Microorganisms often live in symbiosis with their hosts, and some are considered mutualists, where all species involved benefit from the interaction. How free-living microorganisms have evolved to become mutualists is unclear. Here we report an experimental system in which non-symbiotic Escherichia coli evolves into an insect mutualist. The stinkbug Plautia stali is typically associated with its essential gut symbiont, Pantoea sp., which colonizes a specialized symbiotic organ. When sterilized newborn nymphs were infected with E. coli rather than Pantoea sp., only a few insects survived, in which E. coli exhibited specific localization to the symbiotic organ and vertical transmission to the offspring. Through transgenerational maintenance with P. stali, several hypermutating E. coli lines independently evolved to support the host’s high adult emergence and improved body colour; these were called ‘mutualistic’ E. coli. These mutants exhibited slower bacterial growth, smaller size, loss of flagellar motility and lack of an extracellular matrix. Transcriptomic and genomic analyses of ‘mutualistic’ E. coli lines revealed independent mutations that disrupted the carbon catabolite repression global transcriptional regulator system. Each mutation reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that single carbon catabolite repression mutations can make E. coli an insect mutualist. These findings provide an experimental system for future work on host–microbe symbioses and may explain why microbial mutualisms are omnipresent in nature.

微生物通常与宿主共生，有些被认为是互惠互利者，所有参与的物种都从相互作用中受益。尚不清楚自由生活的微生物是如何进化成互惠互利者的。在这里，我们报告了一个实验系统，其中非共生大肠杆菌进化为昆虫互惠生。臭虫Plautia stali通常与其必需的肠道共生体Pantoea sp. 相关联，后者在一个专门的共生器官上定殖。当绝育的新生若虫感染大肠杆菌而不是泛菌时，只有少数昆虫存活，其中大肠杆菌表现出对共生器官的特定定位和对后代的垂直传播。通过P. sali的跨代维持，几个超突变的大肠杆菌系独立进化以支持宿主的高成虫出现和改善的体色；这些被称为“互惠”大肠杆菌。这些突变体表现出较慢的细菌生长、较小的尺寸、鞭毛运动性的丧失和细胞外基质的缺乏。“互惠”大肠杆菌系的转录组学和基因组分析揭示了破坏碳分解代谢物抑制全球转录调节系统的独立突变。当引入野生型大肠杆菌时，每个突变都再现了互惠表型，证实单碳分解代谢物抑制突变可以使大肠杆菌成为昆虫互惠生。这些发现为未来关于宿主-微生物共生的工作提供了一个实验系统，并可以解释为什么微生物共生在自然界中无处不在。