| Vibrio alginolyticus is one of the most common pathogens in the ocean.It not only causes severe vibriosis in marine aquatic animals,but also infects humans who contact with or eat contaminated water or seafood and causes human soft tissue infections,sepsis,and other extraintestinal infections.Bacterial virulence is regulated by environmental factors(such as cell density,temperature,and salinity,etc.),and related virulence regulation systems including biofilm formation,and motility is closely related to the bacterial secretion system and quorum sensing system.The ability of bacteria to resist environmental stress and biofilm formation are the key elements for pathogenic bacteria to exert their virulence,and the regulation of the expression of related genes is crucial.Gene expression is strictly regulated by the sigma factors.The sigma factor combines with RNA polymerase(RNAP)to form RNAP holoenzyme to initiate the transcription of specific genes.rpoS and rpoN are two major members of the sigma family.Research has found that rpoS is the main regulator in the stable phase and may participate in the regulatory network of the quorum sensing system;rpoN is located upstream of the type Ⅵ secretion system(T6SS)gene cluster.Studies have confirmed that the deletion of rpoN affects the function of T6 SS.The regulatory role of rpoS and rpoN in the quorum sensing system and secretion system suggests that they have certain functions in responding to environmental stress and regulating the formation of biofilms.So far,the mechanism by which Vibrio alginolyticus responds to environmental stress and regulates biofilm formation is still unclear.In this study,a pathogenic Vibrio alginolyticus strain HN08155 was used as the research object.rpoS/rpoN single-deletion strain(△rpoS/△ rpoN),rpoS and rpoN double-deletion strain(△ rpoS △ rpoN),as well as the corresponding complement strains,and overexpressed strain were obtained by gene knockout and complement technology.The phenotypic studies of important environmental stress factors and biofilms were carried out on these strains,and the transcriptome sequencing was used to reveal rpoS/rpoN related genes that regulate Vibrio alginolyticus in response to environmental stress,biofilm formation,and possible regulatory mechanisms.This study will lay the foundation for the treatment,prevention,and control of Vibrio alginolyticus diseases during aquaculture.The main results are as follows:(1)Study on the growth characteristics of different strains of Vibrio alginolyticus HN08155 under different salinity conditions.The experimental results show that the wild type strain(WT)is under low salt stress with 10‰ salinity,and under high salt stress with50‰ salinity.Under salinity stress,the growth of △rpoS and △rpoN was significantly reduced compared with WT.The deletion of rpoS and rpoN genes seriously affected the growth of strains under salinity stress.Compared with WT,the growth of overexpression strains WT-p RpoS and WT-p RpoN is still significantly reduced.The above results indicate that the rpoS and rpoN genes play an important role in the resistance of Vibrio alginolyticus to salt stress,both up-regulation and down-regulation of their expression will disable the bacteria’s ability to resist the salinity stress.(2)Determination of polysaccharide content during the growth of different strains of Vibrio alginolyticus HN08155.Under low-salt stress,the polysaccharide content of rpoNdeficient strains was significantly higher than that of WT and the strains aggregated into clusters,while there was no significant difference in the polysaccharide content of the strains under high-salt stress and no strains aggregated into clusters.The results show that Vibrio alginolyticus may use different mechanisms to resist low-salt stress and high-salt stress,and the secretion of polysaccharides may play an important role in the resistance to low-salt stress.(3)Study on the growth characteristics of different strains of Vibrio alginolyticus HN08155 under different temperature conditions.The growth of WT slows down at 37-42°C and is under high temperature stress.Under temperature stress,compared with WT,the growth of △rpoS and △rpoN was further reduced significantly.After supplementing with rpoS and rpoN,the growth of the strain could be promoted.However,overexpression of rpoS and rpoN(WT-p RpoN and WT-p RpoS)in wild strains can also result in a significant reduction in growth.The results show that Vibrio alginolyticus rpoS/rpoN participates in the process of bacteria resisting temperature stress,and there may be feedback inhibition in its regulation process.(4)Determination of biofilm content during the growth of different strains of Vibrio alginolyticus HN08155.During the growth period of the strain,the content of WT biofilm first increased and then decreased,similar to the process of biofilm formation and dissolution during bacterial growth in other related studies.Compared with WT,the formation of biofilm in △rpoN was significantly delayed in the early stage of growth,and the biofilm did not dissolve in the later stage.By measuring the static growth curve of different strains of Vibrio alginolyticus within 24 hours,the growth of the △rpoN strain lags in the early stage,which may be one of the reasons for the decrease of the biofilm content in the early stage.Compared with WT,the formation rate of △rpoS biofilm was faster in the early stage,and the biofilm did not dissolve in the later stage.The above experiments show that the rpoS and rpoN genes have different regulatory effects on the formation of biofilms in the early growth stage of Vibrio alginolyticus HN08155.Moreover,the deletion of rpoS and rpoN genes caused the biofilm of Vibrio alginolyticus HN08155 to be unable to dissolve in the late growth period.(5)Transcriptome data analysis of △rpoS and △rpoN strains.In order to study the regulation of rpoS and rpoN genes to their downstream genes,we used transcriptomics technology to analyze the differential gene expression between knockout strains of Vibrio alginolyticus HN08155 rpoS or rpoN and wild type strain.From the analysis results,it can be concluded that in the absence of rpoS/rpoN,some known stress-related genes have expression differences(transcription up-regulation/down-regulation),including usp family general stress genes,Csp A family cold shock related genes,and trk H membrane signal sensing genes,etc.On the other hand,it verified the role of rpoS and rpoN genes of Vibrio alginolyticus HN08155 in response to environmental stress regulation.However,the diversity of phenotypes and gene expression differences indicates that the environmental stress regulatory mechanism is a diverse and complex regulatory network,in which rpoS and rpoN play different roles and functions.At the same time,in the late growth stage of △rpoN,the expression of flagella-related genes and the main regulator of quorum sensing hap R decreased significantly,while the expression of genes related to polysaccharide synthesis and secretion increased.The expression changes of these genes may cause the △rpoN biofilm to be unable to degrade in the late growth period.However,the transcriptome data of the △rpoS strain did not show differential expression of known biofilm-related genes,and the effect of rpoS on the biofilm may be regulated through currently unknown pathways.In summary,this article confirms that the rpoS and rpoN genes of Vibrio alginolyticus can respond to environmental stress and regulate biofilm growth in Vibrio alginolyticus HN08155,and they have different regulatory pathways,but part of the expression regulation of rpoS is still required participation of rpoN. |
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