| Bacterial resistance has become a global crisis.Screening potential antimicrobial enhancers is one of the measures to relieve the pressure from bacterial resistance.In previous studies,we found that protein kinase Yih E in Escherichia coli is a key regulator of bacterial programmed cell death by antagonizing Maz EF toxin-antitoxin module,which protects bacteria from drug treatment.However,it is not yet understood how Yih E kinase senses the environmental stresses.Based on our previous studies on the expression of yih E,we believe that other transcriptional factor(s)besides the two-component response regulator Cpx R may be involved in the regulation of yih E expression to help bacteria to cope with a variety of environmental pressures.Therefore,the transcriptional factor(s)that regulates the expression of yih E was studied in this work,in order to understand the ways through which environmental siganls transfer to Yih E kinase.In the present work,the transcriptional factors that might regulated the expression of yih E were preliminarily screened: 1)transcriptional factors of yih E were predicted by using three databases(MEME Suite 5.0.2,PRODORIC? Database and Softberry),and a total of 49 possible transcriptional factors were found;2)four transcriptional factors(Deo R,Lac I,Omp R and IHF)that interacted with the promoter of yih E were captured by using DNA pull-down;3)based on the predicted results obtained by bioinformatics and the results of DNA pull-down,35 transcriptional factors were selected for subsequent verification;4)the sensitivity of the mutant strains of the 35 transcriptional factors to nalidixic acid was examined by drug sensitivity test.Among these mutant strains,the sensitivity of △cys B and △tyr R to nalidixic acid significantly reduced,while the sensitivity of △rob,△fur,△fhl A,△fis,△pho P,△mar A and △ula R to nalidixic acid significantly increased;5)the expression of yih E in those35 mutant strains under nalidixic acid treatment at mid-log phase and stationary phase was measured by using Real-time PCR,and it was found that the expression of yih E gene in E.coli was relatively stable at stationary phase,while the expression of yih E gene at mid-log phase phase was easily affected by gene mutations and external pressures;6)four candidate transcriptional factors(Fur,Arc A,Rob and Cys B)from the 35 transcriptional factors that affected the sensitivity of bacteria to nalidixic acid and affected the expression of yih E were selected,and their binding to the promoter region of yih E was further verified by using a bacterial one-hybrid(B1H)system.The negative selection results of bacterial one-hybrid system showed that the three fragments in the yih E gene promoter,p01,p02 and p03,could not self-activate the expression of reporter gene URA3,indicating that the reporter plasmid p H3U3-p01,p H3U3-p02 and p H3U3-p03 could be used in the positive selection of bacterial one-hybrid system to verify whether Rob,Cys B,Arc A and Fur could bind to the promoter region of yih E gene.The construction of expression vectors of the four candidate transcriptional factors and the verification of whether the four candidate transcription factors,as well as the other 31 transcriptional factors,directly regulate the expression of yih E gene by positive selection of bacterial one-hybrid system are in progress.The presented work lays the foundation for searching and verifying transcriptional factors of yih E,which would help us to understand the ways through which environmental siganls transfer to Yih E kinase.Meanwhile,it also provides a new way to screen novel antibacterial enhancers that could enhance the efficacy of existing antibiotics. |
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