1. Activation of the Bacterial Sensor Kinase PhoQ by Acidic pH
Lynne R. Prost, Margaret E. Daley, Valerie Le Sage, Martin W. Bader, Hervé Le Moual, Rachel E. Klevit, Samuel I. Miller 
Molecular Cell 
Volume 26, Issue 2, Pages (April 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
PhoP-Dependent Gene Activation by Acidic pH Is Mediated Directly by PhoQ
(A) PhoP-dependent gene activation increases in acidified media. A reporter fusion between PhoP-dependent acid phosphatase (PhoN) and E. coli PhoA was used to measure activation. Cultures were grown in N-minimal medium buffered with MES and containing 1 or 10 mM MgCl2, as indicated.
(B) The PhoP-dependent pH response requires PhoQ. Activities were measured in the PhoQ wild-type strain CS120 and MB101, which carries a phoQ::tet allele. The response could be complemented in MB106, which has a plasmid carrying PhoQ under the control of the arabinose promoter (pBAD24-phoQ). Cultures were grown in N-minimal medium buffered with MES at pH 7.5 (black bars) and 5.5 (gray bars), and containing 10 mM MgCl2.
(C) PhoQ-mediated phosphorylation of PhoP depends on the acidification of intralumenal pH. The vesicles were formed in the presence of sodium phosphate (black bars) or citrate phosphate (gray bars) adjusted to the appropriate pH. Vesicles were incubated in the presence of [γ-32P]ATP and an 8-fold molar excess of PhoP for 20 min at 22°C. The amounts of [32P]phospho-PhoP were determined with a phosphoimager. All graphed values are mean ± standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Peptides and pH Have an Additive Effect on PhoPQ Activation
(A) PhoP-dependent gene activation relies on Mg2+, peptide, and pH. Cultures were grown in N-minimal medium buffered with MES and containing 1 mM or 10 mM MgCl2. C18G was added to 5 μg/ml where indicated.
(B) The presence of peptide further increases PhoP phosphorylation at all pH values. Vesicles were formed in the presence of sodium phosphate (black bars) or citrate phosphate buffer (gray bars) supplemented or not with 1 μg/ml C18G. Phosphorylation of PhoP was determined as described in Figure 1C. All graphed values are mean ± standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 The PhoQ Sensor Domain Is Sensitive to Acidification
(A) A schematic of the domain organization of PhoQ shows it consists of two transmembrane regions, a periplasmic sensor domain, and a cytosolic domain that contains the catalytic ATP-binding domain and the phosphotransfer domain.
(B) Selected points of the titration of the PhoQ sensor domain in the presence of 20 mM Mg2+. Superimposed two-dimensional HSQC spectra are shown at pH 6.5 (black), 4.9 (green), and 3.5 (red). The insets highlight two regions of the spectrum that are subject to large shifts upon acidification.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 PhoQ Still Binds to Mg2+ and PMNP at pH 5.5
Superimposed HSQC spectra of the PhoQ sensor domain at pH 5.5 in the absence (black) and presence of 4 mM PMNP (green) or 20 mM Mg2+ (red).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Mutations in H157 Result in Divalent Cation Derepression
Strain MB106, which carries a PhoQ null allele and the plasmid pBAD24-PhoQ, was used as the wild-type strain. The indicated mutations were made in pBAD24-PhoQ using QuikChange mutagenesis. Cultures were grown in N-minimal media buffered with MES and supplemented with 1 or 10 mM MgCl2, as indicated. All graphed values are mean ± standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
The H157N Mutation in the Sensor Domain Partly Mimics the Low pH Conformation
The wild-type pH 6.5 (black) and pH 3.5 (red) spectra are compared to the spectra of H157N in the presence of 20 mM Mg2+ at pH 6.5 (dark blue) and pH 3.5 (light blue). The regions shown are the same as the insets shown in Figure 3B. The region in (A) shows peaks that appear in the low pH conformation in the H157N mutant at neutral pH, indicating the region around H157 as one pH-sensitive region. In (B) it is clear that there are other regions of the protein that are unaffected by the mutation at H157 yet are still titrated by low pH.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
The Interaction Network Involving H157 in the Salmonella PhoQ Sensor Domain Dimer
The PhoQ sensor domain (PDB code 1YAX) is colored to illustrate the interaction network surrounding H157. The core of the protein is colored blue and the α4/α5 helices are red. The residues participating in the interaction network comprised of hydrogen bonds and electrostatic contacts are colored green with their side chains displayed in space-filling format. The proposed membrane-facing surface is the bottom of the molecule, with the N and C termini labeled where they would enter the membrane. The gold spheres represent the two Ca2+ ions observed in the crystal structure; a third Ca2+ ion not observed in the crystal structure was found biochemically (Bader et al., 2005) and is not shown. The acidic patch involved in divalent cation binding extends along the bottom of the protein, starting at the end of the α5 helix and including several glutamic acid residues that are also part of the interaction network.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions