| Objective:The transformation of bacteria from planktonic state to biofilm state is an effective strategy for bacteria to response to changes in the external environment.The biofilm formation is regulated by many factors,and s RNAs play an important role in it.Salmonella enterica serovar Typhi(S.Typhi)is an important human intestinal pathogen,also an important prokaryotic model for studying the gene expression and regulation.In our research group,RNA-seq technology was used to perform transcriptome assay on S.Typhi wild strains in plankton and biofilm states,and found multiple s RNAs that were differentially expressed in the two groups.One of them assumed that the expression abundance of S305 in plankton was significantly higher than that of biofilm bacteria.In this study,we focus on S305 and aim to explore its expression characteristics in different periods,and its role and mechanism in biofilm formation and motility of S.Typhi.Methods:1.Molecular identification:(1)Expression conformation: Design specific probes and use northern blot to detect the expression of S305 in S.Typhi,to verify its existence.(2)Full-length identification: The 5' RACE and 3' RACE were used to detect the transcription start site and transcription termination site of S305,and the gene position and sequence length were determined.2.Expression analysis at different stages: qRT-PCR and northern blot were used to detect the expression of S305 in different growth stages of S.Typhi(lag phase,mid-exponential growth phase,the late-exponential growth phase and stationary phase).3.Construction of high-expressing strains: Design specific primers based on the full-length sequence,the S305 high-expression vector was constructed by using pBAD / His A,then transfer positive recombinant plasmids and empty plasmids into S.Typhi wild strain to prepare high-expressing strain WT-pS305 and empty plasmid strain WT-pBAD.4.Phenotypic analysis of each strain: According to the test requirements,WT-pS305 and WT-pBAD strains were cultured to the corresponding conditions,and then performed biofilm formation tests(96-well microplate crystal violet staining method),bacterial swimming test and growth Curve determination.5.Analysis of mechanism of action:(1)qRT-PCR was used to analyze the mRNA levels of genes related to flagella and biofilm formation in WT-pS305 and WT-pBAD strains.(2)Extract the total RNA of WT-pS305 and WT-pBAD at 0 min,4 min,8 min,16 min,32 min after rifampicin treatment,qRT-PCR and northern blot were used to detect the mRNA of csgD and flj B in each group,and then calculate the half-life of the corresponding mRNA.(3)High expression vectors with different fragment lengths of s RNA S305 were constructed by using pBAD/His A plasmid,and transferred electrically into S.Typhi wild strains to prepare high expression strains WT-pS305-60,WT-pS305-120,WT-pS305-180,WT-pS305-240,WT-pS305-300,WT-pS305-360,WT-pS305-420;then qRT-PCR was used to detect the mRNA level of flj B and csgD in each strain.6.Prediction of other target genes: RNAfold website was used to predict the potential target genes of S305 in S.Typhi,and then use qRT-PCR to detect the mRNA levels of some predicted target genes.Results:1.Northern blot results confirmed the existence of S305 and found that it may be derived from the 3' UTR of the repressor protein encoding gene c I;5' RACE results showed that the 5' end of S305 was located 99 nt upstream of the c I stop codon,and 3' RACE results showed The 3' end of S305 is located 396 nt downstream of the c I stop codon.2.The results of northern blot showed that the expression level of S305 showed a waveform change with the growth period,the expression abundance was higher in the mid-exponential growth phase and the stationary phase,and the expression abundance was lower in the lag phase and the late-exponential growth phase;full-length transcripts gradually decreased with growth.3.Phenotypic experiments showed that the biofilm formation and swimming ability of WT-pS305 were significantly lower than WT-pBAD;under normal conditions,the growth rate of WT-pS305 was significantly slower than that of WT-pBAD after entering the lag phase.4.qRT-PCR results showed that the mRNA levels of flj B and csgD in WT-pS305 were significantly lower than those of WT-pBAD,and the mRNA levels of csg A and bcs A decreased slightly compared with WT-pBAD.The half-life test found that the degradation rate of the flj B mRNA in WT-pS305 was significantly faster than WT-pBAD.The half-life of flj B mRNA in WT-pBAD was shortened to about 25 minutes and shortened to about 15 minutes in WT-pS305.5.High expression strains WT-pS305-q60,WT-pS305-q120,WT-pS305-q180,WT-pS305-q240,WT-pS305-q300,WT-pS305-q360,WT-pS305-q420 were successfully constructed.And qRT-PCR results showed that compared with WT-pBAD,the mRNA level of flj B in WT-pS305-q420 decreased significantly,while there was no significant difference in other high-expression strains;compared with WT-pBAD,there was no significant difference in the mRNA levels of csgD in those from WT-pS305-q60 to WT-pS305-q420.6.A variety of S305 potential target genes were found in S.Typhi,functional analysis showed that these genes were involved in bacterial metabolism,substance synthesis,etc.qRT-PCR verification results showed that the expression levels of genes mtg A and glpT in WT-pS305 were significantly lower than those of WT-pBAD.Conclusion:This study found that the small RNA S305 is derived from the 3' UTR of the repressor protein coding gene c I,with a full length of 495 nt and partially overlapping with the c I sequence.The expression level of S305 is different in each growth period of S.Typhi wild strains,with higher expression abundance in the mid-log phase and plateau phase,and lower expression abundance in the slow phase and late log phase;high expression of S305 can inhibit biofilm formation,motility and growth rate of S.Typhi;the high expression of S305 negatively regulates the expression of flj B by affecting the stability of flj B mRNA,while negatively regulating the expression of csgD,mtg A and glpT. |
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