Identifying the specific signals that mediate
correlation-dependent structural plasticity will
be greatly facilitated by exploiting the experimental protocol presented here.
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We thank the laboratory of K. Haas (UBC) for assistance with the
MATLAB analysis tool Dynamo for dynamic morphometric analysis.
We also thank M. Meyer for providing us with the tetanus toxin light
chain plasmid (5UAS-TeNT-EGFP) and D. Freiheit for photography
of tadpoles. We are grateful to S. Glasgow for advice on statistical
analysis and to H. Cline and W. Sossin for useful comments on
our manuscript. This work was supported by grants from the
Canadian Institutes for Health Research and the Natural Sciences and
Engineering Research Council (NSERC) of Canada to E.S.R. and
by fellowships from the Deutscher Akademischer Austausch Dienst
and the NSERC CREATE Neuroengineering Training Program to M.M.,
the Fonds de la recherche en santé du Québec to D.G., and NSERC to
P.S. The authors declare that they have no conflicts of interest.
Materials and Methods
Figs. S1 and S2
Movies S1 to S4
31 January 2014; accepted 25 April 2014
Stop codon reassignments
in the wild
Natalia N. Ivanova,1 Patrick Schwientek,1 H. James Tripp,1 Christian Rinke,1
Amrita Pati,1 Marcel Huntemann,1 Axel Visel,1,2,3 Tanja Woyke,1
Nikos C. Kyrpides,1 Edward M. Rubin1,2†
The canonical genetic code is assumed to be deeply conserved across all domains of life
with very few exceptions. By scanning 5.6 trillion base pairs of metagenomic data for
stop codon reassignment events, we detected recoding in a substantial fraction of the
>1700 environmental samples examined. We observed extensive opal and amber stop
codon reassignments in bacteriophages and of opal in bacteria. Our data indicate that
bacteriophages can infect hosts with a different genetic code and demonstrate phage-host
antagonism based on code differences. The abundance and diversity of genetic codes present
in environmental organisms should be considered in the design of engineered organisms with
altered genetic codes in order to preclude the exchange of genetic information with naturally
Since the discovery of the genetic code and protein translation mechanisms (1), a lim- ited number of variations of the standard assignment between unique base triplets (codons) and their encoded amino acids
and translational stop signals have been found in
bacteria and phages (2–7). Given the apparent
ubiquity of the canonical genetic code, the design
of genomically recoded organisms with noncanonical codes has been suggested as a means to
prevent horizontal gene transfer between laboratory
and environmental organisms (8–10). It is also
predicted that genomically recoded organisms are
immune to infection by viruses, under the assumption that phages and their hosts must share a
common genetic code (6). This paradigm is supported by the observation of increased resistance
of genomically recoded bacteria to phages with a
canonical code (9). Despite these assumptions and
accompanying lines of evidence, it remains unclear
whether differential and noncanonical codon usage
represents an absolute barrier to phage infection
and genetic exchange between organisms.
Our knowledge of the diversity of genetic
codes and their use by viruses and their hosts is
primarily derived from the analysis of cultivated
organisms. This is due to our limited access to
genome sequences from uncultivated organisms,
which are estimated to account for 99% in pro-
karyotes (11). Advances in single-cell sequencing
and metagenome assembly technologies have
enabled the reconstruction of genomes of uncul-
tivated bacterial and archaeal lineages (12–14)
and the discovery of a previously unknown reassign-
ment of TGA opal stop codons to glycine (4, 5, 14).
These initial findings suggest that large-scale
systematic studies of uncultivated microorgan-
isms and viruses may reveal the extent and
modes of divergence from the canonical genetic
code operating in nature.
To explore alternative genetic codes, we carried
out a systematic analysis of stop codon reassignments from the canonical TAG amber, TGA opal,
and TAA ochre codons in assembled metagenomes
and metatranscriptomes from environmental and
host-associated samples, single-cell genomes of
uncultivated bacteria and archaea, and a collection of viral sequences (Fig. 1A) (15). All sequence
data were obtained from the Integrated Microbial Genomes (IMG) database (16). This global
collection of sequences comprised 1776 samples
from 145 studies, including 750 samples obtained
from 17 human body sites (fig. S1) (17). In total,
5.6 terabases of sequence data, including 450 Gb
of contiguous sequences (contigs) greater than
1 kb, were analyzed. All samples were classified
into human-associated, other host–associated,
soil, marine, or freshwater environments according to their metadata (15, 18).
We used a statistic of increased coding potential under alternate genetic codes as calculated
by ab initio gene finder Prodigal (19), which was
selected for its low rate of false-positive predictions
(15). Contigs showing significantly higher coding
potential when annotated with modified translation tables were forwarded to filtering and quality control to confirm stop codon reassignment
through multiple sequence alignments to known
homologs from the National Center for Biotechnology Information protein database (Fig. 1A) (15).
By applying this approach to 450 Gb of contigs
larger than 1 kb in size, we identified 31,415 contigs with evidence of stop coding reassignment,
adding up to a total of 198 Mb of recoded DNA
(Fig. 1A). No recoding was observed in the meta-transcriptome data. Varying ratios of reassigned
to total contigs were observed in samples from
terrestrial and aquatic environments and from
human mouth, throat, and stool microbiomes
1Department of Energy Joint Genome Institute (DOE JGI),
Walnut Creek, CA 94598, USA. 2Genomics Division, Lawrence
Berkeley National Laboratory, Berkeley, CA 94720, USA.
3School of Natural Sciences, University of California, Merced,
CA 95343, USA.
*These authors contributed equally to this work. †Corresponding
author. E-mail: firstname.lastname@example.org