Distribution Of Quorum Sensing System And Transcriptome Of Regulatory Gene Vfls In Vibrio Fluvialis

Vibrio fluvialis is a halophilic,gram-negative bacterium,commonly found in seawater,salty harbor waters and rivers.It causes human enteritis disease,and its clinical symptoms are very similar to cholera-like diarrhea,causing diarrhea outbreaks and outbreaks on a global scale.Previous studies have shown that Vibrio fluvialis regulates the expression of related virulence factors through quorum sensing(QS)system,and the key regulatory protein of quorum sensing system is VflS.To further understand the role of QS systems in the pathogenicity and environmental fitness of V.fluvialis,firstly,the dynamic changes of quorum sensing signal molecules CAI-1,AI-2 and AHLs in Vibrio fluvialis at 30℃ and 37℃ were detected.Then the distribution of quorum sensing system related genes and the production of signal molecules in 70 strains of Vibrio fluvialis from different sources were detected.The dynamic levels of CAI-1,AI-2 and AHLs signal molecules in the culture supernatant of VF85003 were increased in a cell density-dependent way.Generally,the level of AHLs signal molecule at warm environmental temperature(30℃)was higher than that at the host temperature(37℃).On the contrary,the production of CAI-1/AI-2 signal molecules at the host temperature(37℃)was higher than 30 ℃,which suggested the different roles of QS signals in the pathogenicity and environmental fitness.45 strains of Vibrio fluvialis were detected to have quorum sensing system-related genes,the expression levels of the three signaling molecules were mostly concentrated in the range of 100-1000 fold,and 22 strains of Vibrio cholerae were positive for the virulence factor HapA activity.On the other hand,we analyzed the transcriptomes of VF85003 WT and ΔvflS,some differentially expressed genes were detected by RT-PCR.The components of WT and ΔvflS strains with significant differences in gene expression,such as Sec-SRP,curli related genes and CAI-1 synthetase gene cqsA,were selected for luminescence reporter plasmid construction,for further clarify whether vflS plays a regulatory role at the promoter level.The results showed that the promoter activity of these genes was significantly decreased inΔvflS,indicating that these genes are regulated by vflS.To further understand whether csgD and cqsA are directly regulated by VflS protein,we performed an EMSA experiment.The results showed that the VflS protein could bind to csgD and cqsA promoter sequences indicating that these two genes are directly regulated by VflS protein.Through our study,we can understand whether the QS function of Vibrio fluvialis in different periods,regions and sources is complete,and whether their global regulatory protein VflS has functionality,so as to clearly understand the distribution characteristics of quorum sensing system and the importance of pathogenicity and environmental viability of Vibrio fluvialis.Through the mining of RNA-Seq data,we found two important genes directly regulated by VflS regulatory factors,csgD and cqsA,which are important for further understanding of the quorum sensing mechanism of Vibrio fluvialis.However,other aspects of genes regulated by its quorum sensing system still require further research and exploration.