An Expanded Genetic Code Enables Trimethylamine Metabolism in Human Gut Bacteria

Abstract

Links between trimethylamine- N -oxide (TMAO) and cardiovascular disease (CVD) have focused attention on mechanisms by which animal-based diets have negative health consequences. In a meta-analysis of data from foundational gut microbiome studies, we found evidence that specialized bacteria have and express a metabolic pathway that circumvents TMAO production and is often misannotated because it relies on genetic code expansion. This naturally occurring mechanism for TMAO attenuation is negatively correlated with CVD. Ultimately, these findings point to new avenues of research that could increase microbiome-informed understanding of human health and hint at potential biomedical applications in which specialized bacteria are used to curtail CVD development.

Full text

An Expanded Genetic Code Enables Trimethylamine
Metabolism in Human Gut Bacteria
Veronika Kivenson,
a
Stephen J. Giovannoni
a
a
Department of Microbiology, Oregon State University, Corvallis, Oregon, USA
ABSTRACT Cardiovascular disease (CVD) has been linked to animal-based diets,
which are a major source of trimethylamine (TMA), a precursor of the proathero-
genic compound trimethylamine-N-oxide (TMAO). Human gut bacteria in the genus
Bilophila have genomic signatures for genetic code expansion that could enable
them to metabolize both TMA and its precursors without production of TMAO. We
uncovered evidence that the Bilophila demethylation pathway is actively transcribed
in gut microbiomes and that animal-based diets cause Bilophila to rapidly increase
in abundance. CVD occurrence and Bilophila abundance in humans were signifi-
cantly negatively correlated. These data lead us to propose that Bilophila, which is
commonly regarded as a pathobiont, may play a role in mitigating cardiovascular
disease. Human gut microbiomes have been shown to affect the development of a
myriad of disease states, but mechanistic connections between diet, health, and mi-
crobiota have been challenging to establish. The hypothesis that Bilophila reduces
cardiovascular disease by circumventing TMAO production offers a clearly defined
mechanism with a potential human health impact, but investigations of Bilophila cell
biology and ecology will be needed to fully evaluate these ideas.
IMPORTANCE Links between trimethylamine-N-oxide (TMAO) and cardiovascular dis-
ease (CVD) have focused attention on mechanisms by which animal-based diets
have negative health consequences. In a meta-analysis of data from foundational
gut microbiome studies, we found evidence that specialized bacteria have and ex-
press a metabolic pathway that circumvents TMAO production and is often misan-
notated because it relies on genetic code expansion. This naturally occurring mecha-
nism for TMAO attenuation is negatively correlated with CVD. Ultimately, these
findings point to new avenues of research that could increase microbiome-informed
understanding of human health and hint at potential biomedical applications in
which specialized bacteria are used to curtail CVD development.
KEYWORDS cardiovascular disease, microbiome, molecular genetics
The human gut microbiome is increasingly recognized for its influential role in
health. The gut microbiome has been implicated in cardiovascular disease (CVD),
the leading cause of death in developed countries (1). Metabolic end products asso-
ciated with the consumption of animal products have been shown to promote CVD;
dietary sources of proatherogenic compounds include choline, phosphatidylcholine,
and L-carnitine (2–11). Gut microorganisms convert these compounds to trimethyl-
amine (TMA), which in turn is converted to trimethylamine-N-oxide (TMAO) through the
action of host hepatic flavin monooxygenase 3 (12, 13). TMAO is a causative agent of
CVD pathogenesis; therefore, elucidating pathways relevant to this compound is
central to understanding human health (10, 14).
The canonical view of CVD and TMAO involves the action of the gut microbiota in
transforming precursor compounds from a range of animal-based dietary sources to
TMA, which can then be converted to TMAO in the liver (Fig. 1A). While TMAO is
Citation Kivenson V, Giovannoni SJ. 2020. An
expanded genetic code enables
trimethylamine metabolism in human gut
bacteria. mSystems 5:e00413-20. https://doi
.org/10.1128/mSystems.00413-20.
Editor Robert G. Beiko, Dalhousie University
Copyright © 2020 Kivenson and Giovannoni.
This is an open-access article distributed under
the terms of the Creative Commons Attribution
4.0 International license.
Address correspondence to Veronika Kivenson,
kivensov@oregonstate.edu.
Received 18 May 2020
Accepted 24 September 2020
Published
RESEARCH ARTICLE
Molecular Biology and Physiology
crossm
September/October 2020 Volume 5 Issue 5 e00413-20 msystems.asm.org 1
27 October 2020

frequently referred to as the sole breakdown product of TMA, a subset of methano-
genic archaea in the gut have the ability to utilize TMA by an alternative pathway (15,
16). This pathway is enabled by genetic code expansion (GCE), via insertion of the 22nd
amino acid, pyrrolysine (Pyl), in place of a TAG amber codon at conserved sites of the
tri-, di-, and monomethylamine methyltransferase genes, with methane as the end
product (17–19). TMA metabolism that relies on proteins that use an expanded code
has also been described in some bacteria from environmental settings, including
symbionts of gutless marine worms (20), as well as the Firmicutes bacterium Acetoha-
lobium arabaticum isolated from a Crimean lagoon (21). Despite its potential impor-
tance, bacterial metabolism of TMA remains largely unexplored in the gut microbiome.
ADeltaproteobacterium commonly found in the human gut, Bilophila wadsworthia,
also has genes necessary for encoding pyrrolysine (22). First identified in an appendi-
citis infection in 1989, this “bile-loving,” taurine-degrading bacterium is commonly
referred to as a pathobiont associated with abscesses (23, 24). Additionally, Bilophila is
able to produce hydrogen sulfide, and these bacteria may be linked to inflammatory
bowel disease, colorectal cancer, and systematic inflammation (24–28). Despite its
classification as a pathobiont, Bilophila is also commonly present in healthy human
microbiomes (29, 30). There is uncertainty about GCE and TMA metabolic pathway
characteristics in these organisms; notably, whether the pyrrolysine pathway is func-
tional in trans and whether the pathway is expressed. The function of this pathway is
questioned (21) because of organizational differences in the pyrrolysyl-tRNA synthetase
gene (required for encoding Pyl): in archaea, this synthetase is encoded by a single gene,
while in Bilophila, the N- and C-terminal domains are encoded by two distinct genes.
Second, in previous reports of Pyl pathways, the trimethylamine methyltransferase and
Pyl genes are commonly adjacent to one another (21, 22), while this is not the case in
Bilophila. Third, the TMA methyltransferase protein is prematurely truncated at the TAG
codon in public Bilophila data sets available on the NCBI and JGI-IMG webservers.
Despite the ubiquity of this bacterium, and its potential importance in modulating
TMAO levels (and thus CVD risk), these ambiguities remain unresolved, and studies of
the human gut microbiome make no mention of the possibility of a bacterial pathway
for TMA utilization. In this study, we investigate this alternative pathway of trimethyl-
amine metabolism in Bilophila, the change in abundance of this taxon in response to
an animal-based diet, and the correlation between Bilophila abundance and CVD
pathology (Fig. 1B).
FIG 1 (A) Canonical view of TMA: this compound is converted to TMAO in the liver via a host hepatic flavin monooxygenase 3, leading
to increased risk of cardiovascular disease. (B) A new view of TMA metabolism proposed in this study: the specialized gut bacteria,
Bilophila, increase in abundance and use genetic code expansion to augment metabolism, thereby reducing the amount of TMA available
for conversion to TMAO. TMA, trimethylamine; FMO-3, flavin monooxygenase 3; TMAO, trimethylamine-N-oxide; CVD, cardiovascular
disease; DMA, dimethylamine.
Kivenson and Giovannoni
September/October 2020 Volume 5 Issue 5 e00413-20 msystems.asm.org 2

RESULTS
To explore the potential for Bilophila TMA metabolism, we retranslated the protein-
coding sequences from publicly available Bilophila genomes (Table S1) using a custom
translation table with the TAG codon as a readthrough rather than a stop codon (see
Materials and Methods). We found an in-frame TAG codon at a conserved position in
the TMA methyltransferase, corresponding to the pyrrolysine residue of this protein in
archaeal methanogens (17) and bacteria (20, 21)(Fig. 2 and Table S2). All genes known
to have functions specific to pyrrolysine production were also identified in the Bilophila
genomes: PylB, PylC, and PylD and the pyrrolysyl-tRNA synthetase N and C termini, as
well as the Pyl-specific tRNA, with the corresponding CUA anticodon (Fig. 2; Fig. S1).
One exception was that Bilophila wadsworthia ATCC 49260 is missing one component:
a Pyl tRNA was not located. In addition to the Pyl-containing TMA methyltransferase,
accessory genes for the TMA methyltransferase pathway, including ramA (the activating
gene) and the TMA methyltransferase corrinoid protein, are encoded in all of the
Bilophila genomes (Fig. 2 and Table S3). The dimethylamine methyltransferase gene is
not encoded in any of the genomes, and a gene similar to monomethylamine meth-
yltransferase, while present, does not have the conserved in-frame TAG codon that is
a hallmark of this gene. In summary, metabolic inference from genomic data
indicates that Bilophila have the potential to convert TMA to dimethylamine (DMA)
in the human gut.
FIG 2 Mechanistic overview of genetic code expansion augmented metabolism performed by Bilophila in the human gut. Bcct
transporters, betaine/carnitine/choline transporter family proteins; CutD, choline trimethylamine lyase activating enzyme; CutC, choline
trimethylamine lyase; TMA, trimethylamine; DMA, dimethylamine; PylB, 3-methylornithine synthase; PylC, 3-methylornithine-l-lysine ligase;
PylD, 3-methylornithyl-N
6
-L-lysine dehydrogenase; Pyl-tRNA synthetase, pyrrolysyl-tRNA synthetase; Pyl tRNA, pyrrolysine tRNA; ramA,
methylamine methyltransferase corrinoid protein reductive activase; TMA corrinoid, methyltransferase cognate corrinoid protein; SelD,
selenide, water dikinase; SelA, L-seryl-tRNA(Sec) selenium transferase; SelB, selenocysteine-specific translation elongation factor;
Sec-tRNA, selenocysteine tRNA; SECIS Element, selenocysteine insertion sequence element; TMAO, trimethylamine-N-oxide; FMO-3,
flavin monooxygenase 3.
TMA Metabolism in Human Gut Bacteria
September/October 2020 Volume 5 Issue 5 e00413-20 msystems.asm.org 3

The TMA-utilizing pathway we observed may benefit Bilophila by enabling them to
use TMA directly and also TMA produced metabolically within the cell from two
precursor compounds, choline and glycine betaine (Fig. 2). Briefly, Bilophila genomes
encode the glycyl radical choline-TMA lyase and its associated activating protein (CutC
and CutD, respectively), which convert choline to TMA (31). Glycine betaine (sources of
which includes choline [32] and possibly carnitine [33]) can be converted to TMA via the
selenocysteine (Sec)-containing glycine betaine reductase (GRD) pathway (34). The GRD
pathway and the full set of machinery required for the noncanonical amino acid
selenocysteine, encoded with a repurposed UGA stop codon, are also present in the
Bilophila genomes (Table S3). Multiple copies of proteins belonging to the betaine/
carnitine/choline family of transporters are also encoded in the Bilophila genomes. We
conclude that the genomic data indicate that Bilophila has the ability to use choline and
glycine betaine, converting these compounds to TMA, and subsequently to DMA. In
doing so, these bacteria may deplete precursor compounds and TMA that would
otherwise be available for host hepatic processes, thereby reducing or circumventing
production of TMAO (Fig. 2).
Next, we asked whether the pyrrolysine pathway is functional and whether GCE-
enabled TMA metabolism is active in Bilophila from the gut environment. To do so, we
reexamined data sets from a recent human gut metatranscriptomic study (35) and a
mouse model system study (36) (Table S1). Applying a minimum threshold of 98%
amino acid sequence identity with annotated Bilophila proteins, we identified expres-
sion of the TMA methyltransferase and pyrrolysine machinery proteins in both human
fecal and mouse cecum samples (Fig. 2 and Tables S4 and S5). The fraction of TMA
consumed via this bacterial metabolic process in the human gut microbiome remains
uncertain, but expression data support the conclusion that this metabolic process is
active.
DISCUSSION
This survey of published microbiome sequence data uncovered evidence that
bacteria in the genus Bilophila use genetic code expansion in the human gut to
produce a TMA methyltransferase. We hypothesized that this mechanism could be used
to compete with other TMA-utilizing processes, potentially decreasing the production
of TMAO from the proatherogenic precursor trimethylamine. To explore this hypoth-
esis, we reexamined additional publicly available data (Table S1) to determine whether
this naturally occurring mechanism for TMAO attenuation is correlated with CVD. In a
recent study describing the gut microbiome in atherosclerotic cardiovascular disease,
Jie et al. (29) report that Bilophila is one of the 20 most abundant genera in the samples
examined for this project. Their data also show that the abundance of Bilophila is
significantly enriched in the microbiomes of individuals in the healthy/control group
(n⫽187) compared to the CVD group (n⫽218). Second, in a study describing a rapid
diet-induced change in the human gut microbiome, David et al. (37) reveal that
Bilophila significantly increase in abundance in response to an animal-based diet
compared to a plant-based diet. Finally, in a study detailing the transmission of
atherosclerosis susceptibility via gut microbial transplanation, Gregory et al. (38) show
that mice with certain taxa have increased TMAO levels and develop atherosclerotic
lesions and postulate that this is a microbiome-dependent, transmissible trait. Bilophila
is one of only six taxa that are significantly enriched in both the cecal and fecal
microbiome of the healthy group compared to the mice that developed atherosclerotic
lesions. The observations we report may challenge the widely held idea that members
of this taxon act exclusively as pathobionts; their role in the microbiome and human
health may be context dependent, and their potential to mitigate the impacts of animal
products on CVD warrants further study.
MATERIALS AND METHODS
Microbiome data selection and accession numbers. Genomic, metagenomic, and metatranscrip-
tomic data sets used for this study were accessed on 15 January 2020 from publicly available sequencing
projects (see Table S1 in the supplemental material). The Bilophila genomes analyzed are reference
Kivenson and Giovannoni
September/October 2020 Volume 5 Issue 5 e00413-20 msystems.asm.org 4

genomes from the Human Microbiome Project (39) as well as the type strain for this genus, from the
Refseq database (40). All of the available genomes from the genus Bilophila were examined. BioProject
accession no. PRJEB33885 was excluded because it includes a duplicate of a previously published
genome (included in accession no. PRJNA41963). Paired metagenomic-metatranscriptomic data were
accessed from a large cohort study of adult men (35), and additional metatranscriptomic data were used
from a mouse model system (36). Metatranscriptomic and metagenomic read data were obtained using
the SRA-toolkit (https://github.com/ncbi/sra-tools) v2.9.1 data via the fastq-dump option.
Genomic analysis and implementation of a custom protein translation table. Predicted proteins
in each genome were initially identified using Prodigal (42) v.2.6.3 using single genome mode, and
functional annotation was determined using Hmmer (43) v.3.1, with the hmmscan option (1E⫺10 cutoff),
with top hits only, against the Pfam (44) database v.31 and Tigrfam (45) database v.15.0.
For genomes in which enzymes were identified for the synthesis of pyrrolysine, we applied an
alternate protein prediction procedure that reassigned TAG codons from stop to readthrough. To
accomplish this task, we modified the source code for Prodigal by adding a custom translation table that
has TAG readthrough and retains all three canonical bacterial start codons. Proteins with in-frame stop
codons in the relevant genes were then manually inspected to determine whether the region containing
and following the stop codon was conserved in comparisons to homologues containing pyrrolysine at
a similar position (see Fig. S2 in the supplemental material). The modified code and documentation for
the modified translation table are freely available at https://github.com/VeronikaKivenson/Prodigal.
Protein searches for the full-length TMA methyltransferase amino acid sequence from Bilophila were
performed on the NCBI and JGI-IMG webservers on 1 July 2020. For searching and identifying putative
selenocysteine-containing proteins, the bSECIS (46) webserver was used and results were inspected and
compared with previously identified selenoproteins. Protein sequence alignment was performed using
Muscle (47), with Geneious 2020.1 used for visualization of the alignments. The Aragorn (48) web server
was used to locate the tRNA sequences from each genome and to determine the secondary structure of
Sec- and Pyl-specific tRNAs, as well as their corresponding anticodon sequences. tRNAscan-SE (49) v.1.23
was also used to search for the tRNAs for Sec. Chemdraw and Biorender software were used to create
figures.
Metatranscriptomic data analysis. Preliminary mapping of metatranscriptomic sequence data to
Bilophila genomes was done using Bowtie2 (50) v2.3.4.1. This approach identified samples in which these
Bilophila bacteria were present and active at detectable levels. Next, metatranscriptomic reads were
coassembled using SRA data from select samples belonging to each BioProject (Table S4) using Megahit
(51) v.1.1.1, with default parameters. Functional annotation and identification of stop codon readthrough
were performed as described earlier. In addition to Prodigal, FragGeneScan was used to identify partial
genes (52). The computational biology data processing and analysis workflow were completed using the
Extreme Science and Engineering Discovery Environment (XSEDE) Bridges resource at the Pittsburgh
Supercomputing Center (53, 54).
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
FIG S1, PDF file, 0.2 MB.
FIG S2, PDF file, 0.1 MB.
TABLE S1, PDF file, 0.04 MB.
TABLE S2, PDF file, 0.04 MB.
TABLE S3, PDF file, 0.03 MB.
TABLE S4, PDF file, 0.1 MB.
TABLE S5, PDF file, 0.05 MB.
ACKNOWLEDGMENTS
This work was supported by a grant from the Simons Foundation (SFARI 649176 to
V.K.). This work used the Extreme Science and Engineering Discovery Environment
(XSEDE), which is supported by National Science Foundation grant number ACI-
1548562. Specifically, it used the Bridges system, which is supported by NSF award
number ACI-1445606, at the Pittsburgh Supercomputing Center (PSC), through alloca-
tion TG-DEB170007.
Any opinions, findings, and conclusions or recommendations expressed in this
material are those of the author(s) and do not necessarily reflect the views of the
National Science Foundation.
We thank Grace Deitzler and Maude David for helpful discussion of the gut micro-
biome data sets.
We declare that we have no competing interests.
The project was conceived by V.K. and S.J.G. V.K. performed the bioinformatic data
analysis, and interpretation was performed by V.K. and S.J.G. V.K. and S.J.G. wrote the
manuscript.
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September/October 2020 Volume 5 Issue 5 e00413-20 msystems.asm.org 5

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