AphA is an activator of c-di-GMP synthesis and biofilm formation in Vibrio parahaemolyticus

Abstract:

Abstract: ［Objective］ To study the regulation mechanism of biofilm formation c-di-GMP synthesisby AphA in Vibrio parahaemolyticus，by using phenotypic and molecular biochemical experiments．［Methods］ Colony morphology and crystal violet staining assays were used to analyze the phenotypic changes between the aphA null mutant (ΔaphA) and the wide-type (WT) parent strain． The intracellular levels of c-di-GMP in the ΔaphA and WT strains were determined by a chromatography-coupled tandem mass spectrometry (HPLC-MS /MS) method． Total ＲNAs were extracted from ΔaphA and WT．Quantitative ＲT-PCＲ was applied to calculate the transcriptional variation of scrABC and scrG between ΔaphA and WT．The promoter-proximal regions of scrABC and scrG were cloned into the pHＲP309 vector containing a promoterless lacZ gene，respectively．Then，each of the two recombinant LacZ reporter plasmids was transformed into ΔaphA and WT，respectively，to measure the promoter activity (the β-Galactosidase activity) of the target genes in ΔaphA and WT by using the β-Galactosidase Enzyme Assay System．The over-expressed His-AphA was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham)．Then，the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-AphA to scrABC and scrG promoter regions in vitro.［Ｒesults］ The phenotypic experiments disclosed that AphA was an activator of c-di-GMP synthesis and biofilm formation in Vibrio parahaemolyticus．The quantitative ＲT-PCＲ and LacZ fusion results showed that the transcription of scrABC and scrG was under negative control of AphA．However，the purified His-AphA could not bind to the upstream DNA regions of scrABC and scrG，as determined by EMSA．［Conclusion］The fact that AphA represses the transcription of scrABC and scrG will at least partially account for the positive regulation of c-di-GMP synthesis and biofilm formation by AphA in Vibrio parahaemolyticus．