Abstract: Objective To establish a method for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.
Methods The 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. Results opaR and aphA were transcribed in the corresponding
complemented mutant strains, and their mRNA levels were comparable to those detected in the wild-type strains. Conclusion
Amethod has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.