Primers, PCR, and plasmid construct

Primers, PCR, and plasmid construction.All oligonucleotides used for mutant construction are listed in Table 2. All primers were designed to yield melting temperatures of 54 to 56°C and PCR fragments of approximately 1,000 to 1,200 bp. The primers were designed to combine the upstream and downstream regions of the coding regions of the target genes with the kanamycin or erythromycin resistance cassette by overlap extension PCR. Reverse primers for amplification of the upstream and downstream sections contained 25 additional nucleotides at their 5′ ends that were homologous to the downstream region and the antibiotic resistance cassette, respectively. The genomic DNAs purified from A. baumannii strains and pUC4K (or pIL252) for amplification of the kanamycin resistance cassette (or erythromycin resistance cassette) were used as templates for the PCR. PrimeSTAR GXL Taq DNA polymerase (TaKaRa, Shiga, Japan), which exhibits 30 mismatched bases per 486,923 total bases and higher fidelity than Pfu DNA polymerase, was chosen to prevent errors in the PCR products generated by overlap extension PCR and to produce blunt-end PCR products. PCR was performed using 1.25 U PrimeSTAR GXL Taq DNA polymerase, 10 μl of 5× PrimeSTAR GXL buffer, 0.2 mM deoxynucleoside triphosphate (dNTP) mixture, 10 pM of each primer, and template DNA (100 ng). Each mutated fragment was amplified by two-step PCR. For the first step (PCR-1), PCR cycle conditions were as follows: 30 cycles of 98°C for 10 s, 54°C for 40 s, and 68°C for 2 min, with a final elongation at 68°C for 10 min. The products obtained from the first step of PCR were then mixed at equimolar concentrations and were subjected to overlap extension PCR with the forward primers corresponding to the upstream region and the reverse primers corresponding to the antibiotic resistance cassette. PCR cycle conditions were as follows: 30 cycles of 98°C for 10 s, 54°C for 40 s, and 68°C for 4 min, with a final elongation at 68°C for 10 min. The blunt-end PCR products containing each deleted target gene with the antibiotic resistance cassette were ligated with FspI-digested pHKD01 to generate chimeric plasmids (Table 1). The plasmids constructed were confirmed by DNA sequencing.