[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.