Given that CtrA is a global regulatory protein for both essential (e.g. cell division) and non-essential (e.g. polar development) genes, and that the drastic CtrA reduction in YB3558 leads to polar developmental defects but the strain is still viable, we hypothesized that transcription of

CtrA-regulated genes essential for cell survival will be less affected by CtrA reduction in YB3558 than those that are essential for less important cellular functions. Thus we investigated the transcription level of several CtrA-regulated genes in CB15 and YB3558. Plasmids bearing transcriptional lacZ fusions were introduced into both wild type and YB3558 strains. The promoters for the reporter constructs were ctrA (pctrA290, [9]), ctrA P1 (pctrA-P1, [9]) ctrA P2 (pctrA-P2, [9]), ftsZ (plac290/HB2.0BP, [18]), ftsQA (pMSP8LC, [19]), ccrM (pCS148, [20]), fliQ (pWZ162, [21]) and pilA (pJS70, see more [22]) find more as well as lacZ under the control of a xylose

inducible promoter to serve as a negative control (pCS225, [23]). Exponential phase cultures were assayed for β-galactosidase activity (Figure 7). Total transcriptional activity from the ctrA promoter was unaffected, though there was a reduction of activity from the weak P1 promoter, but not the stronger P2. Activity from these promoters is dependent upon many factors, one of them being CtrA protein abundance. It is possible that even though CtrA abundance in YB3558 is severely reduced, it is more than enough to activate the P2 promoter. Figure 7 Expression of CtrA-dependent promoters in wild-type and YB3558 strains. β-galactosidase assays were performed on exponentially growing cultures as described in the Methods. CtrA-dependent promoters of essential cell process genes show little-to-no change between wild-type and YB3558, while the pilA promoter shows a drastic difference in expression between the strains. ftsZ

and ftsQA promoters Beta adrenergic receptor kinase had a moderate reduction in activity, and the ccrM promoter had a slight reduction in activity. These genes are essential for viability. The moderate reduction in transcription for these genes agrees with the hypothesis that genes involved in essential cell cycle processes would not be severely affected by the reduction in CtrA in YB3558. In contrast, the pilA promoter exhibited a drastic decrease in activity, as would be expected given the selection by which this mutant was obtained. However, activity from the fliQ promoter (fliQ is a flagellar biosynthesis gene and not essential) was largely unaffected. It is not clear why this promoter is unaffected while the pilA promoter shows such a difference in activity. It could be that the pilA promoter is much more sensitive to CtrA levels. Regulation of pilA is controlled not only by CtrA, but by SciP.