, 2006). Thus, we defined an extended consensus sequence (CG-N-TAT-N2-G-N6-CTA-N-ATA-N-CG) based on the three strongly repressed Mo-boxes upstream of the morA, mopA, and anfA genes (Fig. 1a; Consensus R). The major difference between MopA/MopB-repressed

Mo-boxes and the MopA-activated mop-Mo-box seems to lie in the right half-site. Therefore, we investigated whether this region of the mop-Mo-box either selectively facilitates binding of MopA and/or discriminates against binding Ferroptosis tumor of MopB. Several rationally designed single-base substitutions were introduced to convert the anfA-Mo-box into the mop-Mo-box and vice versa (Materials and methods). Specifically, mutations T3A, A7G, T17C, A18T, A23T, and C24T converted the anfA-Mo-box toward the mop-Mo-box (Fig. 1b), while mutations A3T, T16C, C17T, T18A, C19T, T23A, and T24C made the mop-Mo-box more similar to the anfA-Mo-box (Fig. 1c). Mutations A18G, T21C, and C24A probed for the principal importance of highly conserved nucleotides, which were exchanged for nucleotides not occurring in any of the Mo-boxes. To prove that the mop-Mo-box was essential for MopA-dependent mop

gene activation, the triple mutation T4A-A5T-G7C was constructed to destroy the conserved left half-site of the mop-Mo-box (Fig. 1c). In addition to these single-base substitutions, the anfA- and mop-Mo-boxes were SB203580 in vitro exchanged against each other (anfAmop and mopanfA). In anfAmop, the entire 25-bp anfA-Mo-box was replaced Staurosporine ic50 by the mop-Mo-box (Fig. 1b). In contrast, in mopanfA, only the first 22 nucleotides were replaced, because nucleotides 23–25 of the mop-Mo-box overlap with the −35 region of the mop promoter and are thus essential for mop gene expression (Fig. 1c). The effects of Mo-box mutations on anfA transcription were examined by lacZ reporter fusions. For this purpose, wild-type and mutant anfA promoter fragments were cloned into the low-copy broad-host-range vector pML5, thus creating transcriptional

fusions to the promoterless lacZ reporter gene (Fig. 1b; Table 1; Materials and methods). These reporter plasmids were transferred into R. capsulatus wild-type and mutant strains defective for mopA, mopB, or both. The resulting reporter strains were grown in minimal medium under Mo-limiting and Mo-replete conditions before determination of β-galactosidase activities (Fig. 2). In addition to these in vivo studies, the in vitro effects of selected anfA-Mo-box mutations on binding by MopA and MopB were analyzed by DNA mobility shift assays (Fig. 3). For this purpose, 209-bp anfA promoter fragments (PanfA; Fig. 1b) were PCR amplified and used for gel-shift assays with increasing amounts of the regulators (Materials and methods). The effects of Mo-box mutations on anfA gene expression and regulator binding may be summarized as follows: (1) In the R.