Toxin-antitoxin systems (TASs) have attracted much attention due to their important physiological functions. These small genetic factors have been widely studied mostly in commensal Escherichia coli strains, whereas the role of TASs in the probiotic E. coli Nissle 1917 (EcN) is still elusive. Here, the physiological role of chromosomally encoded type II TASs in EcN was examined. We showed that gene pair ECOLIN_00240-ECOLIN_00245 and ECOLIN_08365-ECOLIN_08370 were two functional TASs encoding CcdAB and HipAB, respectively. The homologs of CcdAB and HipAB were more conserved in E. coli species belonging to pathogenic groups, suggesting their important roles in EcN. CRISPRi-mediated repression of ccdAB and hipAB significantly reduced the biofilm formation of EcN in the stationary phase. Moreover, ccdAB and hipAB were shown to be responsible for the persister formation in EcN. Biofilm and persister formation of EcN controlled by the ccdAB and hipAB were associated with the expression of genes involved in DNA synthesis, SOS response, and stringent response. Besides, CRISPRi was proposed to be an efficient tool in annotating multiple TASs simultaneously. Collectively, our results advance knowledge and understanding of the role of TASs in EcN, which will enhance the utility of EcN in probiotic therapy.

Key points

• Two TASs in EcN were identified as hipAB and ccdAB.

• Knockdown of HipAB and CcdAB resulted in decreased biofilm formation of EcN.

• Transcriptional silencing of hipAB and ccdAB affected the persister formation of EcN.

• An attractive link between TASs and stress response was unraveled in EcN.

• CRISPRi afforded a fast and in situ annotation of multiple TASs simultaneously.

毒素-抗毒素系统（TASs）由于其重要的生理功能而备受关注。这些小的遗传因素已在共生大肠杆菌菌株中进行了广泛研究，而TAS在益生菌大肠杆菌Nissle 1917（Ec N）中的作用仍然难以捉摸。在这里，检查了Ec N中染色体编码的II型TAS的生理作用。我们显示，基因对ECOLIN_00240 - ECOLIN_00245和ECOLIN_08365 - ECOLIN_08370是分别编码CcdAB和HipAB的两个功能性TAS。CcdAB和HipAB的同源物在大肠杆菌中更保守属于致病菌的物种，表明它们在Ec N中的重要作用。CRISPRi介导的ccdAB和hipAB的阻遏作用显着减少了固定相Ec N的生物膜形成。此外，已证明ccdAB和hipAB负责Ec N中的持久性形成。生物膜和受ccdAB和hipAB控制的Ec N持久性形成。它们与参与DNA合成，SOS反应和严格反应的基因表达有关。此外，CRISPRi被认为是同时注释多个TAS的有效工具。总的来说，我们的结果提高了对TAS在Ec N中的作用的认识和了解，这将增强Ec N在益生菌治疗中的效用。

关键点

• EcN中的两个TAS被识别为hipAB和ccdAB。

•抑制HipAB和CcdAB导致EcN生物膜形成减少。

• hipAB和ccdAB的转录沉默影响EcN的持续形成。

• EcN中没有阐明TAS与压力响应之间的有吸引力的联系。

• CRISPRi同时提供了多个TAS的快速和原位注释。