Translational activity is uncoupled from nucleic acid content in bacterial cells of the human gut microbiota

Abstract

Changes in bacterial diversity in the human gut have been associated with many conditions, despite not always reflecting changes in bacterial activity. Methods linking bacterial identity to function are needed for improved understanding of how bacterial communities adapt and respond to their environment, including the gut. Here, we optimized bioorthogonal non-canonical amino acid tagging (BONCAT) for the gut microbiota and combined it with fluorescently activated cell sorting and sequencing (FACS-Seq) to identify the translationally active members of the community. We then used this novel technique to compare with other bulk community measurements of activity and viability: relative nucleic acid content and membrane damage. The translationally active bacteria represent about half of the gut microbiota, and are not distinct from the whole community. The high nucleic acid content bacteria also represent half of the gut microbiota, but are distinct from the whole community and correlate with the damaged subset. Perturbing the community with xenobiotics previously shown to alter bacterial activity but not diversity resulted in stronger changes in the distinct physiological fractions than in the whole community. BONCAT is a suitable method to probe the translationally active members of the gut microbiota, and combined with FACS-Seq, allows for their identification. The high nucleic acid content bacteria are not necessarily the protein-producing bacteria in the community; thus, further work is needed to understand the relationship between nucleic acid content and bacterial metabolism in the human gut. Considering physiologically distinct subsets of the gut microbiota may be more informative than wholecommunity profiling.

Bioorthogonal Chemistry and Its Applications

“…Taguer et al employed BONCAT to study the human gut microbiome. 92 They optimized BONCAT with HPG for the gut microbiota and combined it with fluorescently activated cell sorting and sequencing (FACS-Seq) to identify the translationally active members of the community. The gut microbiota was isolated from human fecal samples and incubated with HPG.…”

Changes in Gut Bacterial Translation Occur before Symptom Onset and Dysbiosis in Dextran Sodium Sulfate-Induced Murine Colitis

“…These changes are led by the translationally active bacteria, confirming that the gut microbiota is not homogeneous in its functional response to perturbations ( 16 , 27 , 29 , 48 , 49 ). Proportions of bacteria with membrane damage increase early on after DSS administration, indicating that the inflammatory intestinal environment is likely responsible, as this was not a direct cause of the microbial cells being exposed to DSS (see Fig.…”

“…However, this appears to be limited, with no clear phylogenetic bias ( 36 , 50 , 52 , 80 , 81 ). Similarly, the biological complexities associated with nucleic acid content as a proxy for bacterial activity have been discussed elsewhere ( 49 ).…”

Actively replicating gut bacteria identified by 5-ethynyl-2’-deoxyuridine (EdU) click chemistry and cell sorting

“…16 This process of labeling, sorting, and characterizing active bacteria is becoming more and more popular due to its unparalleled ability to link bacterial metabolism and identity. 14,26 One physiological method for labeling replicating bacteria, which has not yet been used with the gut microbiota, are thymidine analogues. New thymidine analogues which use fluorescent probes are easier and safer to use than their older radioactive counterparts, especially in the context of probing environmental communities in situ.…”