Strategy for deleting adeL-adeFGH and adeIJK operons in MDR A. baumannii DB and R2. Panel A, The upstream (UP) and downstream (DOWN) regions (approximately 1 kb) flanking the target genes was cloned into the suicide vector, pMo130-TelR. pMo130-TelR was constructed by inserting a 3.26 kb XmaI-digested tellurite-resistance cassette from pwFRT-TelR into the XmaI site of pMo130. Recombinants obtained after first cross-over were selected for inheritance of tellurite-resistance and xylE+ (yellow colonies). These recombinants also do not produce any amplimers with the primer pair pMo130Tel F and pMo130Tel R. During the second cross-over, mutants with gene deletion (1) were selected for loss of sacB by passaging the first cross-over recombinants in media containing sucrose. The second cross-over could also yield parental genotype (2). Deletion of the adeFGH operon (Panel B) and the adeIJK operon (Panel C) showing the positions of the respective UP and DOWN fragments flanking each deletion (striped and hatched boxes, respectively). The locations of the PCR primers used for amplifying the UP and DOWN fragments and for qRT-PCR analysis of gene expression are indicated by black arrows while P1, P2 and P3 (grey arrows) are the locations of predicted promoters for adeFGH operon, adeL, and adeIJK operon, respectively.
Amin et al. BMC Microbiology 2013 13:158 doi:10.1186/1471-2180-13-158