CRISPRi In Situ Investigates Functions Of Toxin-antitoxin System In Escherichia Coli Nissle 1917

Compared with those pathogenic enterotoxigenic and/or laboratory Escherichia coli strains,currently E.coli Nissle 1917?EcN 1917?is a unique intestinal probiotics for food and drug applications due to its important activities in the treatment of gastrointestinal diseases.In the current study,toxin-antitoxin systems included in EcN 1917 genome were predicted through bioinformatics software.By the heterologous expression in E.coli BL21 and further in situ knockdown in EcN 1917,the structures and activities of toxin-antitoxin genes mazEF-EcN and hipA-EcN were elucidated.Bioinformatics analysis showed that the EcN 1917 genome contained 10 pairs of toxin-antitoxin systems,most of them were Type II.The loci?52351-52901 bp,named PP₀0551?scored the highest value and suggested as the putative mazEF sequence..After further gene cloning and sequencing,the size of PP₀0551 sequence in EcN 1917 was 551 bp,PP₀0240 sequence was upstream adjacent to PP₀0245,and only two CG bases were separated in the middle,sharing one promoter.The PP₀0551 gene sequence was a typical double cistron structure gene structure,and the PP₀0551 gene sequence was mazEF gene sequence of other Escherichia coli.The structure is identical.The res ? Its of cloning and expression of E.coli BL21?growth curve and platelet dot-like growth?also showed that the activity of the gene product was in line with the type II TA system.Therefore,we identified the PP₀0551 sequence as the mazEF toxin antitoxin system and named it mazEF-EcN.By comparing mazEF-EcN with other E.coli mazEF flanking genes,we found that mazEF-EcN had the same gene structure.It can be inferred that mazEF was highly conserved at E.coli species level and had the same level of genetic evolution.In order to further explore the regulatory mechanism of TA system on the probiotic characteristics of EcN 1917,in situ knock-down test was carried out using CRISPRi technique.According to mazEF and hipA gene sequences,gRNA fragments were designed and synthesized by CRISPRdirect,and the transformed plasmids were constructed by connecting with the vector.Then mazEF-gsRNA/dCas9 and hipA-gsRNA/dCas9 plasmids were introduced respectively.After EcN-mazE and EcN-hipA were induced,the expression of mazEF and hipA were inhibited by RT-PCR and PAGE respectively.At the same time,EcN 1917 retention and biofilm formation in Chengdu decreased significantly.When the inducer was removed,the expression of mazEF and hipA returned to normal.CRISPRi technology greatly simplifies the operation process and can quickly compare gene expression with in situ knock-out,which provides a very valuable work idea for studying the physiological function of EcN 1917 TA.In addition,mazEF and hipA reg? late the retention of EcN 1917 and the biofilm of EcN 1917.The probiotic characteristics of EcN 1917,especially its ability to survive and adhere to intestinal tract,have important guiding significance.