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Osmosensing by the bacterial PhoQ/PhoP two-component system

  1. Victor Sourjika,b,1
  1. aMax Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany;
  2. bLOEWE Center for Synthetic Microbiology (SYNMIKRO), 35043 Marburg, Germany
  1. Edited by Susan Gottesman, National Institutes of Health, Bethesda, MD, and approved November 6, 2017 (received for review October 5, 2017)

  1. Fig. 2.

    Characterization of PhoQ-mediated osmosensing. (A) PhoQ activity monitored via the expression of PmgtLA-GFP reporter in E. coli wild-type cells after stimulation with indicated NaCl concentrations. The P values (according to t test) of activation compared with the controls (0 min) are 0.3, 0.05, and 0.002 for stimulations with 75 mM, 150 mM, and 300 mM NaCl, respectively, and less than 0.001 for stimulations with 400 mM, 500 mM, and 600 mM NaCl. (B) PhoQ activity monitored via the expression of PmgtLA-GFP reporter in ?mgrB strain after at least a 30-min incubation without or with indicated ionic (300 mM NaCl) or nonionic (600 mM sucrose, 600 mM sorbitol) osmolytes. The activations are significant at P < 0.01. (C) Activation of PmgtLA-GFP reporter after osmotic upshift (addition of 300 mM NaCl at time 0) in the presence of 10 mM or 1 mM magnesium in the growth media. The activation is significant at P < 0.01. All data points represent the averages of at least three independent experiments and error bars show SDs.

  2. Fig. 3.

    PhoQ-mediated osmosensing relies on the transmembrane domain. (A) Transcriptional response mediated by the wild-type PhoQ or mutants that lack the sensor domain (PhoQ?51–181) or carry mutation in the TM domain (PhoQN202A), expressed from a plasmid at 0.002% arabinose induction in the ?phoQ strain. Transcription of mgtA was monitored upon addition of 300 mM NaCl. The transcription up-regulation is significant for ΔphoQ strains complemented with the wild-type PhoQ or PhoQ?51–181 (P < 0.005). (B) Activity of PmgtLA-GFP reporter in the ?phoQ strains expressing either the wild type or PhoQ mutants. Cells grown to OD600 = 0.4 in the presence of 10 mM magnesium and 0.002% arabinose were stimulated with 300 mM NaCl for 30 min, and their fluorescence was measured before (labeled as 0 mM NaCl) and after the stimulation (labeled as 300 mM NaCl). The P values for the activation of the wild type and PhoQ mutants (PhoQΔ22, PhoQΔ22–23, PhoQΔ22–24, and PhoQΔ22–23+N202A) are 0.002, 0.02, 0.05, 0.08, and 0.02, respectively. Normalized data can be found in SI Appendix, Fig. S3A for clear visualization of error bars. (C) Time course of PmgtLA-GFP reporter activation in ?mgrB cells upon stimulation with 300 mM NaCl, 1% TFE, or both stimuli combined, as indicated. The activation is significant with P < 0.01 for stimulation with 300 mM NaCl or both 1% TFE and 300 mM NaCl. (D) Simulated structures of the wild-type PhoQ TM helices in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) membrane bilayer at 1.0 (green) and 2.5 (blue) atm lateral pressure (see Materials and Methods and SI Appendix, SI Materials and Methods for details of simulations), representing side (Left image) and top (Right image) views. The phosphorus atoms of POPE are represented as orange balls to indicate the interface between solvent and the membrane. (E) Conformational changes in the TM domain of the wild type and PhoQN202A under indicated lateral membrane pressure, represented as the distance between termini of helices at the periplasmic surface. The P values for TM domain conformation change are 0.69 and less than 0.05 for PhoQN202A and the wild type, respectively. Data points in AC represent the averages of at least three independent experiments and error bars show SDs.

  3. Fig. 4.

    PhoQ mediates resistance to hyperosmotic conditions partly by stabilizing RpoS (σS). (A) Growth of indicated E. coli strains in the absence or presence of 600 mM NaCl. PhoQ or PhoP/PhoQ (PhoPQ) were expressed from respective plasmids. Because deletion of phoP has a polar effect on the expression of phoQ, a ΔphoPphoQ strain complemented with PhoQ was used to elucidate the specific effect of phoP. The growth curves are representatives of at least three independent experiments. (B) The amount of σS in the wild-type and ?phoQ strains at indicated time points after 600 mM NaCl addition, monitored via immunoblotting with anti-RpoS primary antibody. Equal amounts of cells were collected at each time point for cell lysate preparation and equal amounts of cell lysate were loaded in each lane. The immunoblot is a representative of three independent experiments. (C) Transcription profiles of mgtA and of σS anti-adaptor genes (iraM, iraP, and iraD), measured by RT-qPCR after addition of 300 mM NaCl to E. coli expressing the wild-type PhoQ or PhoQN202A. All data points represent the averages of three independent experiments and error bars show SDs. The transcription up-regulation is significant at P < 0.0001 for mgtA and iraM in E. coli expressing the wild-type PhoQ. (D) Growth of indicated E. coli strains in the absence or presence of 600 mM NaCl, as in A.

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