16S rRNA gene amplicon sequences from incubations of shallow (0-3 cm) sediment collected from Little Sippewissett Salt Marsh in Cape Cod, MA, USA

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 taxa within 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 (LSSM) microbiome, resulting in fluorescence signals that are significantly higher than background and ncAA-free control experiments. As a proof of concept experiment, we implemented this "Dual-BONCAT" approach in LSSM 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 molecules, 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.