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

Abstract

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 rRNAtargeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescenceactivated 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 sulfatereducing 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.activity-based cell sorting | BONCAT | click chemistry | ecophysiology | single-cell microbiology

Microplastic-Associated Biofilms: A Comparison of Freshwater and Marine Environments

“…3). Stable isotope labeling is increasingly used to characterize microbial activity at the single-cell level, including methods such as heavy water labeling [90] and bioorthogonal noncanonical amino acid tagging (BONCAT) [91]. Heavy water labeling is compatible with Raman spectroscopy and cell sorting using optical tweezers [90], and BONCAT has been combined with fluorescence-activated cell sorting (FACS) [91].…”

Reverse Methanogenesis and Respiration in Methanotrophic Archaea

“…In marine sediments, a distinct zonation occurs where ANME-2a/b dominate upper layers and ANME-2c and/or ANME-1 abundance increases in deeper zones, indicating ecological niche separation [10–15]. ANME also form a versatile partnership with non-SRB such as beta-proteobacteria [16] and Verrucomicrobia [17]. ANME, and especially ANME-1, have also been observed without a (closely associated) bacterial partner [5, 12, 13, 18–22].…”

Diversity decoupled from sulfur isotope fractionation in a sulfate‐reducing microbial community

“…Heterogeneous activity within isolate cultures is well documented (Avery, ; Kopf et al, ). Though a distribution of activity within functional groups is expected in situ, the methods to evaluate this activity on environmental in situ samples are only starting to be developed (e.g., Hatzenpichler et al, ; Berry et al, ). The influence of heterogeneous activity on apparent csSRR has substantial implications for both interpretation of calculations of per cell reduction rates and fractionation signals preserved in the rock record.…”