Multi-omics uncovers nutrient stress-driven interactions in a synthetic Prymnesium parvum holobiont, with vitamin B12-limitation revealing mutualism.

Microalgal-bacterial interactions are central to nutrient cycling and ecosystem functioning in marine habitats, yet the mechanisms structuring these associations under defined nutrient constraints remain poorly resolved. Using a synthetic 15-member bacterial community (SynCom) and controlled nitrogen (N), phosphorus (P), and vitamin B12-limitation, we investigated how nutrient scarcity shapes the physiology, metabolism, and transcriptional activity of the harmful alga Prymnesium parvum and its associated microbiota. Under N- and P-limitation, the SynCom had minimal impact on algal growth despite nutrient-dependent shifts in toxin production and metabolite profiles. In contrast, B12-limitation triggered a strong mutualistic interaction in which the SynCom enabled a three-fold increase in algal biomass and drove restructuring of intracellular and extracellular metabolomes, including the accumulation of ectoine and membrane-associated lipids and the depletion of thiamine-like and stress-associated metabolites. Metabarcoding revealed stable community composition but enrichment of B12-producing taxa under B12-limited conditions, while metatranscriptomics uncovered functional divergence among SynCom members. Bacteria displayed contrasting response strategies, including nutrient-responsive specialists and transcriptionally flexible generalists maintaining gene expression across all nutrient regimes. B12-producers (Marinovum algicola, Roseobacter sp., Halomonas sp.) upregulated cobalamin biosynthesis exclusively under B12-limitation, whereas several B12-auxotrophic taxa induced B12-transport and B12-requiring enzymes, indicating active vitamin exchange within the holobiont. P. parvum displayed transcriptional programs specific to each nutrient limitation, with B12-limited co-cultures shifting toward growth-associated gene expression despite constitutive expression of the B12-dependent metH gene. These results demonstrate that vitamin auxotrophy acts as a key metabolic lever reorganizing holobiont function, driving reciprocal benefits and reprogramming algal-bacterial metabolism in our synthetic system.