Bioorthogonal non-canonical amino acid tagging reveals translationally active subpopulations of the cystic fibrosis lung microbiota

Valentini, T.; Sk, Lucas; Binder, Kelsey A.; Cameron, Lydia C.; Motl, Jason A.; Dunitz, Jordan M.; Hunter, Ryan C.

doi:10.1038/s41467-020-16163-2

Cited by 28 publications

(26 citation statements)

References 76 publications

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“…Using Met analogs to label newly synthesized proteins has expanded to studying bacteria and microbes in environmental and human samples. The fluorescently labeled Met analog can be combined with other techniques such as FISH or FACS to study the translationally active cell populations (Couradeau et al, 2019;Sebastian and Gasol, 2019;Valentini et al, 2020). Thus, labeling of the Met analog shows promise as a tool that can be widely used in different biological systems in combination with other visualization techniques.…”

Section: Protein Visualization Using Fluorescent Moleculesmentioning

confidence: 99%

Using Genetic Code Expansion for Protein Biochemical Studies

Chung

Amikura

Söll

2020

Front. Bioeng. Biotechnol.

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Protein identification has gone beyond simply using protein/peptide tags and labeling canonical amino acids. Genetic code expansion has allowed residue-or site-specific incorporation of non-canonical amino acids into proteins. By taking advantage of the unique properties of non-canonical amino acids, we can identify spatiotemporal-specific protein states within living cells. Insertion of more than one non-canonical amino acid allows for selective labeling that can aid in the identification of weak or transient protein-protein interactions. This review will discuss recent studies applying genetic code expansion for protein labeling and identifying protein-protein interactions and offer considerations for future work in expanding genetic code expansion methods.

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Section: Protein Visualization Using Fluorescent Moleculesmentioning

confidence: 99%

Using Genetic Code Expansion for Protein Biochemical Studies

Chung

Amikura

Söll

2020

Front. Bioeng. Biotechnol.

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“…Azide and alkyne modifications are considered biologically inert: they do not interfere with biological processes and do not naturally exist in most biological systems, including bacteria. [30][31][32] Previously, BONCAT has been used in bacterial isolates, natural assemblages in aquatic systems, [31][32][33] soil, 34 and sputum from cystic fibrosis patients, 35 and typically combined with fluorescent in-situ hybridization (FISH) with 16S rRNA probes to identify the protein-producing bacteria. To our knowledge, ours is the first study to apply BONCAT to the gut microbiota, while other gut microbiota studies have focused on selective uptake and incorporation.…”

Section: Introductionmentioning

confidence: 99%

“…36 As 16S rRNA-FISH offers limited taxonomic resolution and requires designing probes for specific taxa of interest a priori, 37 we instead combine BONCAT with fluorescenceactivated cell sorting (FACS) and subsequent 16S rRNA gene sequencing (FACS-Seq) as previously done elsewhere. 34,35,38 This allows us to identify the diversity of the BONCAT+ and BONCAT-communities, linking together bacterial identity to activity and increasing throughput. Assessing protein production through BONCAT yields similar results to assessing protein production through nano-SIMS 30 and MAR-FISH, 31 yet it is faster and less expensive.…”

Section: Introductionmentioning

confidence: 99%

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

2021

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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.

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“…Previously, BONCAT has been used in bacterial isolates, natural assemblages in aquatic systems[30–32], soil[33], and sputum from cystic fibrosis patients[34], and typically combined with fluorescent in-situ hybridization (FISH) with 16S rRNA probes to identify the protein-producing bacteria. To our knowledge, ours is the first study to apply BONCAT to the gut microbiota.…”

Section: Introductionmentioning

confidence: 99%

“…To our knowledge, ours is the first study to apply BONCAT to the gut microbiota. As 16S rRNA-FISH offers limited taxonomic resolution and requires designing probes for specific taxa of interest a priori [35], we instead combine BONCAT with fluorescence-activated cell sorting (FACS) and subsequent 16S rRNA gene sequencing (FACS-Seq) as previously done elsewhere[33, 34, 36]. This allows us to identify the diversity of the BONCAT+ and BONCAT- communities, linking together bacterial identity to activity and increasing throughput.…”

Section: Introductionmentioning

confidence: 99%

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

Taguer

Shapiro

Maurice

2020

Preprint

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BackgroundChanges in bacterial diversity in the human gut microbiome, characterized primarily though DNA sequencing methods, have been associated with many different adverse health conditions. However, these changes do not always reflect changes in bacterial activity, and thus how the gut microbiome is implicated in disease is still not often understood. New methods that link together bacterial function to bacterial identity are needed to further explore the role of the gut microbiome in health and disease. 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 and contrast to other methods of bulk community measurements of activity and viability: physiological staining of relative nucleic acid content and membrane damage. Relative nucleic acid content has previously been linked to metabolic activity, yet remains currently undefined for the human gut microbiota.ResultsTen healthy, unrelated individuals were sampled to determine the proportion and diversity of distinct physiological fractions of their gut microbiota. The translationally active bacteria represent about half of the gut microbiota, and are not distinct from the whole community. The high nucleic acid content (HNA) bacteria also represent about 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.ConclusionsBONCAT is a suitable method to probe the translationally active members of the human 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, and so further work is needed to understand the relationship between nucleic acid content and bacterial metabolism in the human gut. Taking into account physiologically distinct subsets of the gut microbiota may be more informative than relying on whole community profiling.

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Bioorthogonal non-canonical amino acid tagging reveals translationally active subpopulations of the cystic fibrosis lung microbiota

Cited by 28 publications

References 76 publications

Using Genetic Code Expansion for Protein Biochemical Studies

Using Genetic Code Expansion for Protein Biochemical Studies

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

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

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