Identification And Functional Study Of Cell Density-Related SRNA In Vibrio Alginolyticus

Vibrio alginolyticus is a Gram-negative bacterium belonging to the Vibrio genus,which is widely found in lakes and seawater.It can cause ulcer disease in fish and cause death in severe cases.It is an important pathogen in China’s aquaculture industry.In addition,it can also harm human health through the consumption of seafood,causing food safety problems.At present,the known virulence factors mainly include lipopolysaccharide,flagella,biofilm,iron carrier,extracellular protease and outer membrane protein.Now,the prevention and treatment of Vibrio alginolyticus disease is still dominated by antibiotics,but it is easy to produce drug resistance,and there are problems such as drug residues.Therefore,it is urgent to seek a green safe prevention and treatment method.A large number of non-coding sRNAs exist in bacteria,which can be rapidly synthesized and post-transcriptionally affect the stability and translational activity of target mRNA by base pairing.They are widely involved in the physiological process of bacteria.Except for the five kinds of cell density dependent sRNAs,Qrr1-5,no other cell density-related sRNAs have been reported.This study screened and identified the differentially expressed sRNA molecules in Vibrio alginolyticus at different cell densities.Five of the sRNA molecules with the most significant differential expression at different cell densities were selected,named 655,662,665.669,678.Then the deletion mutant strains were constructed using the label-free deletion method,and the corresponding complement strains using the complement plasmid pBAD33 were also generated.In further,its effects on different physiological functions in Vibrio alginolyticus were determinde and its intrinsic molecular regulation mechanism was also revealed at the same time.It provides a theoretical basis for safe and effective green control method development.The main results are as follows:(1)the transcriptome sequencing analysis of Vibrio alginolyticus at different cell densities was carried out by RNA-Seq,and the sRNA molecules underdifferent cell densities in Vibrio alginolyticus were identified by bioinformatic method.The results showed that 146 sRNA molecules were identified in Vibrio alginolyticus.Under high-density growth conditions,82 sRNA molecules were differentially expressed compaired to that under low-density growth conditions,47 sRNA molecules were up-regulated,and 35 sRNA molecules were down-regulated.Five novel sRNAs with the most significant change level,three of which were up-regulated and two down-regulated,were selected as the research objects.They all had the promoter region and terminator region and the presence of them were verified by real-time fluorescent quantitative PCR.Subsequently,MFold was used to predict the secondary structure of five sRNA molecules.The results showed that all five sRNA molecules had a typical stem-loop structure of RNA molecules,a stretch region rich in AU region for Hfq bindign adjacent to the stem-loop.Finally,the Target RNA2 was utilized to predict the target mRNA for each sRNA and the results demonstrated that all the sRNA had various potential targets in Vibrio alginolyticus and involved in different physical processes.(2)five mutant strains were constructed in this experiment with deletion of114 bp,298bp,142 bp,221bp,106 bp in Δ678,Δ665,Δ669,Δ655,Δ662respectively using the marker-free deletion mutant method.The complemented strains were also successfully constructed.Then the growth curve,motility,biofilm formarion,exopolysacchride were assayed to characterized the functions of sRNAs in Vibrio alginolyticus.The results showed that the five cell density-related sRNA molecules had different effects on the growth of Vibrio alginolyticus,among which 655 was the most significant.The loss of 655 resulted in significant inhibition of the growth of Vibrio alginolyticus,followed by 665,678,669,while 662 had relatively weak effects.Five kinds of sRNAs can promote the swimming and swarming ability of Vibrio alginolyticus.After the gene is deleted,its mobility is obviously limited.After the gene is replenished,its mobility ability has a certain recovery,but it has not returned to the wild type level.The regulation of swarming ability mainly depends on the activation of lateral flagellum synthesis proteins LafK and LafA,while the effect of swimming ability mainly depends on the action of polar flagellum synthesis related proteins FliS and FlaK.Among them,669 plays the most significant rolein regulating the motility of Vibrio alginolyticus.They also promoted the formation of biofilm of Vibrio alginolyticus,by the post-transcriptional regulation of the biofilm regulator SypG.Besides,they also had a positive regulatory effect on the synthesis of extracellular polysaccharides of Vibrio alginolyticus,of which 665 has a very significant regulatory effect on the production of extracellular polysaccharides of Vibrio alginolyticus.In addition,these five sRNAs also have different regulatory effects on antibiotic susceptibility of Vibrio alginolyticus,and no effect on environmental stress.(3)the five sRNAs have different regulatory effects on LuxR and AphA,which are the core elements of the quorum sensing system of Vibrio alginolyticus.First,665,662 and 655 have no obvious inhibitory effects on LuxR protein expression,669 and 678 can promote LuxR protein expression by Western blot.The promotion effect was confirmed by RT-PCR.In the strains of Δ655,Δ665and Δ662,the mRNA level of LuxR was up-regulated by 1.17,1.57 and 0.57 times,comparing with wild type,while 669 and 678 had higher mRNA levels of LuxR.In the Δ669 and Δ678 strains,the mRNA level of LuxR was down-regulated by 5.18 and 5.65 times,comparing with the wild type.In the strains of Δ655,Δ665 and Δ662,the mRNA level of AphA was down-regulated compared with wild type.In Δ669 and Δ678 strains,the mRNA level of AphA was up-regulated compared with wild-type,indicating that 669 and 678 have inhibited effects on AphA.At the same time,the five sRNA molecules have a certain positive regulation effect on the upstream elements LuxU and LuxO,that is,the sRNA achieves precise regulation of the target gene through the quorum sensing system.This study will further enrich people’s understanding of the virulence regulation mechanism of Vibrio alginolyticus,and provide a theoretical basis for the development of new control methods of Vibrio alginolyticus-related diseases.