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.

Fig. 1.

Hierarchical clustering of bacterial SR1 OTU abundance (Bray-Curtis similarity matrices) by body sites and corresponding frequency distribution of the six major SR1 OTUs at those sites. The skin SR1 sequences (indicated by “s”) were combined for OTU abundance calculation due to their low numbers. SR1-OR1 belongs to OTU#1.

Fig. 2.

Maximum-likelihood phylogenetic tree of SR1 and related bacteria based on RNA polymerase protein sequences (β-β′ subunits). A phylogeny representing all Bacteria is shown in Fig. S5. Node labels denote branch support. The cluster of candidate phyla including SR1 is highlighted.

Fig. 3.

Codon use in SR1. (A) Frequency of internal TGAs in protein coding genes from SR1-OR1 and SR1 metagenomic scaffolds. (B) Box-and-whisker plot comparison of Gly codon use in the SR1-OR1 (♦) with that of predicted genes in all SR1 HMP metagenomic scaffolds (SR1 “pangenome”). Outliers are indicated with an asterisk.

Fig. 4.

Distribution of reprogrammed TGA codons in SR1 RubisCO genes. The type of Gly codon, TGA (black) or canonical GGA (white), at the four reprogrammed positions in the RubisCO SR1 pangenome is overlaid on the gene phylogeny, based on full-length sequences from HMP metagenomes and SR1-OR1. Each HMP number defines a different human donor. For four human subjects, two SR1 phylotypes were identified or genes were present in samples collected at different times (-2). Multiple sequences from donors 159814214 (*), 809635352 (grey rectangle), 370425937 (#), and 764447348 (oval) are indicated by superscript symbols. The average dN/dS values for the two main clades are indicated.

Fig. 5.

Biochemical characterization of SR1 GlyRS and tRNA^Gly_UCA. (A) To assay UGA translation in vivo in E. coli, β-galactosidase was expressed from a lacZ reporter gene with either Methionine 3 (wild-type) or M3 mutated to TGA. β-Galactosidase activities, shown relative to the wild-type activity level, were measured in the presence or absence of tRNA^Gly_UCA variants (SR1, ACD78, and ACD80) and SR1 GlyRS. (B) In vitro glycylation activity of SR1 GlyRS with tRNA^Gly_UCA (SR1: □; ACD78: +; ACD80: ♢) and canonical tRNA^Gly (SR1 ○, E. coli △) variants. The data are based on triplicate experiments with 10 μM GlyRS and 0.2 μM tRNA.