[AphA is an activator of c-di-GMP synthesis and biofilm formation in vibrio parahaemolyticus].

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 (delta aphA) and the wide-type (WT) parent strain. The intracellular levels of c-di-GMP in the delta aphA and WT strains were determined by a chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) method. Total RNAs were extracted from delta aphA and WT. Quantitative RT-PCR was applied to calculate the transcriptional variation of scrABC and scrG between delta aphA 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 delta aphA and WT, respectively, to measure the promoter activity (the beta-Galactosidase activity) of the target genes in AaphA and WT by using the beta-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 e-di-GMP synthesis and biofilm formation by AphA in Vibrio parahaemolyticus.