coli lacZ gene. The resulting reporter plasmids

(listed in Table 1) were conjugationally transferred to R. capsulatus wild-type and mutant strains defective for mopA, mopB, or both. Rhodobacter capsulatus reporter strains were grown in a molybdenum-free AK-NL minimal medium containing 9.5 mM serine as the sole source of nitrogen. When required, Na2MoO4 was added to a ZD1839 ic50 final concentration of 10 μM. Following growth to the late exponential phase, β-galactosidase activities were determined as described previously (Miller, 1972; Sicking et al., 2005). Purification of His-tagged MopA and MopB proteins from E. coli, and gel-shift assays were carried out as described previously (Wiethaus et al., 2006). Escherichia coli BL21(DE3) strains carrying either plasmid

pJW32 (mopAhis) or pJW33 (mopBhis) were used to overexpress recombinant regulator proteins. Primer pairs 5′-ACGGGCAGGCGCGGGGTTCT-3′/5′-CCGGCATTCGCCGGTGAAGCACTG-3′ and 5′-GGCACTGACCGACCTTTTGACC-3′/5′-CCAGTGTTAACCTTTGCTACCCCTTTG-3′ were used to PCR amplify 209-bp anfA (Fig. 1b) and 138-bp mop promoter fragments (Fig. 1c), respectively, with the pBluescript derivatives carrying the respective anfA and mop promoter variants (Table 1) as templates. The 5′ ends of PCR products were 32P-labeled with T4 polynucleotide kinase (Fermentas, St. Leon-Rot, Germany). Up to 150 pmol of regulator proteins were preincubated in buffer B [40 mM NaH2PO4 (pH 8.0), 500 mM NaCl] at room temperature in a total volume of 16 μL. After 10 min, a mixture of 1 μL 32P-labeled DNA (5 fmol μL−1), 1 μL poly(dI-dC) (1 μg μL−1),

http://www.selleckchem.com/products/DAPT-GSI-IX.html and 2 μL binding buffer [25 mM HEPES (pH 8.0), 50 mM K-glutamate, 50 mM MgSO4, 1 mM DTT, 0.1 mM EDTA, 0.05% Igepal CA-630] was added. Samples were incubated at 30 °C for 20 min, before free and bound DNAs were separated on 6% polyacrylamide gels. 32P-labeled bands were documented using an Amersham Hyperfilm™ MP (GE Healthcare, Freiburg, Germany). To date, five molybdate (Mo)-regulated promoters have been described Ribonucleotide reductase for R. capsulatus (Wiethaus et al., 2006) (Fig. 1a). In the presence of Mo, the transcription of morC, morAB, mopA-modABCD, and anfA is repressed by either MopA or MopB, while mop is exclusively activated by MopA. In line with reporter gene studies, both regulators bind all Mo-repressed promoters in vitro, while only MopA (but not MopB) binds the Mo-activated mop promoter. All five promoters contain conserved sequences of dyad symmetry called Mo-boxes (Fig. 1a). Deletion of one or five nucleotides from the anfA-Mo-box completely abolished Mo repression of anfA (Kutsche et al., 1996), strongly suggesting that the anfA-Mo-box is essential for binding of MopA and MopB. A core consensus sequence (CG-N-TAT-N13-ATA-N2-G) is strictly conserved in all Mo-repressed and Mo-activated Mo-boxes (Fig. 1a; Consensus C). In addition to these key nucleotides, further bases are conserved between strongly repressed Mo-boxes.