Small RNA RybB and chaperone protein Hfq of Salmonella Typhimurium regulate expression of porin OmpD

Abstract:

[Objective] To investigate the role of the small RNA (sRNA) RybB and the chaperone protein Hfq in regulating the expression of porin OmpD in Salmonella. [Methods] In this study, Salmonella Typhimurium (STM) was used as the research object. The pCE40 plasmid carrying the reporter gene lacZ encoding β-galactosidase was transferred into the single mutant lacking ompD to obtain the lacZ reporter strain. On this basis, we employed P22 phage-mediated transduction to construct the double mutants lacking full-length rybB, full-length hfq, partial sequence of hfq, or truncated hfq sequence and the triple mutantlacking full-length rybB and full-length hfq. The regulatory effects of RybB and Hfq on the expression of OmpD were probed by β-galactosidase activity assay and RT-qPCR. [Results] We successfully constructed the double and the triple mutant. Compared with that in the wild type (WT), the OmpD activity was down-regulated by 2.16% in the lacZ reporter strain with truncated sequence (87 residues) of hfq, and the β-galactosidase activity of OmpD increased in the rest strains. Compared with WT, except for STM LT2∆ompD::lacZ∆hfq6, all the mutants showed up-regulated transcript level of ompD (P<0.05), with the most significant up-regulation of 1.83-folds in the triple mutant. [Conclusion] The transcription and translation of ompD are mainly regulated by the negative feedback of hfq and RybB. The distal end of Hfq plays a key role in the transcriptional repression of ompD. By construction of several mutants, this article illustrated the interactions of RybB and Hfq with OmpD and explored the key regions of Hfq in regulating ompD, which enriched the theory of sRNA regulation.