[Establishment of a method for gene complementation in Vibrio parahaemolyticus].
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.


CONCLUSIONS
A method has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.
Nanlou Department of Respiratory Diseases, General Hospital of PLA, Beijing 100853, China. E-mail: zhenhong_chen@hotmail.com.
Zhenhong
Z
Chen
Li
L
Wang
Yiquan
Y
Zhang
Jiao
J
Feng
Ruifu
R
Yang
De
D
Chang
Li
L
An
Changting
C
Liu
Dongsheng
D
Zhou
D001426:Bacterial Proteins / Q000235:genetics; D015870:Gene Expression; D005816:Genetic Complementation Test / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D014736:Vibrio parahaemolyticus / Q000235:genetics*
D001426:Bacterial Proteins