Transcriptional regulation of type VI secretion system 1 genes by QsvR in Vibrio parahaemolyticus

Abstract:

[Objective] To study the transcriptional regulation of type VI secretion system 1 (T6SS1) genes by QsvR in Vibrio parahaemolyticus. [Methods] Total RNA was extracted from the wild type (WT) and qsvR mutant (ΔqsvR). Quantitative real-time PCR (qPCR) was employed to investigate the transcriptional regulation of target genes by QsvR. Primer extension was carried out to detect the transcription initiation site and core promoter for each target gene and calculate the transcriptional variations between WT and ΔqsvR. The regulatory DNA region of each target gene was cloned into the restriction endonuclease sites of pHRP309 harboring a promoterless gene lacZ, and then each recombinant plasmid was transferred into WT and ΔqsvR, respectively. A β-Galactosidase Enzyme Assay System (Promega) was used to measure the β-galactosidase activity in cell lysates. The recombinant pHRP309 vector containing the regulatory DNA region of one of the target gene was transferred into Escherichia coli 100λpir harboring an empty pBAD33 or pBAD33-qsvR to test whether QsvR can regulate the target genes in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and His-QsvR was over-expressed and then purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Electrophoretic mobility shift assay (EMSA) was employed to determine the DNA-binding activity of His-QsvR to each target DNA fragment in vitro. [Results] The mRNA levels of T6SS1-associated genes, VP1388 (the first gene of VP1388-1390 operon) and hcp1 (the first gene of VP1393-1406 operon), were significantly up-regulated in ΔqsvR relative to those in WT, indicating that QsvR activated the transcription of VP1388 and hcp1. Only one transcription initiation site was detected for VP1388 or hcp1, locating at 64 bp upstream of VP1388 and 62 bp upstream of hcp1, respectively, and their transcriptional activities were all repressed by QsvR. QsvR repressed the promoter activities of VP1388 and hcp1 in both V. parahaemolyticus and E. coli 100λpir. His-QsvR was able to bind to the regulatory DNA regions of VP1388 and hcp1. [Conclusion] QsvR directly repressed the transcription of T6SS1-associated operons, VP1388-1390 and VP1393-1406, in V. parahaemolyticus.