Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea
Fig 1
Schematic of inactivation and complementation of luxS.
(A) Inactivation of luxS by using an ermFermAM cassette. The sequences that flanked the 5′- and 3′- ends of luxS (Coch_1216) were amplified with primers Capno1 and 2, and primers Capno3 and 4, respectively. The ermFermAM cassette was inserted between the amplified fragments and cloned. The plasmid was linearized and introduced into C. ochracea ATCC 27872 by electroporation. The resultant luxS∷ermFermAM strain was named TmAI2. (B) Inactivation of luxS by using tetQ. The tetQ fragment was inserted between the sequences that flanked the 5′- and 3′- ends of luxS by using the PCR-based overlap extension method and self-ligattion of the fragment, and then amplified in E. coli. The resultant plasmid was linearized and introduced into C. ochracea ATCC 27872 by electroporation. The resultant luxS∷tetQ strain was named LKT7. (C) Complementation of luxS. An ermF-luxS fragment was constructed by using the PCR-based overlap extension method to express both genes under control of the ermF promoter, and the fragment was cloned into the center of the tetQ gene in pAITQ. The resultant plasmid was linearized and introduced into C. ochracea LKT7 by electroporation. The resultant tet∷ermF-luxS strain was named luxS-C3.