Results 
Mild acid pH induces transcription of PmrA-regulated genes To examine the mild acid pH induction of PmrA-activated genes, we grew Salmonella cells harbouring chromosomal lacZYA transcriptional fusions to the PmrA-regulated genes pbgP, pmrC and ugd (Wosten and Groisman, 1999) in N-minimal media buffered at pH 5.8 or 7.7.
This medium lacked Fe3+ or Al3+, the only known PmrB ligands (Wosten et al., 2000), and contained 10 mM MgCl2, which represses expression of PmrA-activated genes (Soncini and Groisman, 1996; Kox et al., 2000).
All three genes were expressed when cells were grown in media buffered at pH 5.8 but not at pH 7.7 (Fig. 2A-C), in agreement with previous results (Soncini and Groisman, 1996).
A similar induction of pbgP transcription was found when MES was used as the buffering agent in the media at pH 5.8 instead of Bis-Tris (data not shown), indicating that the mild acid effect on gene expression was not due to a particular buffering system.
The transcriptional activation of PmrA-regulated genes taking place at pH 5.8 could be due to trace amounts of metals such as Fe3+, which is more soluble at acidic pH.
To rule out this possibility, we treated the culture medium with Chelex 100 resin, an agent known to chelate polyvalent metal ions that does not affect Salmonella growth.
We determined that Chelex 100 was effective at chelating iron because expression of the pmrA-independent iron-repressed iroA gene (Hall and Foster, 1996) was induced to higher levels in cultures treated with Chelex 100 (Fig. 2D).
Expression of pbgP was still induced when Salmonella was grown in the Chelex-treated medium (Fig. 2D) or in media containing the specific Fe3+ chelator deferoxamine mesylate (data not shown) supporting the notion that mild acid pH is responsible for the observed induction.
We determined that the regulatory protein PmrA is required for the transcriptional activation in response to mild acid pH because there was no induction of the three investigated genes in a pmrA mutant (Fig. 2A-C).
Moreover, a mutant expressing a derivative of the PmrA protein that cannot be phosphorylated due to substitution of the putative phosphorylation residue aspartate 51 by alanine (Kato and Groisman, 2004) completely failed to promote transcription of PmrA-activated genes in response to pH 5.8, in a similar fashion to the pmrA strain (A. Kato and E.A. Groisman, unpubl. results).
From these results we conclude that Salmonella harbours a signalling pathway that responds to mild acid pH by activating the PmrA protein through phosphorylation.
