Prediction And Verification Of Transcription Factor Interacting With SmpB Promoter In Aeromonas Veronii

As a human-fish comorbid pathogen,Aeromonas veronii seriously endangers the development of the aquatic industry and human health.As an important part of the trans-translation system,SmpB protein can play a role in translational monitoring and rescue of retained ribosomes during the translation of Aeromonas veronii,and regulate the survival and virulence factors of Aeromonas veronii.Searching for transcription factors which regulate the smpB promoter will facilitate the in-depth study of SmpB protein and trans-translation systems,clarify the pathogenic mechanism of Aeromonas veronii,and provide a theoretical basis for the development of attenuated vaccines for Aeromonas veronii.In this paper,a convolutional neural network model capable of predicting and regulating the smpB promoter was constructed by machine learning in bioinformatics,and transcription factors that presumably bound to the smpB promoter were predicted.Experimental results such as bacterial one-hybridization,bacterial gene knockout,real-time quantitative PCR(Real-time Quantitative PCR),and green fluorescent protein reporter gene assay demonstrated that the transcription factor ArgR directly bound to the smpB promoter region and functioned to negatively regulate the smpB promoter.And the N-terminal ArgR played a major role in the interaction with the smpB promoter.The specific results were as follows:(1)A crawler program was written in Python to collect information on 6242 bacterial transcription factor binding sequences in published papers.(2)Using the data collected by the crawler program and machine learning techniques,a convolutional neural network model was constructed to predict the binding of transcription factors with bacterial promoters at an accuracy rate of 90%.By inputting the Fasta file of the smpB promoter sequence,the binding probability was calculated,and the five transcription factors with the highest probability were ArgR(0.213),CueR(0.155),OxyR(0,141),IscR(0.076)and OmpR(0.059).(3)The promoter of smpB was inserted to pBXcmT vector to obtain a pBXcmT-PsmpB vector.The five transcription factors argR,cueR,oxyR,iscR,and ompR,which were predicted to interact with the promoter of smpB,were inserted into pTRG vectors,to obtain pTRG-ArgR?pTRG-CueR?pTRG-OxyR?pTRG-IscR?pTRG-OmpR,respectively.(4)The pBXcmT-PsmpB vector was co-transformed into the E.coli XL 1-Blue MR(Reporter)strain with pTRG-ArgR?pTRG-CueR?pTRG-OxyR?pTRG-IscR?and pTRG-OmpR,respectively,for bacterial one-hybrid experiments.The results of the screening plate showed that the reporter strain co-transformed with the pTRG-ArgR vector and the pBXcmT-PsmpB vector could grow in the presence of 12 mM 3-AT selective medium,which proved that the transcription factor ArgR could bind strongly to the smpB promoter region.(5)The argR gene was knocked out successfully in the wild-type Aeromonas veronii(WT)genome,and designated as Aeromonas veronii argR knockout type(?argR).The argR complemented type(::argR);the knockout type and the complement type were sequenced and confirmed.(6)The growth curves of WT,AargR.and::argR were determined.The results showed that the growth rate of AargR was faster compared to WT,while the growth rate of::argR was slower.(7)In order to study the regulation of SmpB expression by ArgR,real-time quantitative PCR(qPCR Real-time Quantitative PCR)was performed in the LB medium,5 mM Mg2+ LB medium and 5 mM Ca2+ LB medium.The expression levels of smpB in WT,?argR and::argR showed that the expression of smpB was significantly higher than that of WT in the log phase,while the differences were not significant in stationary phase.(8)In order to detect whether the transcription factoi ArgR had a direct effect on the smpB promoter,the smpB promoter and enhanced green fluorescent protein(eGFP Enhanced Green Fluorescent Protein)were constructed on pUC19 vector to obtain the fusion fluorescent vector pUC19-PsmpB-eGFP.After that,pUC19-PsmpB-eGFP was co-transformed into the Reporter strain with the pBT-ArgR.The fluorescence decreases significantly,demonstrating that the transcription factor ArgR negatively regulated the smpB promoter.(9)In order to further elucidate the key region of ArgR interacting with the smpB promoter,the C-and N-terminal truncations pBT-ArgRAC246 and pBT-ArgRAN245 were contructed and co-transformed with fluorescent vector pUC19-PsmpB-eGFP into the Reporter strain.The fluorescence of pBT-ArgRAC246 was lower than that of pBT-ArgR?N245,demonstrating that N-terminus played a important role in the binding to the smpB promoter sequence.(10)In order to further investigate the key sites of ArgR acted on smpB promoter,the smpB promoter and eGFP were inserted into pACYCDuet-1 vector to obtain pACYCDuet-l-PsmpB-eGFP,and co-transformed with pTRG-ArgR-G134?pTRG-ArgR-D121?pTRG?ArgR-SR54,55 into the Reporter strain.However,the transformants revealed no significant differences in fluorescence values,indicating that there might be other key residues for interaction.This study used convolutional neural networks to predict transcription factors that bound to promoters in Aeromonas veronii and experimentally verified them,avoiding the huge workload in the traditional library screening process.Subsequent studies of bacterial transcription factor regulatory promoters are of great significance.