Disruption of methionine synthesis repressor makes <i>Escherichia coli</i> mutualistic to host stinkbug

Degenerative genome evolution is widely found among obligatory bacterial mutualists, as observed in plant-sucking hemipteran insects whose symbiont genomes are highly reduced and specialized for provisioning of essential amino acids. Originally, such symbionts must have been derived from environmental free-living bacteria. It is elusive, however, what evolutionary changes are involved in the early stages of such elaborate mutualistic associations. Here, we addressed this evolutionary question using the experimental symbiotic system consisting of the stinkbug <i>Plautia stali</i> and the model bacterium <i>Escherichia coli</i>. In <i>E. coli</i>, <i>metJ</i> encodes a repressor of the methionine synthesis pathway, and its disruption upregulates production of the essential amino acid methionine. We found that, when <i>metJ</i>-disrupted <i>E. coli</i> was inoculated to <i>P. stali</i>, the insects exhibited significantly elevated hemolymphal methionine levels and improved adult emergence rates, demonstrating that the single-gene mutation makes <i>E. coli</i> mutualistic to <i>P. stali</i>. In comparison with mutualistic <i>E. coli</i> single-gene mutants that upregulate another essential amino acid tryptophan, the phenotypic effects on <i>P. stali</i> were somewhat different: the adult emergence rate was improved by both the methionine-overproducing and tryptophan-overproducing <i>E. coli</i> mutants, whereas the adult body color was improved by the tryptophan-overproducing <i>E. coli</i> mutant only. When we generated a double mutant <i>E. coli</i> Δ<i>metJ</i>Δ<i>tnaA</i> and inoculated it to <i>P. stali</i>, the adult emergence rate was not improved but rather attenuated, uncovering non-additive fitness consequences of these single-gene mutations. These results provide insights into what genetic changes may have facilitated the early evolution of the insect-microbe mutualism.IMPORTANCEWhat is the evolutionary origin of elaborate bacterial mutualists entailing drastic genome reduction, specialized metabolism, and uncultivability? This question is important but challenging to address, because the evolution of such symbiotic associations occurred in the past and cannot be observed directly. However, the recent development of an experimental symbiotic system consisting of the stinkbug <i>Plautia stali</i> as host and the model bacterium <i>Escherichia coli</i> as symbiont has opened an avenue to empirically investigate the evolution of host-microbe mutualism. We demonstrated that, strikingly, single-gene mutations of <i>E. coli</i> that upregulate the production of methionine and tryptophan make the non-symbiotic bacterium mutualistic to <i>P. stali</i>, plausibly via provisioning of the essential amino acids that complement the nutritional requirements of the plant-sucking insect host. Our finding provides insight into what genetic changes of the symbiont side can be involved in the early evolution of the host-microbe mutualism.