Fig. 1.

The Salmonella and Yersinia PhoP proteins differ in their ability to promote gene transcription. (A) The Salmonella mgtA and ugtL promoters differ both in the orientation and the distance of the PhoP box to the promoter −10 region. The PhoP box is located in the place that corresponds to the promoter −35 region in the mgtA promoter but is 20 bp further upstream in the ugtL promoter. (B and C) GFP expression driven by the Salmonella mgtA and ugtL promoters in Salmonella and Yersinia. Approximately 150-nt DNA fragments corresponding to the mgtA and ugtL promoter regions (covering ≈130 nucleotides upstream and ≈20 nucleotides downstream of the transcription start sites) were cloned in front of a promoterless gfp gene in the low-copy plasmid pMS201. Organisms were grown in defined medium containing 50 μM MgSO₄, inducing conditions for the PhoP/PhoQ system. GFP expression was normalized to cell density. Shown are the ratios of the normalized GFP values between wild-type Salmonella (14028s) or Yersinia (KIM6) and their respective isogenic phoP mutant strains (EG15598 and EG14737, respectively). Values shown are mean plus SD of at least 3 independent experiments. (D) Schematic of the genomic context of the phoPQ locus in Salmonella strain EG13918 coding for the Salmonella PhoP-HA and PhoQ proteins and its derivative harboring the Yersinia phoP-HA and phoQ genes (in blue) (EG17569). (E) Expression of the Salmonella and Yersinia PhoP proteins in the Salmonella strains EG13918 and EG17569 depicted in D. Western blot analysis was performed with anti-HA and anti-RpoB antibodies (to detect the PhoP-HA and RpoB proteins, respectively) on cell extracts prepared from bacteria grown as described in B and C in medium containing 10 mM (H) or 50 μM (L) MgSO₄. (F and G) mgtA and ugtL expression in the Salmonella strains depicted in D. Cells were grown as described in B and C. Transcript levels were determined by quantitative real-time PCR and normalized to ribosomal RNA levels. Shown are the ratios of the normalized transcript levels present in the strains described in D relative to those produced by the phoPQ mutant EG15598. Values shown are mean plus SD of at least 3 independent experiments. (H–M) Single-round in vitro transcription assays with linear templates corresponding to the mgtA (H–J) and ugtL (K–M) promoters, E. coli RNA polymerase, and increasing amounts of phosphorylated Salmonella or Yersinia PhoP proteins. The upper band in H–I corresponds to treR, a PhoP-repressed transcript going in the reverse orientation (22). The upper band in K and L corresponds to a spurious transcript observed in vitro but not in vivo (21, 45). Quantification of the in vitro transcription assays is shown in J for mgtA and in M for ugtL.