Stop codon reassignment to tryptophan in members of the bacterial phylum Actinomycetota.

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 reassignment 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 reassigned genomes are paraphyletic, forming two distinct groups separated by a third lineage with strains that 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. Organisms representing these genera have reduced genomes and complete or partial loss of biosynthetic pathways, suggesting increasing host dependency and a transition to obligate symbiosis. This likely facilitated stop codon reassignment in Equivita and Gorillivita and suggests Tapirivita is primed for reassignment. 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.