The DNA fragment containing the 6× his-tagged-irr

fusion was amplified from pQE30IRR using primers BT3157 and BT3158. The PCR product was digested with EcoRI and then cloned into pBBR1MCS-4 (Kovach et al., 1995), which was digested with EcoRI and SmaI, generating a plasmid named pHIRR. The full-length A. tumefaciens manganese uptake Galunisertib in vitro regulator gene (mur, Atu0354) (Wood et al., 2001) without the start codon was amplified by PCR using primers BT3321 and BT693. A similar protocol as described above was used to construct pQE30MUR and pHMUR. The plasmids pHIRR and pHMUR were transferred to A. tumefaciens cells to produce the 6× His-tagged fusion proteins His-Irr and His-Mur. Site-directed mutagenesis was performed on the irr coding sequence using pIRR or pHIRR as the template and a QuikChange XL mutagenic PCR kit (Stratagene) following the manufacturer’s instructions. Amino acid residues in the candidate metal- and haem-binding sites of Irr protein, including H38, H45, H65, D86, H92, H93, H94, D105 and H127, were mutated to alanine individually or in combination (Table 1). The primers for site-directed mutagenesis are listed in Supporting Information, Table S1. The

mTOR inhibitor mutations were confirmed by DNA sequencing. Exponential growth phase cells were washed and resuspended in minimal Agrobacterium (AB) medium (Cangelosi et al., 1991). The cells were grown for another 1 h and were then harvested. Crude bacterial lysates were prepared as previously described (Kitphati et al., 2007). Protein concentrations were determined using the Bradford Bio-Rad protein assay. The total protein (75 μg) from lysate samples was separated on 12.5% SDS-polyacrylamide gels and transferred onto Hybond-P PVDF membranes (Amersham Pharmacia Biotech) using a Bio-Rad semi-dry blotting apparatus. The recombinant 6× His-tagged proteins were detected using mouse anti-RGS-His monoclonal antibody (Qiagen) and sheep anti-mouse

IgG-HRP conjugate (Qiagen). Proteins were visualized using the Lumi-Lightplus chemiluminescent peroxidase (POD)-substrate (Roche). DNA fragments (378 bp) containing the promoter region of mbfA (Atu0251) GNE-0877 (Wood et al., 2001) were amplified by PCR with primers BT1707 and BT1665. The PCR products were cloned into a promoter probe vector pUFR027lacZ, as previously described (Kitphati et al., 2007), to generate plasmid pPNLZ01. Exponential phase cells were washed and resuspended in minimal AB medium to an OD600 nm of 0.1. Cells were untreated or treated with 50 μM FeCl3, 100 μM 2,2′-dipyridyl (Dipy) or 50 μM haem. The cells were incubated at 28 °C with shaking for 18 h. The cells were harvested and β-galactosidase activity was measured as described previously (Miller, 1972). Specific activity is defined as the units per mg of protein (U mg protein−1) and is expressed as the mean of triplicate samples ± SD. Cells grown on LA for 48 h were washed and resuspended in fresh LB medium.