, 1990) were used for DNA cloning and were grown aerobically at 37 °C in LB medium supplemented with 100 μg mL−1 ampicillin (Ap), 30 μg mL−1 Gm or 15 μg mL−1 Km, as required. Bacteria grown overnight in LB medium were subcultured into fresh LB medium to give an OD600 nm of 0.1. Exponential-growth phase cells (OD600 nm of 0.5 after incubation for 4 h) were used as indicated. The A. tumefaciens mbfA gene (Atu0251) (Wood et al., 2001)
was disrupted by a single homologous recombination method. The internal DNA fragment of the mbfA coding region was amplified by PCR with primers BT1666 (5′-AAGCTCCTGGGTGATCTGGC-3′) and BT1667 (5′-CGCTTCAACGGTGATCCACG-3′), using genomic wild-type NTL4 as the template. The 343-bp PCR product was cloned into the unique SmaI site of the pKNOCK-Km suicide plasmid (Alexeyev, 1999), generating pKNOCKNR114. The pKNOCKNR114 plasmid was transferred to wild-type NTL4
by conjugation (Cangelosi find more et al., 1991). The recombinants were selected on LB agar plates containing 25 μg mL−1 Cm and 30 μg mL−1 Km. Correct integration of the pKNOCKNR114 into the mbfA locus (the NR114 mutant strain) was confirmed by Southern blot analysis. The A. tumefaciens irr gene (Atu0153) was inactivated using the protocol described earlier. Primers BT696 (5′-GCCAGCGCGTTGCTTTGGGT-3′) and BT697 (5′-AAGAAGTGATGGTGATCCGA-3′) were used. The Alectinib ic50 PCR product was cloned into pKNOCK-Gm, generating plasmid pKNOCKWK074. The pKNOCKWK074 was transferred to the wild-type NTL4 generating the irr mutant strain (WK074) that was selected on LB agar plates containing 25 μg mL−1 Cm and Tenoxicam 90 μg mL−1 Gm. The NRSB111 strain (disruption of both irr and mbfA genes) was created by transferring pKNOCKNR114 into the WK074 mutant strain. The NRSB111 mutant was selected on LB agar plates containing 25 μg mL−1 Cm, 90 μg mL−1 Gm and 30 μg mL−1 Km. The DNA fragment containing the full-length A. tumefaciens mbfA gene and its native promoter was amplified from NTL4 genomic DNA by PCR with primers BT1707 (5′-CCTGAATTTCCGCATTGTGG-3′) and BT1677 (5′-TTCACGCGTTGCCGATGATA-3′). The 1174-bp PCR products
were cloned into the unique SmaI site of the expression vector pBBR1MCS-4 (Kovach et al., 1995), creating the recombinant plasmid pNR114C. An H2O2 sensitivity test was performed as previously described (Kitphati et al., 2007). Exponential-growth phase cells were adjusted, diluted and spotted onto the LB agar plates containing 200, 350 and 375 μM H2O2 in the absence or presence of 50 μM 2,2′-dipyridyl (Dipy). Plates were incubated at 28 °C for 48 h. Each strain was tested in duplicate, and the experiment was repeated a minimum of two times to ensure the reproducibility of the results. Exponential-growth phase cells grown in LB medium were treated with 50 μM FeCl3, 200 μM Dipy or 250 μM H2O2 for 15 min. Total RNA extraction, cDNA preparation and RT-PCR analysis were conducted as described previously (Ngok-ngam et al., 2009).