Bioenergetics and the Role of Soluble Cytochromes c for Alkaline Adaptation in Gram-Negative Alkaliphilic Pseudomonas

Figure 6

Scheme of producing unmarked cytochrome c-552-deficient mutant of P. alcaliphila AL15- by homologous recombination. The mutant fragment generated by overlap extension PCR is cloned into the pDONR221 and then recombined into the pEX18ApGW plasmid by gateway-recombinational cloning. The resulting suicide vector is transferred to P. alcaliphila AL15-. The plasmid-borne deletion mutation is exchanged with the corresponding part in the chromosome. The merodiploid state is resolved by sacB-mediated sucrose counterselection in the presence of gentamycin. Finally, the gentamycin resistance marker (Gm^R) is deleted by Flp/FRF homologues DNA recombination. sacB: Bacillus subtilis levansucrase-encoding gene, Cd: carbenicillin resistance marker.