Fig. 1. Model for the activation of the PmrA–PmrB two-component system by the PhoP–PhoQ two-component system. Transcription of PmrA-activated genes can be induced by growth in high iron independently of the PhoP–PhoQ two-component system (right) or by growth in low magnesium in a PhoP–PhoQ-dependent manner (left). During growth in low magnesium, the PhoP–PhoQ system promotes expression of the pmrD gene. The PmrD protein controls the activity of the PmrA–PmrB system at a post-transcriptional level. The seven-gene pbgP/E operon has also been designated the pmrF locus (Gunn et al., 1998).

Fig. 2. The pmrD gene is necessary for transcription of PhoP-activated PmrA-dependent genes, but does not affect transcription of PhoP-activated PmrA-independent genes. β-galactosidase activities (Miller units) expressed by strains grown in N-minimal medium pH 7.7 with 10 µM Mg²⁺ were determined for mutants harboring a lac transcriptional fusion to the PhoP-activated, PmrA-dependent genes pbgP, pmrC and ugd (A) and the PhoP-activated, PmrA-independent genes mgtA, mgtC and pcgL (B). The transcriptional activity was investigated in four genetic backgrounds: wild type, phoP, pmrD and pmrA. Data correspond to mean values of four independent experiments performed in duplicate. Error bars correspond to the standard deviation (and are only shown if greater than the resolution of the figure).

Fig. 6. Transcripton of pbgP in a strain grown in low magnesium expressing the constitutive pmrA505 allele is independent of pmrD. β-galactosidase activities (Miller units) expressed by transformants grown in N-minimal medium pH 7.7 with 10 µM Mg²⁺ (A), 10 µM Mg²⁺ and 100 µM Fe²⁺ (B) or 10 mM Mg²⁺ (C) were determined for a mutant harboring a lac transcriptional fusion to pbgP. The transcriptional activity was investigated in four genetic backgrounds: wild type, pmrD, pmrA505 and pmrD pmrA505. The data correspond to mean values of four independent experiments performed in duplicate. Error bars correspond to the standard deviation (and are only shown if greater than the resolution of the figure).

Fig. 5. PmrD acts downstream of the PhoP–PhoQ system and upstream of the PmrA–PmrB system. β-galactosidase activities (Miller units) expressed by transformants grown in N-minimal medium pH 7.7 with 10 µM Mg²⁺ (A) or 10 mM Mg²⁺ (B) were determined for mutants harboring a lac transcriptional fusion to pbgP. The transcriptional activity was determined in six genetic backgrounds: wild type, phoQ, phoP, pmrD, pmrB and pmrA. The strains were transformed with a plasmid harboring the pmrD gene expressed from the lac promoter (pLK24) or the cloning vector (pUC19). The data correspond to mean values of four independent experiments performed in duplicate. Error bars correspond to the standard deviation (and are only shown if greater than the resolution of the figure).

Fig. 4. PmrD is essential for transcription of the PmrA-activated gene pbgP during growth in low magnesium. Bacteria were grown in N-minimal medium pH 7.7 with 10 µM Mg²⁺ (A and C) or 10 µM Mg²⁺ and 100 µM Fe²⁺ (B). β-galactosidase activities (Miller units) were determined for mutants harboring a lac transcriptional fusion to pbgP. The transcriptional activity was determined in six genetic backgrounds: wild type, phoQ, phoP, pmrD, pmrB and pmrA (A and B) or wild-type and pmrD backgrounds for the complementation assay (C). The pmrD– strains used for this assay were transformed with a plasmid harboring the pmrD gene expressed from its own promoter (pLK23) or the cloning vector (pUC19). The data correspond to mean values of four independent experiments performed in duplicate. Error bars correspond to the standard deviation (and are only shown if greater than the resolution of the figure).

Fig. 3. Transcription of pmrD requires PhoP, but is PmrA independent. (A) S1 mapping of pmrD transcripts produced in wild-type (14028s), phoP (MS7953s) and pmrA (EG7139) bacteria grown in N-minimal medium pH 7.7, containing 10 mM Mg²⁺, 10 µM Mg²⁺ or 10 µM Mg²⁺ and 100 µM Fe²⁺ and harvested during the logarithmic phase. The S1 protection assay was performed as described in Materials and methods. Lanes G, A, T and C correspond to dideoxy chain termination sequence reactions corresponding to this region. The sequence spanning the transcription start site is shown, and the transcription start site is marked with an arrow. (B) DNA sequence of the promoter region of the pmrD gene. The arrow corresponds to the +1 transcription start site, the underlined sequence is the predicted –10 region for the pmrD promoter and the sequence in bold matches the consensus found as a direct repeat in the promoter region of PhoP-activated genes in E.coli K-12 (Kato et al., 1999).

Fig. 7. PmrD activates the PmrA–PmrB system by a post-transcriptional mechanism. (A) Expression of the pmrAB genes from a heterologous promoter does not rescue pbgP transcripton in a pmrD mutant. β-galactosidase activities (Miller units) expressed by bacteria harboring a lac transcriptional fusion to the pbgP gene grown in N-minimal medium pH 7.7 with 10 µM Mg²⁺. The transcriptional activity was investigated in three genetic backgrounds: wild type, pmrD and pmrA, the latter carrying either a plasmid with the pmrAB genes under the control of a derivative of the lac promoter or the plasmid vector. The data correspond to mean values of four independent experiments performed in duplicate. (B) pmrD-mediated activation is dependent on the putative site of PmrA phosphorylation.β-galactosidase activities (Miller units) expressed by bacteria harboring a lac transcriptional fusion to the pbgP gene grown in N-minimal medium pH 7.7 with 10 µM Mg²⁺. The transcriptional activity was investigated in three genetic backgrounds: a pmrA null mutant harboring plasmid pEG9102 with the pmrAB genes under the control of a derivative of the lac promoter, plasmid pLK39 with the pmrA(D51A)B genes under the control of a derivative of the lac promoter or the plasmid vector. Similar results were obtained with plamids harboring only the pmrA gene under the control of a derivative of the lac promoter: the D51A mutation abolished pbgP transcription. The data correspond to mean values of four independent experiments performed in duplicate. Error bars correspond to the standard deviation (and are only shown if greater than the resolution of the figure).

Fig. 8. The pmrD gene is required for polymyxin resistance during growth in low magnesium. Wild-type (14028s), pbgP (EG9241), ugd (EG9524), pmrA (EG7139) and pmrD (EG11491) bacteria were grown to logarithmic phase in N-minimal medium pH 5.8 with 10 µM Mg²⁺ (A and C) or 10 µM Mg²⁺ and 100 µM Fe²⁺ (B), washed, and incubated in the presence of polymyxin (a final concentration of 2.5 µg/ml) for 1 h at 37°C. Samples were diluted in phosphate-buffered saline and plated on LB-agar plates to assess bacterial viability. Survival values are relative to the original inoculum. Complementation experiments were carried out with the pmrD mutant transformed with a plasmid harboring the pmrD gene expressed from its own promoter (pLK23) or the cloning vector (pUC19) (C). Data correspond to mean values from three independent experiments performed in duplicate. Error bars correspond to the standard deviation (and are only shown if bigger than the resolution of the figure).