Molecular Insights Into Mechanism Of Gene Regulating CRISPR/CAS Expression In Escherichia Coli

Escherichia coli can cause a variety of diseases in humans and animals,posing a huge threat to public health and animal farming.Due to the severe resistance of clinical strains to antibiotics,the application of antibiotics faces challenges.Bacteriophage,as a virus that infects bacteria,can specifically lyse bacteria.Therefore,it has application potential to fight drug-resistant E.coli.However,during the evolution of E.coli,a variety of immune mechanisms have been formed to resist phage infection.Among the immune mechanisms,CRISPR(Clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated proteins)is a highly efficient adaptive immune system.The activation of the CRISPR/Cas system will inhibit phage infection,as well as antibacterial application of phage.The CRISPR/Cas of E.coli belongs to Type I-E.The Cas proteins are encoded by the cas3 operon and the cse1 operon.The deletion of any operon will cause the silence of the immune function of the CRISPR/Cas system.Therefore,targeting the cas3 or cse1operon and exploring the functional protein that regulates the operon will not only help to accurately analyze how E.coli can effectively and appropriately use the CRISPR/Cas system to defend invasive DNA,but also can provide more auxiliary molecules for breaking through the CRISPR/Cas defense barrier and enhancing the efficacy of bacteriophage theropy.In this study,the strain E.coli K-12 MG1655 was used as the model,and the regulatory genes were screened by using random transposon mutation technology and DNA pull-down technology.This research provided theoretical and application basis for preventing and controlling drug-resistant E.coli infection through the identification of functional molecules that regulate the cas3 operon,systematically analyzing the molecular basis and mechanism of functional molecules to regulate the expression of cas3 and the bacteriophage infection,and verifying its regulatory effect on the pathogenic E.coli CRISPR/Cas system.In order to screen the functional molecules that indirectly regulate the cas3 operon of the CRISPR/Cas system,the transposon random mutation technology was used in this study.Through antibiotic pressure induction,E.coli MG1655 was induced to produce nalidixic acid resistance,and the ORF of the lac Z reporter gene was used to replace the ORF of cas3 to successfully construct a nalidixic acid resistant E.coli reporter strain.Using?-galactosidase and q RT-PCR method,the cas3 promoter activity different strains caused by Tn5 transposon insertion were screened.Finally,among about 2820 mutants,4 mutants with higher differences in cas3 promoter activity were selected,and the inactivated genes of these 4 mutants were successfully identificated as gcv P,atp D,kdp A,and waa R using the genome walking method.It is suggested that the above genes can directly or indirectly regulate the expression of cas3,which provides a basis for further research on the regulatory mechanism of the above molecules on the cas gene and the molecular mechanism of resistance to phage infection.In order to screen the functional molecules that directly regulate the cas3 operon,DNA pull-down technology was used to screen the interacting proteins of the cas3 promoter.A total of 71 proteins potentially binds to the cas3 promoter were obtained.Based on the function and relative amount of the protein,Icl R transcription factor regulatory family Kdg R protein and c AMP receptor protein(CRP)were selected for further research.The Kdg R protein can bind to the cas3 promoter,but the regulation ability is weak.The CRP protein can bind to the cas3 promoter and activate the expression of cas3.The screening of these two direct regulatory proteins provide a basis for the subsequent research on regulatory mechanisms.Based on the results of transposon random mutation and DNA pull-down screening,in order to clarify the specific mechanism of gcv P gene regulation of cas3 expression,this study constructed a vairety of gene deletion and complemented strains.Glycine decarboxylase is encoded by the gcv P gene and belongs to the glycine cleavage system(GCS).It was found that adding N⁵,N¹⁰-methylene tetrahydrofolate(N⁵,N¹⁰-m THF),which is the product of GCS-catalyzed glycine,was able to activate cas3 expression.In addition,a c AMP receptor protein(CRP)encoded by crp activated cas3 expression via binding to the cas3 promoter in response to c AMP concentration.Since N⁵,N¹⁰-m THF provides one-carbon unit for purine,we assumed GCS regulates cas3 through associating with CRP.It was evident that the mutation of gcv P failed to further reduce the cas3expression with the crp deletion.These results illustrated a novel regulatory pathway which GCS and CRP co-regulate cas3 of the CRISPR/Cas system and contribute to the defence against invasive genetic elements,where CRP is indispensable for GCS regulation of cas3expression.In addition,due to intracellular differences in the c AMP level which is necessary for the function of CRP protein at different bacterial concentrations and different expression levels of cas genes at different bacterial density.There may be a correlation between the Quorum sensing(QS)system and the CRISPR/Cas system.In order to verify whether the CRISPR/Cas system in E.coli is regulated by the QS system,we analyzed the regulatory function of the signalling molecule AI-2 synthesizing gene lux S and the regulatory gene lsr R in the QS system.The study found that the deletion of lsr R in the QS system resulted in a decrease in the expression level of the cse1 operon in the CRISPR/Cas system.In addition,under the culture conditions without glucose,the deletion of lsr R leads to an increase in the expression level of gene hns which is the cse1operon suppressor.The results suggested that lsr R in the QS system can affect the expression of cse1 in the CRISPR/Cas system by regulating the expression of hns.In order to further expand the study of the cas3 operon of the CRISPR/Cas system and clarify the related mechanism between phage infection and atp D,which is another gene selected by the transposon random mutation,we analyzed the regulatory function of the Atp D protein belonging to the?subunit of ATP synthase.It was found that ATP synthase can significantly affect the expression levels of cse1 and cas3 in the CRISPR/Cas system,but the direct effect of ATP synthase on the infection ability of E.coli bacteriophage is more significant.Further research found that ATP synthase affects phage adsorption by regulating the expression of the E.coli LPS core polysaccharide synthesis gene waa C,and affects the proliferation of phage by regulating the injection of phage DNA,the expression of the pressure stress factor rpo S,and intracellular ATP content.The results show that ATP synthase affects the proliferation of bacteriophage by regulating bacteriophage adsorption,DNA injection,and assembly.To verify whether the CRP protein with the regulatory mechanism resolved regulates the CRISPR/Cas system in pathogenic E.coli O157:H7,the bacteriophage of O157:H7was isolated and used to verify the function of CRISPR/Cas and the regulation of CRP in O157:H7.The results showed that we successfully isolated and identified a lytic phage that could effectively lyse O157:H7.The homology of cas3 protein in O157:H7 was only 29%compared to that in K-12.In addition,the transformation of the CRISPR/Cas system targeted plasmid into O157:H7 can activate the CRISPR/Cas system to exhibit an anti-phage function.Furthermore,overexpression of CRP protein led to a decrease in the expression level of cas3 and an increase in phage proliferation capacity.The results suggested that CRP protein can regulate the CRISPR/Cas system in O157:H7.In summary,we successfully screened the proteins that potentially regulate the CRISPR/Cas system in E.coli MG1655.The regulating mechanism of GCS,CRP,and ATP synthase on E.coli CRISPR/Cas system and the molecular mechanism of ATP synthase to regulate phage infection were described in detail,and the regulatory effect of QS system on CRISPR/Cas system is explored and confirmed,which provides a theoretical basis for breaking through the defense system of CRISPR/Cas.It was further verified that the application value of CRP protein on CRISPR/Cas system in O157:H7 as a research target for breaking through the pathogenic E.coli CRISPR/Cas system defense barrier.