First report of stop codon reassignment to tryptophan in members of the bacterial phylum Actinomycetota

Abstract Reassignment of stop codons is a significant evolutionary event with recoding of UGA to tryptophan being previously identified in only three bacterial phyla, the Bacillota, Pseudomonadota, and Verrucomicrobiota. Here, we present genomic evidence of this recoding in a fourth bacterial phylum, the Actinomycetota, specifically in the family Eggerthellaceae. We identify the UGA stop-to-tryptophan reassignment in 34 metagenome-assembled genomes recovered from the stool samples of diverse mammalian hosts, including equids and primates. Canonical markers for this reassignment are consistently observed including conserved UGA codons aligning to tryptophan, loss of release factor 2 (prfB), and presence of a tRNATrp(UCA) gene. We infer that this reassignment occurred at least twice as the lineages containing recoded genomes are paraphyletic, forming two distinct groups separated by a third lineage with strains that continue to use UGA as a stop codon. These lineages represent three new Eggerthellaceae genera for which we propose the type species Equivita altericodex, Gorillivita intestinalis, and Tapirivita inops reflecting isolation source and genomic properties. Genomes representing these genera, including the non-recoded Tapirivita lineage, have reduced genomes and complete or partial loss of biosynthetic pathways, suggesting a transition to obligate symbiosis. Increasing host dependency may have facilitated stop codon reassignment in Equivita and Gorillivita species. This work expands the known phylogenetic diversity of UGA stop-to-tryptophan reassignment in the bacterial domain and establishes the Eggerthellaceae as a new focal point for understanding the evolutionary drivers of genetic code plasticity. Competing Interest Statement The authors have declared no competing interest. Footnotes The manuscript has been corrected to reflect that Actinomycetota is the fourth bacterial phylum with an identified UGA-to-tryptophan reassignment, rather than the third as previously stated.