First report of stop codon reassignment to tryptophan in members of the bacterial phylum <i>Actinomycetota</i>

Abstract Reassignment of stop codons is a significant evolutionary event with recoding of UGA to tryptophan being previously identified in only two bacterial phyla, the Bacillota and Pseudomonadota . Here, we present genomic evidence of this recoding in a third bacterial phylum, the Actinomycetota , specifically in the family Eggerthellaceae . We identify the UGA stop-to-tryptophan recoding in 34 metagenome-assembled genomes recovered from the stool samples of diverse mammalian hosts, including equids and primates. Canonical markers for this recoding are consistently observed including conserved UGA codons aligning to tryptophan, loss of release factor 2 ( prfB ), and presence of a tRNA Trp (UCA) gene. We infer that this recoding 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.