Analysis Of The Function Of Small RNA T64 In Salmonella Enterica Serover Typhi

Objective: With the development of bioinformatics prediction and molecular biotechnology, more and more sRNAs have been found in many bacteria. As regulatory factors of gene expression, they play an important role in the life activity of bacteria. In this work, we want to study the function of a novel sRNA T64, whose gene location and expression characteristics have been studied previously in Salmonella enterica serovar Typhi(S. Typhi). Methods: 1. The construction of T64-overexpressing strain. The upper and lower primers were designed at the transcriptional start site and stop site respectively based on the sequence information of T64 from previous study. The gene fragment coding T64 was amplified using this pair of primers. After digestion by Nco I and Hind III, the fragment was linked with the vector pBAD/Myc-His A, which was also digested by these two restriction enzymes, and then transformed into E. coli DH5α. After screening, the recombinant vector was extracted from the positive clone and then transformed to the wild type strain of S. Typhi by electroporation to obtain the T64-overexpressing strain. 2. Overexpression of T64 in the hfq deletion mutant. The recombinant vector containing the gene fragment coding T64 was extracted and transformed to the hfq deletion mutant by electroporation. Overexpression of T64 was induced in the hfq deletion mutant. 3. The growth curves of T64-overexpressing strain. The T64-overexpressing strain and the control strain were incubated in TSB with shaking. The OD600 value was measured every hour for 10 hours. To compare the growth of T64-overexpressing strain and the control strain, growth curves were drawn by using growth time as the abscissa and value of OD600 as the ordinate. 4. The motility assay of T64-overexpressing strain. The T64-overexpressing strain and the control strain were incubated to log-phase(OD600 0.4), then 0.2%(w/v) arabinose was added to induce T64 expression for 0.5 h. Each 1 μl of bacterial cultures of the T64-overexpressing strain and the control strain was spotted into semi-solid LB or TSB plates(0.3% agar) and incubated at 37°C for 12 h. Motility was assessed by measuring the diameter of the motility circle. 5. The analysis of transcriptional level of genes by quantitative real-time PCR. Bacteria were incubated before total RNAs were extracted using TRIzol reagent. Specific primers were used for reverse transcription and quantitative real-time PCR to analyze the difference of T64 expression when the wild-type strain was grown in different mediums. The transcriptional level of biofilm-related target genes in T64-overexpressing strain was also measured. 6. Biofilm formation assay. Bacteria were incubated to log-phase(OD600 0.4), then 0.2%(w/v) arabinose was added to induce T64 expression for 0.5 h. Bacterial cultures were transferred to a 96-well microtiter plate. After growing at 30°C for 4 d without shaking, adherent biofilm was stained with crystal violet. The OD570 was measured to quantify biofilm formation. 7. Congo Red assay. Overnight cultures of the T64-overexpressing strain and the control strain were pelleted by centrifugation. Then the bacteria were resuspended with PBS and adjusted to the same amount. 5 μl of bacterial cell suspensions were spread on Congo Red-agar plates. After incubated at 28°C for 3 d, the colony morphology was observed. Results: 1. By the analysis of PCR, restriction digestion and sequencing, the construction of the recombinant vector pBAD-T64 was confirmed. The recombinant vector was transferred into the wild-type strain of S. Typhi. So the T64-overexpressing strain WT+pBAD-T64 was successfully constructed. 2. The results of PCR showed that the recombinant vector pBAD-T64 was transformed into the hfq deletion mutant successfully to produce the strain Δhfq+pBAD-T64. 3. The results of growth curves indicated that the T64-overexpressing strain grew faster than the control strain markedly during a certain period of time. 4. The result of motility assay showed that the motility of T64-overexpressing strain decreased a little in comparison to the control strain when they were incubated in semi-solid TSB plates, while there was no significant difference in motility between them when incubated in semi-solid LB plates. 5. The result of quantitative real-time PCR indicated that T64 expression by the wild-type strain incubated in TSB differed from that in LB broth. The T64 expression increased obviously when bacteria were incubated in TSB. And the transcriptional level of biofilm-related target genes fimY, pilP, stcB and stgD were up-regulated in T64-overexpressing strain. 6. The result of biofilm formation assay showed that the T64-overexpression strain had an increase in biofilm formation compared with the control strain. After the T64 gene was complemented, the T64 deletion mutant restored its ability of biofilm formation, while the hfq deletion mutant still couldn’t form biofilms. 7. The result of Congo Red assay revealed that both the T64-overexpressing strain and the control strain produced a smooth and white colony, and the colony morphology corresponding to biofilm formation was not shown. Conclusions: The expression of s RNA T64 in S. Typhi is affected by the growth conditions. After overexpression of T64, the growth of S. Typhi is promoted and the motility is repressed in some degree. T64 may be beneficial to biofilm formation in S. Typhi, but it’s not adequate to erase the repression of biofilm formation by hfq deletion.