Inhibition Of Plasmid-mediated Conjugative Transfer In Foodborne Antibiotic-resistant Bacteria Based On CRISPRi Technology

The horizontal transfer of food-borne resistance genes mediated by conjugative transfer units between different species of bacteria through the food chain is one of the main reasons for the wanton spread of drug-resistant bacteria around the world.Among them,self-replicating conjugative plasmids are the most important contributor to conjugative transfer unit.The ribose import binding protein(Rbs B)could be used as a potential signaling molecule receptor in the quorum sensing system to participate in the regulation of bacterial physiological behavior.The physiological changes are closely related to the occurrence of conjugation transfer.Therefore,we used the CRISPRi system to suppress the expression of Rbs B to develop an effective strategy to inhibit plasmid transfer between bacteria of the same genus.In our study,the main findings are as follows:(1)Plasmid RP4 was used to establish the stable conjugative transfer model,and it was found that plasmid RP4 was easier to transfer based on solid-phase medium,which could be used to evaluate the effect of CRISPRi system on plasmid conjugation transfer.The results showed that plasmid RP4 could be completely transformed into competent cells by electroporation,and the donor strain E.coil HB101(RP4)was successfully constructed;plasmid RP4 could be more efficiently and stably conjugated into the recipient bacteria E.coil J53 in solid-phase reaction medium.(2)Based on the CRISPRi system,an effective system could inhibit the conjugative transfer of plasmid RP4 was established.The results showed that all CRISPRi recombinant plasmids targeting the rbs B gene,namely plasmid plv-d Cas9-Bn(n=0,1,2,3),could be successfully transformed into competent cells and stored stably in the culture environment supplemented with appropriate chloramphenicol.After induction with anhydrotetracycline,the recombinant strain E.coil HB101(RP4,plv-d Cas9-Bn)were at a similar growth level so could be used as a donor strain to participate in the conjugation transfer experiment.In addition,the induced CRISPRi recombinant plasmid plv-d Cas9-B1 not only effectively and reversibly silenced the expression of the target gene-the relative expression was down-regulated by nearly2 times,but also significantly inhibited the conjugative transfer of plasmid RP4 between E.coli-the inhibition rate was about 50%.More importantly,the correlation analysis confirmed that there was a significant positive correlation between the relative expression level and the frequency of conjugation transfer.These data indicated that silencing the expression of the target gene rbs B could effectively hinder the conjugation transfer of plasmid RP4.(3)At the biomolecular level,the mechanism of the CRISPRi system to inhibit the conjugative transfer of plasmid RP4 was discussed.The results showed that after inducing the expression of the CRISPRi system containing the plasmid plv-d Cas9-B1,the expression levels of the characteristic gene lux S of the quorum sensing signal molecule AI-2,the representative gene sdi A of the quorum sensing receptor protein,and the main genes qse B and qse C of the two-component regulatory system Qse B/Qse C all showed a significant upward-regulated trend,indicating that the quorum sensing system was promoted at this time.Subsequently,the correlation analysis showed that the expression of quorum-sensing-related genes was significantly negatively correlated with the frequency of conjugation transfer,presumably because the Rbs B protein could act as a potential receptor protein for small signaling molecules,regulating the amount of biofilm formation and bacterial motility.In conclusion,this study has developed a technique to effectively cut off the conjugative transfer of plasmids based on CRISPRi system,which opened a new way to prevent the rapid spread of food-borne drug-resistant bacteria.Meanwhile,the effect of Rbs B protein on conjugative transfer was discussed with the help of the CRISPRi system,which further enriched people’s cognition on the relationship between Rbs B protein and quorum sensing system,thus providing theoretical basis and technical support for the regulation mechanism of plasmid transfer and diffusion by quorum sensing system.