UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota

Abstract

The composition of the human microbiota is recognized as an important factor in human health and disease. Many of our cohabitating microbes belong to phylum-level divisions for which there are no cultivated representatives and are only represented by small subunit rRNA sequences. For one such taxon (SR1), which includes bacteria with elevated abundance in periodontitis, we provide a single-cell genome sequence from a healthy oral sample. SR1 bacteria use a unique genetic code. In-frame TGA (opal) codons are found in most genes (85%), often at loci normally encoding conserved glycine residues. UGA appears not to function as a stop codon and is in equilibrium with the canonical GGN glycine codons, displaying strain-specific variation across the human population. SR1 encodes a divergent tRNA Gly UCA with an opal-decoding anticodon. SR1 glycyl-tRNA synthetase acylates tRNA Gly UCA with glycine in vitro with similar activity compared with normal tRNA Gly UCC . Coexpression of SR1 glycyl-tRNA synthetase and tRNA Gly UCA in Escherichia coli yields significant β-galactosidase activity in vivo from a lacZ gene containing an in-frame TGA codon. Comparative genomic analysis with Human Microbiome Project data revealed that the human body harbors a striking diversity of SR1 bacteria. This is a surprising finding because SR1 is most closely related to bacteria that live in anoxic and thermal environments. Some of these bacteria share common genetic and metabolic features with SR1, including UGA to glycine reassignment and an archaeal-type ribulose-1,5-bisphosphate carboxylase (RubisCO) involved in AMP recycling. UGA codon reassignment renders SR1 genes untranslatable by other bacteria, which impacts horizontal gene transfer within the human microbiota.

Insights into the phylogeny and coding potential of microbial dark matter

“…13a). The same recoding was found and biochemically validated in candidate phylum SR1 very recently 41 , suggesting that this codon reassignment may be phylogenetically widespread in uncharacterized lineages. This expands the known alternative coding for UGA, which has previously been reported for selenocysteine 42 and tryptophan 43,44 .…”

Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

“…For example, we demonstrated that single-cell genomes from uncultivated phyla substantially improve the phylogenetic anchoring of metagenomic reads 1 . Mapping of metagenome sequences to single-cell genomes has not only been applied to validating metagenome assembly and binning 7 but also to subsequently improving the single-cell assemblies, as has been shown for SR1 bacteria, atribacteria and ammoniaoxidizing archaea [8][9][10] . Other studies successfully used fragment recruitment by single-cell genomes to investigate biogeographic distribution of uncultivated, marine taxa 11,12 .…”

Identifying contamination with advanced visualization and analysis practices: metagenomic approaches for eukaryotic genome assemblies

“…To estimate the number of bacterial genomes in a given collection of scaffolds in a raw assembly or in a curated genome bin, and to visualize the distribution of HMM hits for each bacterial single-copy gene, we used the anvi'o script 'gen-stats-for-single-copy-genes', which reports the most frequent number in the list of number of hits per single-copy gene as the estimated number of bacterial genomes in a collection of scaffolds. The script uses HMMer v3.1b2 [39] to search for Hidden Markov Profiles (HMMs) of 139 bacterial single-copy genes identified by Campbell et al [11], and the R library 'ggplot' v1.0.0 [40,41] to plot results.…”

“…Although Boothby et al reported the lack of 16S rRNA genes in their assembly [29], anvi'o estimated that it contained at least 10 complete bacterial genomes (Fig. 2) using a bacterial single-copy gene collection [11]. This simple yet powerful step could identify cases of extensive contamination, and alert researchers to be diligent in identifying scaffolds originating from bacterial organisms.…”

“…Due to the extensive diversity of bacteria and archaea in most environmental samples [6,7], the field of metagenomics has rapidly evolved to accurately delineate genomes in assembly results. Today, microbiologists often exploit two essential properties of bacterial and archaeal genomes to improve the "binning" step: (1) k-mer frequencies that are somewhat preserved throughout a single microbial genome [8], to identify contigs that likely originate from the same genome [9], and (2) a set of genes that occur in the vast majority of bacterial genomes as a single copy, to estimate the level of completion and contamination of genome bins [10,11,12]. These properties, along with differential coverage of contigs across multiple samples when such data exist, are routinely used to identify coherent microbial draft genomes in metagenomic assemblies [13,14,15,16].…”