A. baumannii R2 and DB harboring the inserted pMo130-TelR-adeFGH (Up/Down) construct was cultured in LB broth containing 10% sucrose and passaged daily to select for deletion of adeFGH operon and loss
of the sacB gene by a second cross-over and allelic replacement. Such bacteria, which were white when sprayed with 0.45 M pyrocathechol and were susceptible to 30 mg/L tellurite, usually selleck chemicals appeared after the second passage. If the desired gene deletion had occurred, PCR of genomic DNA from these bacteria would produce only a 2 kb amplimer with the primer pair AdeGUp(Not1)F and AdeGDwn(Sph1)R. Selleckchem LEE011 The same genomic DNA would not give any amplimer using the primer pair: AdeG RTF and AdeG RTR which annealed to the DNA that has been deleted (Figure 1B). The suicide plasmid for deleting the adeIJK operon was constructed as described above but by first ligating the 1 kb UP fragment and a 0.9 kb DOWN fragment flanking the deletion before inserting into the pMo130-TelR vector (Figure 1C). The UP and DOWN fragments were amplified from R2 genomic DNA using the primer pairs, AdeJ(UP) PstI F and AdeJ(UP)BamHI R, and AdeJ(DWN)BamHI F and AdeJ(DWN)SphI R, respectively (Figure 1C and Additional file 1: Table S1). The UP and DOWN fragments were digested with BamHI and
ligated together in a 1:1 ratio. The ligated product was amplified using AdeJ(UP) PstI and AdeJ(DWN)SphI R to give a 1.9 kb amplimer which was then digested with PstI Abiraterone nmr and SphI and ligated with pMo130-TelR linearized with PstI and SphI to give pMo130-TelR-adeJ(Up/Down). The plasmid see more construct was introduced into E. coli S17-1 and used for the two-step selection for deletion of the adeIJK operon as described above. Verification of genomic deletions Genomic deletions of the adeFGH and adeIJK operons in the mutants were verified by comparing the PCR amplimers obtained from the parental isolates and corresponding pump gene deletion mutants. For the pump gene deletions, PCR using primers flanking the deletion produced a 2-kb amplimer corresponding to
the UP and DOWN fragments (Figure 2, lanes 3, 7, 11, 15, 17, 19, 21 and 23) while a larger wild-type amplimer was obtained using genomic DNA from the parental isolates, R2 and DB (Figure 2, lanes 1, 5, 9 and 13). For the ΔadeFGH constructs, the deletion was also confirmed using PCR primers that annealed to the deleted region in adeG, whereby a 474 bp amplimer was obtained using genomic DNA from parental isolates (Figure 2, lanes 2 and 6), but no amplimer was obtained using genomic DNA from the ΔadeFGH deletion mutants (Figure 2, lanes 4, 8, 18 and 22). For the ΔadeIJK constructs, the deletion produced a 0.26-kb amplimer using the primers AdeJ F and AdeK R and genomic DNA from the ΔadeIJK mutants (Figure 2, lanes 12, 16, 20 and 24) and a longer 3.