OBJECTIVETo establish a method for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.

METHODSThe entire coding region of opaR or aphA was amplified by PCR and cloned into pBAD33. The recombinant plasmid was transformed into δopaR and δaphA (the opaR or aphA null mutant strain, respectively) separately to construct the complemented mutant strain C-δaphA and C-δopaR, respectively. RT-PCR was used to verify the transcription of opaR and aphA in the corresponding complemented mutant strains. Primer extension experiments were performed to determine the relative mRNA levels of mfpA (a gene previously characterized to be negatively regulated by AphA and positively by OpaR) in the wild-type strain, δopaR, δaphA, C-δaphA, and C-δopaR.

RESULTSopaR and aphA were transcribed in the corresponding complemented mutant strains, and their mRNA levels were comparable to those detected in the wild-type strains.

CONCLUSIONA method has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.