Culturing the ubiquitous freshwater actinobacterial acI lineage by supplying a biochemical ‘helper’ catalase

Unlike the ocean from which abundant microorganisms with streamlined genomes such as Prochlorococcus, Pelagibacter, and Nitrosopumilus have been isolated, no stable axenic bacterial cultures are available for the ubiquitous freshwater actinobacterial acI lineage. The acI lineage is among the most successful limnic bacterioplankton found on all continents, often representing more than half of all microbial cells in the lacustrine environment and constituting multiple ecotypes. Dilution-to-extinction culturing followed by whole-genome amplification recently yielded 20 complete acI genomes from lakes in Asia and Europe. However, stably growing pure cultures have not been established despite various efforts at cultivation using growth factors predicted from genome information. Here, we report two pure cultures of the acI lineage successfully maintained by supplementing the growth media with catalase. Catalase was critical for stabilizing growth by degrading hydrogen peroxide, irrespective of the genomic presence of the catalase-peroxidase (katG) gene, making the acI strains the first example of the Black Queen hypothesis reported for freshwater bacteria. The two strains, representing two novel species, displayed differential phenotypes and distinct preferences for reduced sulfurs and carbohydrates, some of which were difficult to predict based on genomic information. Our results suggest that culture of previously uncultured freshwater bacteria can be facilitated by a simple catalase-supplement method and indicate that genome-based metabolic prediction can be complemented by physiological analyses.