Global transcriptional rewiring provides recurrent evolutionary routes to insect– <i>Escherichia coli</i> mutualism

<jats:title>Abstract</jats:title> <jats:p> Microbial symbionts often evolve specialized functions that compensate for host nutritional deficiencies, yet the evolutionary routes by which such mutualisms arise remain poorly understood. Here, using an experimental symbiosis between the stinkbug <jats:italic>Plautia stali</jats:italic> and <jats:italic>Escherichia coli</jats:italic> , we investigated the early evolution of insect–bacterium mutualism. Among 144 independently evolved bacterial lineages, six acquired the ability to improve host performance. Four lineages carried independent disruptive mutations in <jats:italic>cyaA</jats:italic> , disabling carbon catabolite repression and thereby increasing the essential amino acid tryptophan. The remaining two lineages evolved through nonsynonymous mutations in <jats:italic>rpoB</jats:italic> or <jats:italic>rpoD</jats:italic> , encoding core components of the transcriptional machinery. Transcriptomic analyses revealed extensive regulatory rewiring in both mutants, including marked upregulation of biosynthetic pathways for branched-chain essential amino acids. Our results identify global transcriptional regulatory systems as major evolutionary targets for the emergence of mutualism and demonstrate how large-effect regulatory mutations can rapidly generate host-beneficial traits. (146 &lt; 150 words) </jats:p>