Transcriptional regulation of vp1667 by H-NS in Vibrio parahaemolyticus
10.7644/j.issn.1674-9960.2017.06.008
- VernacularTitle:拟核结合蛋白H-NS调控副溶血弧菌vp1667的转录
- Author:
Minghua ZHAN
;
Wei ZHANG
;
Dongsheng ZHOU
;
Xinxiang HUANG
;
Huiying YANG
;
Zhe YIN
;
Yiquan ZHANG
- Keywords:
Vibrio parahaemolyticus;
H-NS;
vp1667;
transcriptional regulation
- From:
Military Medical Sciences
2017;41(6):445-448
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the transcriptional regulation of vp1667 by H-NS in Vibrio parahaemolyticus.Methods Total RNAs were extracted from Δhns and WT strains.Quantitative RT-PCR was carried out to calculate the transcriptional variation of vp1667 between Δhns and WT.Primer extension assay was also employed to detect the transcription start site and the promoter activity (i.e.the amount of primer extension products) of vp1667 in Δhns and that in WT.The promoter DNA region of vp1667 was amplified, purified, and cloned into the corresponding restriction endonuclease sites of pHRP309 that harbors a gentamicin resistance gene and a promoterless lacZ reporter gene.The recombinant pHRP309 plasmid was transformed into Δhns and WT, respectively, while β-galactosidase activity in cellular extracts was measured using a β-galactosidase enzyme assay system.The over-expressed His-H-NS was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns.The electrophoretic mobility shift assay (EMSA) and DNaseⅠ footprinting were then applied to analyze the DNA-binding activity of His-H-NS to vp1667 promoter region in vitro.Results and Conclusion The primer extension assay detected one transcription start site for vp1667, which was located at 28 bp upstream of vp1667, and its transcribed activity was under the negative control of the H-NS.The EMSA and DNaseⅠ footprinting assay results showed that His-H-NS was unable to bind to the promoter-proximal DNA region of vp1667, suggesting that H-NS indirectly inhibits the transcription of vp1667.
