It is possible that the dramatic decrease in pilA promoter activity in YB3558 is not from CtrA abundance itself, but an indirect effect of reduced CtrA abundance leading to increased SciP activity. However, CtrA positively regulates transcription of sciP and the strong reduction of CtrA activity in the YB3558 mutant should lead to a decrease in SciP levels, not an increase. In agreement MLN0128 mouse with this hypothesis it has been shown that a site-directed mutation that abolishes transcription from the ctrA P1 promoter caused a strong reduction of CtrA abundance [25], similar to that of the YB3558 mutation in this study, and this lead to significantly reduced expression

of SciP, down to 19% of wild-type level [25]. The ctrA P1 mutant also had morphological and growth defects similar to those found here, and several assays demonstrated

that CcrM transcription and translation was largely unaffected, agreeing with our results. Therefore it is Selleckchem MM-102 unlikely that the effects observed on gene expression are the result of increased SciP activity. Some CtrA-dependent promoters appear more resilient to changes in CtrA concentration than others. It has been shown previously that promoters that deviate from the canonical TTAA-N7-TTAA CtrA binding site have a lower CtrA binding affinity [26, 27]. It is possible promoters that are more susceptible to changes in CtrA concentration have more divergent CtrA binding sites, causing them to have lower CtrA affinity and thus lower binding site occupancy at lower CtrA concentrations such as found in YB3558. A list of CtrA binding

sites from each of the transcriptional fusions used in Figure 7 (excluding the xyl control) is shown in Table 2. The CtrA binding region for each gene was determined experimentally by DNA footprinting (see references in Table 2). The ctrA-P2, ccrM and fliQ reporters displayed the least Adavosertib molecular weight change in YB3558 compared to wild-type, indicating expression from these promoters is more resilient to changes in CtrA concentration. ALOX15 The ctrA-P2 site is well characterized as TTAA-N6-TTAA with an additional TTAA half site 1 bp downstream. This binding site is relatively close to the canonical structure. The binding sites for ccrM and fliQ are TTAA-N7-CTAA and CTAA-N7-TTAA respectively. Each binding site differs from the canonical structure by a single base pair substitution. Therefore, the promoters displaying little change in YB3558 all are relatively similar to the known CtrA binding sequence. The ctrA-P1, ftsZ, pilA and to a lesser extent ftsQA fusions all displayed noticeable changes in expression in YB3558 compared to wild-type. The ctrA-P1 binding site consists of a single TTAA half site and obviously diverges greatly from the consensus CtrA binding site. The ftsQA site is TTAA-N7-CTAA, the same as the ccrM binding site, though ftsQA only displays a moderate change in transcription inYB3558.