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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.

Functional investigation of the chromosomal ccdAB and hipAB operon in Escherichia coli Nissle 1917