The PmrD protein is necessary for normal PmrA activation at pH 5.8 
The PhoP-activated PmrD protein favours the phosphorylated state of the PmrA protein (Fig. 1) (Kato and Groisman, 2004).
Thus, we tested the possibility of PmrD participating in the PmrA-dependent response to acidic conditions, and thus contributing to the pbgP transcription remaining in a pmrB mutant.
Expression of the pbgP gene was abolished in a pmrB pmrD double mutant (Fig. 3) indicating that both genes are necessary to activate PmrA under acidic conditions.
In contrast to the phenotype of the pta ackA double mutant, pbgP transcription was reduced in the pmrD mutant (Fig. 3).
These results imply that the pmrD gene was being expressed even though the media contained 10 mM MgCl2, a concentration known to repress transcription of PhoP-activated genes (Soncini et al., 1996).
We examined transcription of the pmrD gene using RNA isolated from organisms grown at pH 5.8 or 7.7.
Growth at pH 5.8 resulted in pmrD transcript levels that were approximately3.5-fold higher than in organisms grown at pH 7.7 (Fig. 4A).
This acid pH-promoted increase appears to be specific to a subset of PhoP-activated genes (our unpublished results) that includes pmrD because expression of the PhoP-regulated slyA gene and the PhoP-independent corA gene was not affected by the pH of the medium (Fig. 4A).
In agreement with the gene transcription data, Western blot analysis of crude extracts using anti-PmrD antibodies showed that the PmrD protein was produced in cells grown in N-minimal medium pH 5.8 and 10 mM MgCl2 but not in cells grown in the same medium buffered at pH 7.7 (Fig. 4B).
The acid-promoted expression of the PmrD protein was phoPQ-dependent, which is in agreement with the fact that PhoP is the only known direct transcriptional activator of pmrD (Kox et al., 2000).
