Transcriptional regulation of hcp1 by H-NS in Vibrio parahaemolyticus

Abstract:

[Objective] To study the transcriptional regulation of the structural components Hcp1 by H-NS in Vibrio parahaemolyticus.[Methods] Expression of Hcp1 in the wide-type(WT) strain and hns mutant(Δhns) were detected by Western blot using rabbit anti-Hcp1 polyclonal antibodies. Total RNAs were extracted from WT and Δhns strains. Quantitative RT-PCR was carried out to calculate the transcriptional variation of hcp1 between WT and Δhns strains, and then primer extension assay was used to detect the transcription start site and the promoter activity(the amount of primer extension product) of hcp1 in WT and Δhns. The entire promoter region of hcp1 was amplified by PCR with ExTaq^TM DNA polymerase using WT genomic DNA as the template. The over-expressed His-H-NS was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns(Amersham). Electrophoretic mobility shift assay(EMSA) was applied to analyze the DNA-binding activity of His-H-NS to hcp1 promoter region in vitro.[Results] Western blot and quantitative RT-PCR results showed that expression of Hcp1 was inhibited by H-NS. The primer extension assay detected only one transcription start site located at 62 bp upstream of hcp1, whose transcription was H-NS and σ⁵⁴-dependent. EMSA result indicated that His-H-NS was able to bind the promoter DNA region of hcp1.[Conclusion] The expression of hcp1 was directly repressed by H-NS in V. parahaemolyticus.