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Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

Significance One of the biggest challenges in environmental microbiology is to determine the activity of uncultured cells directly in their habitat. We report on the application of bioorthogonal noncanonical amino acid tagging (BONCAT), a high-throughput approach to detecting protein synthesis in individual cells by fluorescence staining, on deep-sea methane seep sediments. By combining BONCAT with fluorescence in situ hybridization, we visualized active archaeal−bacterial consortia catalyzing the anaerobic oxidation of methane. We further developed a novel approach that combines BONCAT with fluorescence-activated cell sorting (FACS) to separate translationally active cells from complex samples. BONCAT-FACS enabled us to directly link the identities of anaerobic methane-oxidizing archaea with their partner bacteria for individual active consortia, uncovering previously unknown interactions between these archaea and Verrucomicrobia. To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.