Regulation Of CpsQ And MfpABC By Quorum Sensing System Core Regulators OpaR And AphA In Vibrio Parahaemolyticus

Background Vibrio parahaemolyticus a foodborne bacterium whichcauses gastrointestinal illness in human beings, has a strongbiofilm-forming ability, the process of which is mainly regulated by the QSsystem. OpaR and AphA are two core regulators of the QS system of Vibrioparahaemolyticus, which regulate biofilm formation at different bacterialdensities.The cpsQ and mfp genes, whose locus are adjacent to each other,are related to cell surface characteristics. Both genes are regulated bymultiple regulators, e.g.the transcription of cpsQ is regulated by CpsR andCpsQ itself. Whether there is a direct regulation of cpsQ and mfp by OpaRand AphA needs to be verified.Objective To study the regulation mechanism of the QS system coreregulators OpaR and the AphA on cpsQ and mfpA genes in Vibrioparahaemolyticus.Methods The Vibrio parahaemolyticus mutants of opaR of aphA wereconstructed using the suicide plasmid m. Total RNA of the wild-type strainand the mutants were extracted, which was applied to primer extensionassay to analyse the transcription start sites and relative abundance of targetgenes. The promoter regions of cpsQ and mfpA were cloned and ligated to the upstream of the beta-galactosidase gene of pHRB309, and thebeta-galactosidase activities were tested to further confirm the regulation ofregulators on target genes. The DNA promoter regions of cpsQ and mfpAwere amplified and the fusion protein opaR of aphA were expressed, andElectrophoretic Mobility Shift Assay (EMSA) and DNase I footprintingwere conducted to study the direct interaction between regulator proteinsand target genes.Results AphA was expressed at low cell density while opaR wasexpressed at high cell density, and the expression of both genes wasdensity-dependent. At low cell density, the mfpA transcription was inhibitedby AphA directly, and AphA was able to bind the promoter-proximalregions of mfpA. The cpsQ transcription was also repressed by AphA, butin an indirect way. At high cell density, both mfpA and cpsQ transcriptionwas induced by OpaR, with the same manners as by AphA in low celldensity.Conclusion AphA and OpaR, both in a direct manner, positively andnegatively regulated mfpABC, respectively. In contrast, the regulation ofcpsQ-mfpABC by AphA and OpaR achieved most likely in an indirectmanner, since no binding of both AphA and OpaR was detected.