The Mechanism Of Qrr1-5 On The Alkaline Serine Protease Synthesis In Vibiro Alginolyticus

Vibrio alginolyticus is an important pathogen in marine aquaculture and its main virulence factor is the extracellular alkaline serine protease,Asp.s RNA,the small non-coding RNA,is widespread in bacteria and modulates the target genes at post-transcriptional level by base-pairing to participate in various physiological processes.The previous work has characterized five s RNAs(Qrr1-5),which influence the production of alkaline serine protease in Vibrio alginolyticus.In order to illustrate the molecular regulation mechanism of Qrr1-5 on alkaline serine protease production,the influence of Qrr1-5 on the activity of Asp was detected by HPA assay,and then the underlying regulation mechanism of Qrr1-5 on Asp production was further studied by utilizing the methods of Western Blot,Realtime PCR,translational fusion,EMSA,and so on.The main results were as follows:(1)The activities of Asp in different Vibrio alginolyticus strains were detected by HPA assay and the results showed that all of Qrr1-5 molecules repress the activity of Asp to some extent,while Qrr1 and Qrr3 play the most important roles in the regulation of Asp activity.Then,the expressi on levels of Lu x R were detected in the wild-type,the mutant and the complement strains.The results demonstrated that the regulation mode of Qrr1-5 on Lux R expression is down-regulated,which indicated that at high cell density the repression of Qrr1-5 on Asp synthesis is mainly through its negative control of Lux R.Besides,the translational fusion strain of Aph A-Flag/?qrr1-5 was successfull y constructed on the basis of the strain of Aph A-Flag/WT.Aph A was expressed at low cell densityculture but not at hi gh cell density.Qrr2 and Qrr3 repress the expression of Aph A,while otherstake no obviou s effect.(2)The crosstalk between Qrr1-5 and quorum sensing system was studied by Realtime PCR.All Qrr1-5 molecules have no influence on the m RNA level of Lux R but severe repression on the Lux R protein expression.Also all Qrr1-5 molecules have n o influence on the m RNA level of Aph A.Only Qrr1 and Qrr2,but not Qrr3,Qrr4,and Qrr5,repress the expre ssion of Aph A protein.Besides,all Qrr1-5 molecules can stimulate the expression of both lux O and lux U genes.For the three autoinducers synthesase,Lux M,Lux S,and Cqs A,the influences of Qrr1-5 were different.Qrr1 and Qrr3 can promote the expression of all the three genes,Qrr5 can only positively regulate the expression o f cqs A gene.Qrr2 and Qrr4 play take no effect on the expression of these three genes.Furthermore,Lu x R protein can bind directly to the promoter regions of all five qrr1-5genes in quantity-dependent manner to activate the transcriptions of these five s RNAs.Aph A protein can only bind directly to the promoter regions of qrr2,qrr3,qrr4 genes but not qrr1 and qrr5 genes in quantity-dependent manner to repress th e transcriptions of these three s RNAs.Also there are two Aph A binding domains in both qrr3 and qrr4 genes but only one in qrr2 gene.When the genes of lux O or lux U were depleted,the transcriptional levels of qrr1、qrr2、 qrr3、 qrr4、 qrr5 genes were all down-regulated contrast to th e wild-type strain,and the effects were most obvious on qrr2 and qrr3.The three autoinducer synthesases take different effects on the transcriptional levels of qrr1-5 genes respectively.In further,Lux R repressed the expression of Hfq protein while Aph A had no influence.All the Qrr1-5 molecules can negatively control the expression level of Hfq protein,and the obvious influence for Qrr5 and least for Qrr1.(3)The target genes of Qrr1-5 were predicted by the informatics software,Target RNA2.The results showed that there are various target genes taking part in kinds of ph ysiological processes for all five Qrr molecules in Vibrio alginolyticus.The target genes can encode different proteins,like type III secre tion system related protein,heat shock protein,exopolysaccharide,and so on.Then,the production o f exopolysaccharide in different strains were detected and the results showed that Qrr1 and Qrr3 can repress the exopolysaccharide synthesis,while Qrr2,Qrr4,and Qrr5 can positively regulate the exopolysaccharide synthesis.The results also confirmed the realiabilit y of the informatics prediction.