Vitamin B1 (B1 herein) is a vital enzyme cofactor required by virtually all cells, including bacterioplankton, which strongly influence aquatic biogeochemistry and productivity and modulate climate on Earth. Intriguingly, bacterioplankton can be de novo B1 synthesizers or B1 auxotrophs, which cannot synthesize B1 de novo and require exogenous B1 or B1 precursors to survive. Recent isolate-based work suggests select abundant bacterioplankton are B1 auxotrophs, but direct evidence of B1 auxotrophy among natural communities is scant. In addition, it is entirely unknown if bulk bacterioplankton growth is ever B1-limited. We show by surveying for B1-related genes in estuarine, marine, and freshwater metagenomes and metagenome-assembled genomes (MAGs) that most naturally occurring bacterioplankton are B1 auxotrophs. Pyrimidine B1-auxotrophic bacterioplankton numerically dominated metagenomes, but multiple other B1-auxotrophic types and distinct uptake and B1-salvaging strategies were also identified, including dual (pyrimidine and thiazole) and intact B1 auxotrophs that have received little prior consideration. Time-series metagenomes from the Baltic Sea revealed pronounced shifts in the prevalence of multiple B1-auxotrophic types and in the B1-uptake and B1-salvaging strategies over time. Complementarily, we documented B1/precursor limitation of bacterioplankton production in three of five nutrient-amendment experiments at the same time-series station, specifically when intact B1 concentrations were ≤3.7 pM, based on bioassays with a genetically engineered Vibrio anguillarum B1-auxotrophic strain. Collectively, the data presented highlight the prevalent reliance of bacterioplankton on exogenous B1/precursors and on the bioavailability of the micronutrients as an overlooked factor that could influence bacterioplankton growth and succession and thereby the cycling of nutrients and energy in aquatic systems.

The Baltic Sea is one of the world’s largest brackish water bodies and is characterised by pronounced physicochemical gradients where microbes are the main biogeochemical catalysts. Meta-omic methods provide rich information on the composition of, and activities within, microbial ecosystems, but are computationally heavy to perform. We here present the Baltic Sea Reference Metagenome (BARM), complete with annotated genes to facilitate further studies with much less computational effort. The assembly is constructed using 2.6 billion metagenomic reads from 81 water samples, spanning both spatial and temporal dimensions, and contains 6.8 million genes that have been annotated for function and taxonomy. The assembly is useful as a reference, facilitating taxonomic and functional annotation of additional samples by simply mapping their reads against the assembly. This capability is demonstrated by the successful mapping and annotation of 24 external samples. In addition, we present a public web interface, BalticMicrobeDB, for interactive exploratory analysis of the dataset.