Prokaryotic microorganisms in freshwater habitats are centrally involved in carbon turnover processes, yet individual microbial populations greatly differ in their respective metabolic and ecological features. The pelagic zone of freshwater systems is typically dominated by freely suspended ultramicrobacteria. The proposed project will study the functional differences and similarities of three monophyletic groups of freshwater ultramicrobacteria (P. necessarius, actinobacteria from the Ac1 clade and alphaproteobacteria affiliated with the LD12 lineage). It will furthermore investigate the sorting of physiologically and genotypically distinct ecotypes within each group by their specific habitats. Single-cell approaches will be combined with (meta) genomic and transcriptomic analyses both on available isolates and on uncultured ultramicrobacteria in experimental studies and comparative in situ investigations in different habitats, and the diversity and expression of functional genes (e.g., bacterial rhodopsins) will be assessed. The different levels of interactions and common activities envisaged in the proposed project will for the first time allow a synoptic view of the functional roles and phylogeographic distribution patterns of freshwater ultramicrobacteria that could not be achieved otherwise. It also offers a chance to European scientists to take a leading role in freshwater microbiology through the concerted development and application of novel functional genomic research tools.