrient utilisation by Trichodesmium characterisation of molecular and physiological Processes

The activity of photosynthetic cyanobacteria capable of nitrogen (N2) fixation (diazotrophs) strongly influences oceanic primary production and global biogeochemical cycles. The niche of these organisms extends mainly across low latitude oligotrophic oceans, largely deficient in nitrate, where they introduce ‘new’ nitrogen (N) to the system. In these regions the abundant marine cyanobacterium Trichodesmium spp. accounts for a significant proportion of the fixed N flux. Despite fixation of N, the availability of phosphorus (P) and iron (Fe) remain a constraint to the activity and biogeography of diazotrophs. The genome of Trichodesmium has therefore been shaped to provide intricate adaptive strategies optimising growth under both P and Fe depletion. Characterisation of these strategies can provide information that will enhance the understanding of the organism’s biogeography in the contemporary and future ocean. In this work, molecular and physiological techniques are employed to study nutrient uptake pathways, and the metabolic response of Trichodesmium erythraeum IMS101 (Trichodesmium hereafter) to nutrient limitation. The current lack of an established system for genetic manipulation of this organism inhibits direct functional characterisation of proteins. To circumvent this, the model cyanobacteria Synechocystis sp. PCC 6803 (Synechocystis hereafter) is used as a vehicle for the heterologous expression of Trichodesmium genes. Using this technique, the suggested contribution of Trichodesmium to an emerging oceanic P redox cycle is first explored. A four-gene cluster (ptxABCD), that encodes a putative ABC transporter (ptxABC) and NAD-dependent dehydrogenase (ptxD), is demonstrated to be responsible for the organism’s ability to utilise the reduced inorganic compound phosphite. The presence and expression of this gene cluster is also confirmed in diverse field metagenomic and metatranscriptomic datasets further confirming its role in Trichodesmium species. Pathways of Fe utilisation are also investigated. Through heterologous expression the function of a currently employed Fe stress biomarker, protein Tery_3377 (IdiA), which is homologous to both Fe3+ transporters (FutA2-like) and intracellular proteins with protective function under Fe stress (FutA1-like), is elucidated. Fusing the signal sequence of this protein to GFP revealed its periplasmic localisation, and its expression in Synechocystis mutants of both futA1 and futA2 paralogues further supported involvement in Fe3+ uptake, providing evidence for its function as an Fe transporter in Trichodesmium. Finally, a physiological experiment was performed to determine the significance of direct physical contact with Saharan desert dust for acquisition of Fe by Trichodesmium. It is demonstrated that cell surface processes are fundamental in dust-Fe utilisation by this organism and transcriptomic analysis identifies a number of unique genes regulated under different Fe and dust regimes including putative cell-surface proteins not previously studied in Trichodesmium. Combined, these studies have revealed a diverse array of molecular and physiological strategies potentially employed by Trichodesmium to survive and thrive on the ephemeral supplies of nutrients encountered in oligotrophic oceans, an attribute that facilitates its significant contribution to biogeochemical cycles.