The difficulty in cultivating many microbial taxa vexes researchers intent on understanding the contributions of these organisms to natural systems, particularly when these organisms are numerically abundant, and many cultivation attempts recover only rare taxa. Efforts to improve this conundrum with marine bacterioplankton have been successful with natural seawater media, but that approach suffers from a number of drawbacks and there have been no comparable artificial alternatives created in the laboratory. This work demonstrates that a newly developed suite of artificial seawater media can successfully cultivate many of the most abundant taxa from seawater samples and many taxa previously only cultivated with natural seawater media. This methodology therefore significantly simplifies efforts to cultivate bacterioplankton and greatly improves our ability to perform physiological characterization of cultures postisolation.
Highlights d Development of a model microbiome with high strain-level diversity for C. elegans d Host genetic variation drives distinct microbiome community types in wild C. elegans d Adult gut microbiome composition is driven by insulin signaling networks d Gut microbiome alters host growth and activates insulin signaling pathways
Cultivated bacterioplankton representatives from diverse lineages and locations are essential for microbiology, but the large majority of taxa either remain uncultivated or lack isolates from diverse geographic locales. We paired large scale dilution-to-extinction (DTE) cultivation with microbial community analysis and modeling to expand the phylogenetic and geographic diversity of cultivated bacterioplankton and to evaluate DTE cultivation success. Here, we report results from 17 DTE experiments totaling 7,820 individual incubations over three years, yielding 328 repeatably transferable isolates. Comparison of isolates to microbial community data of source waters indicated that we successfully isolated 5% of the observed bacterioplankton community throughout the study. 43% and 26% of our isolates matched operational taxonomic units and amplicon single nucleotide variants, respectively, within the top 50 most abundant taxa. Isolates included those from previously uncultivated clades such as SAR11 LD12 and Actinobacteria acIV, as well as geographically novel members from other ecologically important groups like SAR11 subclade IIIa, SAR116, and others; providing isolates in eight putatively new genera and seven putatively new species. Using a newly developed DTE cultivation model, we evaluated taxon viability by comparing relative abundance with cultivation success. The model i) revealed the minimum attempts required for successful isolation of taxa amenable to growth on our media, and ii) identified possible subpopulation viability variation in abundant taxa such as SAR11 that likely impacts cultivation success. By incorporating viability in experimental design, we can now statistically constrain the effort necessary for successful cultivation of specific taxa on a defined medium.
Importance Even before the coining of the term “great plate count anomaly” in the 1980s, scientists had noted the discrepancy between the number of microorganisms observed under the microscope and the number of colonies that grew on traditional agar media. New cultivation approaches have reduced this disparity, resulting in the isolation of some of the “most wanted” bacterial lineages. Nevertheless, the vast majority of microorganisms remain uncultured, hampering progress towards answering fundamental biological questions about many important microorganisms. Furthermore, few studies have evaluated the underlying factors influencing cultivation success, limiting our ability to improve cultivation efficacy. Our work details the use of dilution-to-extinction (DTE) cultivation to expand the phylogenetic and geographic diversity of available axenic cultures. We also provide a new model of the DTE approach that uses cultivation results and natural abundance information to predict taxon-specific viability and iteratively constrain DTE experimental design to improve cultivation success.
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