Marlow, J. | bioRxiv Science

bioRxiv2024

Dual-BONCAT Reveals Distinct Day- and Night-Active Microbial Populations in Salt Marsh Sediments

Bio-Orthogonal Non-Canonical Amino Acid Tagging (BONCAT) has emerged as a prominent molecular technique that enables microbial ecologists to visualize and identify metabolically active cells in cultures and complex microbial communities. To date, researchers have used just one non-canonical amino acid (ncAA) in a given experiment; here, we validate a novel approach using two different ncAAs in a single experiment. This advancement facilitates the detection of differentially active subpopulations within the same experimental context, thereby reducing the uncertainty and variability associated with parallel treatments, and providing precise spatial information about organisms that are active under distinct conditions or at different times. We show that both ncAAs can be taken up by E. coli cultures and by constituents of the Little Sippewissett Salt Marsh microbiome, resulting in fluorescence signals that are significantly higher than background and ncAA-free control experiments, as well as differential labeling patterns reflective of distinct subpopulations. As a proof of concept, we implemented this "dual-BONCAT" approach in salt marsh sediments, adding one ncAA during daytime hours and the other at night. Subpopulations of cells that were anabolically active during the day and/or night were distinguishable by both fluorescence microscopy, and by fluorescence-activated cell sorting. Subsequent high-throughput 16S rRNA gene amplicon sequencing of active subpopulations revealed that Methylobacterium, potentially feeding on plant exudate carbon, was preferentially active during the day, while sulfur-cycling taxa dominated the night-active population. Dual-BONCAT offers an important advancement in multiplexing substrate-analog probing techniques, providing a more realistic understanding of metabolic activity under distinct environmental conditions. ImportanceMicrobial communities are complex and dynamic, with different groups of microbes active under distinct conditions. Bio-Orthogonal Non-Canonical Amino Acid Tagging (BONCAT) uses synthetic amino acids to tag newly made proteins, allowing researchers to see and identify the active subset of a community. While BONCAT studies to date have used a single synthetic amino acid to evaluate cell activity in a single experimental context, here we introduce a new approach, "dual-BONCAT," using two synthetic amino acids to track differential responses to changing conditions. After validating the approach with E. coli, we deployed it in a salt marsh sediment community, finding that organisms potentially feeding on plant root sugars were more active during the day, and microbes likely metabolizing sulfur were more active at night. We believe dual-BONCAT will prove useful in many studies, as it illuminates microbial community responses to changing conditions, which has important implications for ecosystem dynamics.

microbiology↗

bioRxiv2020

Spatially-resolved correlative microscopy and microbial identification reveals dynamic depth- and mineral-dependent anabolic activity in salt marsh sediment

Coastal salt marshes are key sites of biogeochemical cycling and ideal systems in which to investigate the community structure of complex microbial communities. Here, we clarify structural-functional relationships among microorganisms and their mineralogical environment, revealing previously undescribed metabolic activity patterns and precise spatial arrangements within salt marsh sediment. Following 3.7-day in situ incubations with a non-canonical amino acid that was incorporated into new biomass, samples were embedded and analyzed by correlative fluorescence and electron microscopy to map the microscale arrangements of anabolically active and inactive organisms alongside mineral grains. Parallel sediment samples were examined by fluorescence-activated cell sorting and 16S rRNA gene sequencing to link anabolic activity to taxonomic identity. Both approaches demonstrated a rapid decline in the proportion of anabolically active cells with depth into salt marsh sediment, from [~]60% in the top cm to 10-25% between 2-7 cm. From the top to the bottom, the most prominent active community members shifted from sulfur cycling phototrophic consortia, to sulfate-reducing bacteria likely oxidizing organic compounds, to fermentative lineages. Correlative microscopy revealed more abundant (and more anabolically active) organisms around non-quartz minerals including rutile, orthoclase, and plagioclase. Microbe-mineral relationships appear to be dynamic and context-dependent arbiters of biogeochemical cycling. Statement of SignificanceMicroscale spatial relationships dictate critical aspects of a microbiomes inner workings and emergent properties, such as evolutionary pathways, niche development, and community structure and function. However, many commonly used methods in microbial ecology neglect this parameter - obscuring important microbe-microbe and microbe-mineral interactions - and instead employ bulk-scale methodologies that are incapable of resolving these intricate relationships. This benchmark study presents a compelling new approach for exploring the anabolic activity of a complex microbial community by mapping the precise spatial configuration of anabolically active organisms within mineralogically heterogeneous sediment through in situ incubation, resin embedding, and correlative fluorescence and electron microscopy. In parallel, active organisms were identified through fluorescence-activated cell sorting and 16S rRNA gene sequencing, enabling a powerful interpretive framework connecting location, identity, activity, and putative biogeochemical roles of microbial community members. We deploy this novel approach in salt marsh sediment, revealing quantitative insights into the fundamental principles that govern the structure and function of sediment-hosted microbial communities. In particular, at different sediment horizons, we observed striking changes in the proportion of anabolically active cells, the identities of the most prominent active community members, and the nature of microbe-mineral affiliations. Improved approaches for understanding microscale ecosystems in a new light, such as those presented here, reveal environmental parameters that promote or constrain metabolic activity and clarify the impact that microbial communities have on our world.

microbiology↗