Screening And Functional Research Of Transcription Factor SEN3610 Regulatory Genes In Salmonella Enteritidis

Salmonella enterica serovar Enteritidis(S,Enteritidis)is one of the most important foodborne zoonotic pathogens,which can cause food poisoning,diarrhea and even death in humans,and have caused serious harm to public health and food safety.The DeoR family transcriptional regulator is involved in the regulation of glucose metabolism and plays an important role in bacterial infection,proliferation and response to environmental stress.In our previous studies,a DeoR-type transcriptional factor SEN3610 in S,Enteritidis was upregulated greatly in S.Enteritidis-infected macrophages by selective capture of transcribed sequences(SCOTS),and the role of this gene played in Salmonella infection was primarily studied using iTRAQ.However,it remains unclear about the genes directly regulated by SEN3610 and its function in the regulating metabolic pathway.In this study,we identified the genes potentially regulated by SEN3610 using RNA-Seq analysis of SEN3610 deleted and overexpressed strains.The differentially expressed genes were further verified by qRT-PCR analysis.The SEN3610 was further cloned into the prokaryotic expression vector pET28a(+)and expressed for purification of the SEN3610-His6 recombinant protein.The EMSA assay was then used to detect the binding between gene promoter and protein in vitro,so as to screen out the genes directly regulated by SEN3610.The function of SEN3610 in regulating the glycerol metabolic pathway of S,Enteritidis was further determined by qRT-PCR analysis of target genes and detection of the growth ability between wild type and mutant strains.1 Screening of genes regulated by SEN3610In this study,we analyzed the differentially expressed genes in the SEN3610-deleted strain and its complementary strain using RNA-Seq.The results showed that deletion of SEN3610 caused significant downregulation of the SPI-1 and SPI-2 genes compared to the complementary strain.qRT-PCR results further confirmed that these genes were downregulated in the SEN3610-deleted strain,which was consistent with the results of RNASeq.Therefore,we constructed a prokaryotic expression strain of SEN3610 and successfully purified the recombinant protein SEN3610-His6.EMSA was then used to detect whether SEN3610-His6 binds to the gene promoter probes in vitro,and the results revealed that SEN3610 could not directly regulate the expression of the SPI-1 and SPI-2 genes by binding to their promoters.To identify the genes directly regulated by SEN3610,we analyzed the upregulated genes in the SEN3610-deleted strain based on the RNA-Seq results,indicating that the glp and fim genes were upregulated in the SEN3610-deleted strain.qRT-PCR was also used to verify the transcriptional expression of glp and fim genes in the SEN3610-deleted strain combined with the promoter-binding characteristics of DeoR-type transcriptional factors.The results showed that glpABC,glpD and glpT were significantly upregulated in the deletion strain,and this trend was consistent with the results of RNA-Seq,while there was no significant difference in other glp and fim genes,indicating that SEN3610 may act as a repressor of glpD,glpT and glpABC genes.2.The regulation mechanism of glp genes by SEN3610Because of the transcriptional regulation of SEN3610 on glp genes,we predicted the potential function of SEN3610 in regulating glp genes involved in bacterial glycerol metabolism.Simultaneously,the promoters of glp genes were compared with the conserved binding sites of DeoR family transcriptional factors,and a completely conserved binding motif was identified in the promoters of glpT,glpD,and glpABC genes.Therefore,EMSA was used to determine whether SEN3610-His6 binds to these glp gene promoters.The results showed that the recombinant protein could bind to each type of probes,indicating that SEN3610 can directly regulate the expression of these genes by binding to the promoters.GlpR is a confirmed transcriptional repressor encoded by glpR in the glp gene cluster;and it can downregulate the expression of various glp genes,including glpT,glpD,and glpABC.To further analyze the correlation between SEN3610 and GlpR-regulated glp genes and the bacterial glycerol metabolic pathway,we constructed deletion mutants of glpA,glpD,glpT,and glpR using the X-Red homologous recombination system.We also used qRT-PCR to compare the transcriptional levels of the glp genes in each deletion strain cultured with different concentrations of glycerol as the sole carbon source.The results showed that the change in glycerol concentration from 1%to 2%had no significant effect on the transcriptional expression levels of glpD,glpT,and glpABC.Therefore,we measured the growth rate of these mutants by using 2%glycerol as the sole carbon source.The results showed that deletion of SEN3610 or glpR had no significant effect on the growth rate of S.Enteritidis,whereas deletion of both genes could cause a significant increase in the growth rate,indicating the enhancement of glycerol utilization capacity in the ΔSEN3610ΔglpR strain,which is consistent with the increased expression of glp genes.Our results revealed that both SEN3610 and GlpR could inhibit the transcriptional expression of glp genes and,thus,jointly regulate the metabolism of glycerol in S.Enteritidis.The SEN3610 is a new regulator repressing the expression of glp genes involved in the metabolism of glycerol.