Study On The Mechanism Of Regulation Of The Type I-E Crispr-Cas System By Histone-like Protein StpA In Escherichia Coli

To cope with environmental stresses,bacteria and archaea acquire exogenous genes from genomes and plasmids of the other strains via horizontal gene transfer(HGT).Simultaneously,prokaryotes are challenged by invasion of ‘harmful genes'.To reduce the risk the acquisition of exgenous DNA,prokaryotes have evolved an adaptive immunity that is mediated by CRISPR-Cas systems.CRISPR-Cas systems digesting foreign DNA efficiently and specifically.Working mechanisms of CRISPR-Cas systems have been intensively studied.However,little work of regulating mechanisms has been carried out.It has been reported that H-NS can repress the activity of type I-E CRISPR-Cas system in Escherichia Coli,the function of StpA in regulating the CRISPR-Cas system remains unidentified.We focus on the mechanism of regulation of the type I-E CRISPR-Cas system by histone-like protein StpA in Escherichia coli.Firstly,we established a fluorescent reporting system to detect the activity of the cas promoter and an experimental system to detect the function of the CRISPR-Cas system cutting the target DNA.It was found that StpA can activate the cas promoter in the absence of hns and promote the increase of the cr RNA content.Thus,it enhanced the ability of type I-E CRISPR-Cas system cleave the target plasmid.Secondly,to confirm that StpA and H-NS regulate cas promoter by binding to the predicted DBS,we mutated the conserved sites of the binding sites.Result showed that H-NS repress transcription of the cas promoter by binding to the predicted DBS.In the mean time,StpA promotes the activity of cas promoter by binding to the predicted DBS of H-NS.Otherwise,further study showed that low expression of StpA on the chromosome promoted the expression of cas promoter increase CRISPR-Cassystem activity,whereas,high expression of StpA failed to stimulate cas promoter in hns mutant.It is reported that magnesium ion can affect the binding structure of H-NS and StpA with DNA in vitro.Our research explores the effect of magnesium ions on StpA regulation of cas gene expression.We found that the addition of magnesium ions in M9 medium significantly improves the stability of StpA regulation cas promoter,which enable its activate cas in a wider p H range gene expression.In summary,StpA can not only activate the cas gene expression of the CRISPR-Cas system,but also promote the production of cr RNA,thereby improving the ability of the CRISPR-Cas system to prevent foreign DNA invasion.StpA combines the predicted binding sites of H-NS regulate the expression of cas gene.The expression level of StpA affects its regulation on cas gene expression: high-level expression of StpA inhibits cas gene expression,while low-level expression of StpA activates the cas promoter.In addition,the preliminary results of this paper show that magnesium ions can improve the stability of StpA regulation cas promoter.