| Objective: Small RNA SbfR is a non-coding RNA of Salmonella enterica serovar Typhi(S.Typhi)which was first found by our research group through the use of Solexa high throughput sequencing technology.According to the results of the molecular structure,SbfR is likely a trans-coding RNA.This paper aims at preliminarily exploring the regulatory effect and mechanism of SbfR in S.Typhi and to laying the foundation for subsequent research into the target genes of SbfR and its mechanism of action.Methods: 1.Analysis of the transcriptome of S.Typhi: The SbfR deletion mutant(?SbfR+p BAD/Myc-His A)and the SbfR complementary strain(?SbfR+p BAD/Myc-His A-SbfR)were cultivated to log-phase(OD600?0.4),with the addition of L-arabinose to induce the expression of SbfR and total RNA extracted.Fluorescein was added to the total RNA and reverse transcribed.The labelled c DNAs were hybridized with the genomic microarray.The scanning signals of fluorescence were digitized to analyze the gene expression difference between the SbfR deletion mutant and the SbfR complementary strain.In addition,part of the differential expression genes were chosen to do q RT-PCR experiment to verify the results.2.q RT-PCR analysis of gene expression: Total RNAs of bacteria were extracted for reverse transcription and q RT-PCR experiments carried out to detect gene m RNA levels.3.The growth curves of bacteria under different temperature: The SbfR deletion mutant and the SbfR complementary strain were both cultivated at 37°C and 42°C,and the OD600 measured hourly.The growth curves were determined with the OD600 value as Y-axis and the time as X-axis.4.The construction of different SbfR part complementary strain: The plassmid carrying the former 69 nt or later 69 nt of SbfR were constructed using p BAD/Myc-His A.Then the recombinant plasmid were cloned into ?SbfR to construct ?SbfR-p BAD-F69 and ?SbfR-p BAD-L69 respectively.5.The motility assay: The empty vector strain(WT+p BAD),the SbfR deletion mutant,the SbfR complementary strain and the SbfR partly complementary strain were cultivated to log-phase and were stab inoculated through a LB motility-swim agar plate at 37°C overnight.After that,the diameter of the circular zones were measured to evaluate the motility.6.Western-blot to detect the Flj B level: The empty vector strain,the SbfR deletion mutant and the SbfR complementary strain were cultivated to log-phase and total protein were collected.Western-blot analysis using rabbit anti-Flj B antiserum was done to detcet the Flj B level.7.The He La cells invasion assay: The empty vector strain,the SbfR deletion mutant and the SbfR complementary strain were cultivated to log-phase and incubated with He La cells for 90 min.Then we discarded the culture medium.Half of the cell holes were added with Triton,then they were coated on the plates to growth.We counted the clump counts for adhesion level;Half of the cells were cultured for another 90 min with gentamicin,then they were added with Triton,they were coated on the plates to growth.We counted the clump counts for invasion level.8.Analysis of biofilm formation: The empty vector strain,the SbfR deletion mutant,the SbfR complementary strain and the SbfR partly complementary strain were cultivated to log-phase in TSB medium and transferred into 96-well plate for another 4 days incubation under 30°C.After that,the medium was discarded and the biofilm were stained with crystal violet.30% glacial acetic acid was used to dissolve the crystal violet and the amount of biofilm formation was quantified at 595 nm.Results: 1.According to gene chip assay results,there were 99 differentially expressed genes between the SbfR deletion mutant and the SbfR complementary strain.The results of q RT-PCR were the same as gene chip.2.The the growth curve results showed that the growing ability of the SbfR deletion mutant were the same as the SbfR complementary strain under 37°C,however,the SbfR complementary strain grew slower than the SbfR deletion mutant at 42°C.3.?SbfR+p BAD-F69 and ?SbfR+p BAD-L69 were constructed successfully.4.Compared to the empty vector strain,the motility of the SbfR deletion mutant was declining,however,the SbfR complementary strain recovered.In addition,the motilities of the two SbfR partly complementary strains recovered to the levels of the empty vector strain and the SbfR complementary strain.5.Compared to the empty vector strain,the Flj B expression level of the SbfR deletion mutant declined while the SbfR complementary strain recovered.6.Compared with the empty vector strain,the ability of He La cells invasion of the SbfR deletion mutant declined while the SbfR complementary strain recovered.7.Compared to the empty vector strain,the biofilm formation ability of the SbfR deletion mutant declined while the SbfR complementary strain recovered.In addition,the biofilm formation ability of the two SbfR partly complementary strains recovered to the levels of the empty vector strain,but was weaker than the SbfR complementary strain.8.Compared to the SbfR complementary strain,the transcription levels of hsl S,hsl T,htp G,fli A,flj B and sop E were lower in the SbfR deletion mutant;the transcription level of flh D was higher;the transcription levels of prg J?fim A?csg A?yih P?rfa D?bcs A?wca A and bap A were the same.Conclusins: The sRNA SbfR is involved in the regulation of S.Typhi growth under heat stress,motility,invasion and bioflim formation.It inhibits growth under heat stress and promotes motility,invasion and bioflim formation.Both the SbfR former 69 nt and later 69 nt complementary strains can accomplish the promotion of motility like the SbfR complementary strain,however they only partly play a role in biofilm formation. |
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