| Bacteria will survive in different ways in the face of complex living environments,and the formation of biological membranes(BF)is one of the ubiquitous survival modes of bacteria in nature.Due to the particularity of the BF structure,BF formed by pathogenic microorganisms can easily cause a series of related chronic or refractory microbial infections in clinical medicine.In addition,during food production and processing,some major foodborne pathogens such as E.coli and Staphylococcus aureus can form BF on the surface of food or food production equipment,causing food corruption and infection with foodborne diseases.A series of food safety issues,eliminating or inhibiting the formation of BF is a huge challenge facing the medical and food industries.As the formation of BF undergoes the "movement-rest-movement" complex dynamic process,flagella-mediated movement and the regulation of bacterial two-component signaling system(TCS)are inseparable.Therefore,this subject focuses on three aspects of bacterial formation,movement,and TCS system regulation and research to find a target to effectively eliminate the formation of BF,which has very important practical significance for the subsequent development of BF inhibitors.In this paper,Escherichia coli MG1655 is used as the experimental object.First,CRISPR/Cas9 editing technology and ?-Red recombination system are used to knock out key genes of the QseBC system to construct ?qse C and ?qse BC strains.Second,the crystal violet staining method(CV)and laser co-extraction Quantitative and structural qualitative analysis of BF formed by focusing microscope(CLSM)and electron scanning microscope(SEM)on wild type(WT)and mutant strains under different time conditions(6,12,24,and 36h);Strains were analyzed for bacterial motility in different agar concentration media(0.1-0.6%)and at different times(6,12,24,36,48,and 72 h),and determined using the Bioscreen automatic microbial growth curve analyzer The growth curve and growth kinetic parameters were analyzed.Finally,qRT-PCR was used to detect the expression of related genes that caused phenotypic differences between WT and mutant strains.The main research contents and results of this article are as follows:1.Construction of mutant strains of key genes of E.coli QseBC system using CRISPR/Cas9 systemThe CRISPR/Cas9 system(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein9)is an acquired immune defense mechanism that can use its own special immune function to mediate genes for editing.It is highly targeted,convenient and efficient.Based on the ?qse B mutants previously obtained in this laboratory,the subject continued to use the CRISPR/Cas9 technology and the ?-Red recombination system coupled with a dual plasmid system(p Cas/p Target)to perform the non-Trace knockout editing.Analysis of the transformant colony PCR identification and sequencing verification results showed that,based on the successful construction of the repair template and the plasmid containing the 20 bp recognition sequence,the qse C and qse BC gene knockouts were verified by colony PCR identification and sequencing.2.QseBC two-component system affects phenotype changes of E.coli motility and biofilm formationNext,the functions of qse C and qse B genes in BF formation and motility were further verified.In the determination of BF formation,the CV method was first used for quantitative analysis of WT and mutant BF.The results showed that E.coli BF formed the most in 24 h.?qse C caused an increase in the amount of early biofilm formation.?qse B did not affect the change in BF formation.?qse BC increased the BF formation at 12 h,and BF in the early,mature and dispersed phases.There were no significant differences.Then the qualitative analysis of the BF structure of WT and mutant strains was performed using CLSM and ISA software.The results showed that the BF structure parameters ADD and TE values of ?qse C increased,indicating that colony formation was more dense in the early BF.Then,the early BF formed by WT and mutant strains were observed by SEM.The results further showed that compared with WT,the early BF formation of ?qse C was more dense and the cell length was significantly longer.In order to further explore the influencing factors of the difference in the formation of BF caused by the absence of qse C,the analysis was performed from the aspects of exercise and growth.The results show that the QseBC system is involved in regulating the swimming movement of E.coli;compared with WT,?qse C exercise is significantly reduced by about 0.55 times,?qse B movement is significantly increased by about 1.98 times,and there is no difference in ?qse BC movement;in addition,the growth kinetic parameters of WT and mutant The analysis found that the maximum growth rate of ?qse C was reduced compared to WT.In general,?qse C promotes early BF formation and inhibits swimming exercise;?qse B does not affect BF formation,but promotes swimming exercise;?qse BC does not affect BF formation and motility.This shows that the QseBC system is closely related to exercise and BF formation,and there is no strict negative regulation of bacterial movement and BF formation.3.qRT-PCR detection of relative expression of QseBC system related genesIn order to further investigate the reasons for the difference between ?qse C and ?qse B in the formation of BF and swimming,qRT-PCR was used to detect the relative expression of flagella,pili,metabolism,and TCS system genes.The results showed that the expression of qse B in the ?qse C strain increased by about 196 times,indicating that when the external signal stimulated the QseBC system,due to the lack of the membrane protein Qse C to receive the signal for transformation,the intracellular homologous protein Qse B has been in a "ready" state,causing Qse B expression.In addition,combined with the phenotypic results,it can be concluded that Qse B protein has a negative regulatory relationship with bacterial movement,qse B is over-expressed and inhibits movement,?qse B promotes movement.In addition,?qse C and ?qse BC flagellar gene expression increased but did not promote exercise,indicating that one of the key factors regulating movement and biofilm formation is the QseBC system.According to the measurement results of the metabolic gene dna A expression,it is known that there is a close regulatory relationship between qse C and dna A.When qse C expression decreases,dan A expression increases.The QseBC-related TCS system gene expression results showed that in ?qse C,?qse B and ?qse BC,the gene expression of the TCS system cell membrane proteins Bas S and Pho Q decreased;in ?qse BC,the gene expression of the two-component system intracellular protein Kdp E increased 5.7 times.In this study,the function verification of the QseBC system was conducted,and the bacterial movement combined with the TCS system was used to conduct targeted research on the elimination of BF formation,providing an effective target for the subsequent development of BF inhibitors or antimicrobial infections. |
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