4b) because no or little mop expression was observed under Mo-limiting conditions (Fig. 4a). (3) Activation of the wild-type mop promoter was enhanced in the mopB mutant as compared with the wild-type background (Fig. 4b; Wiethaus et al., 2009). As MopB represses mopA transcription (Kutsche et al., 1996; Wiethaus et al.,

2006), MopA is likely to accumulate to higher amounts in the mopB mutant than in the wild type. In addition, the formation of MopA–MopB heteromers in the wild-type background may interfere with mop activation by MopA homodimers (Wiethaus et al., 2009). Similar to the wild-type mop promoter, activation of mutant mop promoters containing different single-base substitutions was enhanced in the mopB mutant (Fig. 4b). (4) The requirement of the mop-Mo-box for MopA binding was verified using triple mutation T4A-A5T-G7C, buy Buparlisib which destroyed the highly conserved left half-site of the mop-Mo-box (Fig. 1a and c). This mutation completely abolished mop expression (Fig. 4b) and binding by MopA (Fig. 5), thus demonstrating that the mop-Mo-box is essential for mop gene activation. (5) Two mutations (T16C and C17T) increased MopA-activated mop expression (Fig. 4b). Accordingly, MopA bound the T16C mutant promoter at least as well as the wild-type promoter (Fig. 5). ABT737 This finding suggests that the wild-type mop-Mo-box

is not optimized for MopA binding. Most likely, moderate Mop production is physiologically relevant, because Mop specifically interacts with MopB (Wiethaus et al., 2009), and thus, Mop–MopB complex formation might affect MopB-dependent gene regulation. (6) Mutation T23A completely abolished mop expression and mutation T24C greatly diminished it (Fig. 4b). Although the mutant promoters T23A and T24C were bound, they could not or could barely be activated by MopA (Fig. 5). Presumably, RNA polymerase was unable to recognize these promoters because nucleotides 23–25 (TTG) of the mop-Mo-box overlap with the putative −35 region (TTGTCA) located upstream of the experimentally determined

Immune system mop transcription start site (Wiethaus et al., 2006). (7) None of the single-base substitutions in the Mo-box facilitated mop activation (Fig. 4b) or binding of the mop promoter by MopB (Fig. 5), suggesting that more than one substitution in the weakly conserved right half-site of the mop-Mo-box (Fig. 1a) is required to confer recognition by MopB. (8) The mopanfA mutant promoter, in which the mop-Mo-box is exchanged against the anfA-Mo-box, was bound (Fig. 5) and activated (Fig. 4b) by MopA, showing that the anfA-Mo-box, which normally serves as a repressor-binding site, may well act as an activator-binding site. This finding demonstrates that the function of Mo-boxes as either a repressor- or activator-binding site essentially depends on its position relative to the −35/−10 regions. Although MopA and MopB shifted the wild-type anfA promoter with similar efficiency (Fig.