1. Microbiology and Infectious Disease
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A lipoprotein/β-barrel complex monitors lipopolysaccharide integrity transducing information across the outer membrane

  1. Anna Konovalova
  2. Angela M Mitchell
  3. Thomas J Silhavy  Is a corresponding author
  1. Princeton University, Lewis Thomas Laboratory, United States
Research Article
Cite this article as: eLife 2016;5:e15276 doi: 10.7554/eLife.15276
5 figures and 1 additional file

Figures

Figure 1 with 1 supplement
PMB causes a specific OM defect.

(A) PMB at 0.5 µg/ml causes a slight OM permeability defect based on increased uptake and fluorescence of NPN dye. Graphs represent mean normalized end-point fluorescence +/-SD, n=3. (B) This concentration of PMB does not cause depolarization of the IM. Unlike gramicidin, PMB is unable to release PMF-dependent DiSC3(5) dye. Graphs represent mean normalized end-point fluorescence +/-SD, n=3. (C) Kinetics of Rcs induction upon PMB and Glb treatment at the mRNA (upper panel) and protein level (lower panel). Induction was monitored using a chromosomal PrprA-lacZ reporter by qRT-PCR or β-galactosidase assays. For mRNA quantification, graphs represent relative expression values normalized to a no treatment control for each time point +/- SD. β-galactosidase activity represent mean Vmax normalized to OD600, +/- SEM, n=3. (D) PMB induces Rcs but not the Cpx or SigmaE stress responses. Induction was monitored by following the relative expression of PrprA-lacZ (Rcs), cpxP (Cpx) or rpoE (SigmaE) by qRT-PCR. Graphs represent mean +/- SEM, n=3. (E) PMB does not cause OMP assembly defects based on immunoblot analysis.

https://doi.org/10.7554/eLife.15276.003
Figure 1—figure supplement 1
Glb induces Rcs but not the SigmaE stress response.

Induction was monitored by following the expression of PrprA-lacZ (Rcs) and rpoE (SigmaE) by qRT-PCR. Graphs represent relative expression values normalized to a no treatment control for each time point, mean +/- SEM, n=3

https://doi.org/10.7554/eLife.15276.004
The PMB-induced Rcs response is independent of de novo protein synthesis.

Cell cultures were pretreated with Ksg to inhibit protein synthesis for 15 min prior to addition of PMB or Glb. Rcs induction was then monitored by qRT-PCR. Graphs represent relative expression values normalized to a no treatment control for each time point, mean +/- SEM, n=3

https://doi.org/10.7554/eLife.15276.005
Figure 3 with 2 supplements
RcsF/OMP complexes are required for sensing OM stress.

(A) Topology and assembly pathway of RcsF/OMP complexes (based on (14)). The lipidated N-terminus of RcsF is anchored in the outer leaflet of the OM exposing residues 16–49 on the cell surface. The transmembrane segment (residues 50–65) of RcsF is threaded through the lumen of the OMP exposing the C-terminal domain in the periplasm. RcsF/OMP complexes are assembled by the Bam machine. Not all OMPs are complexed with RcsF. The effect of different mutants used in this study on the assembly of RcsF/OMP complexes is shown. (B) The effect of ompA, bamE and rcsF_A55Y mutations on RcsF crosslinking to BamA and OmpA (upper panel) and the total RcsF, BamA and OmpA levels based on immunoblot analysis. (C) The ompA, bamE and rcsF_A55Y mutants do not respond to PMB (upper panel) but respond to Glb (lower panel) treatment based on expression of PrprA-lacZ. Graphs represent mean β-galactosidase activity +/- SEM, n=3 (D) The bamE and rcsF_A55Y mutants result in decreased PrprA-lacZ. Graphs represent mean β-galactosidase activity +/- SEM, n=3. Corresponding OD600 graphs and untreated controls are shown in Figure 3—figure supplement 1.

https://doi.org/10.7554/eLife.15276.006
Figure 3—figure supplement 1
Growth and kinetcs of Rcs induction in the assembly mutants including untreated cotrols.

(A) MIC values for the RcsF/OMP assembly defective strains. (B) Kinetics of PrprA-lacZ expression in RcsF/OMP assembly defective strains upon treatment with PMB, Glb (same as Figure 3C) and untreated controls together with corresponding growth curves. Graphs represent mean β-galactosidase activity or OD600 +/- SEM, n=3

https://doi.org/10.7554/eLife.15276.007
Figure 3—figure supplement 2
Growth phenotype of the assembly mutants.

(A) Growth curves of the assembly mutants in waaP background. Strains were grown at 37° C in 1 ml of LB in 24 well plate and OD600 was monitored with a Biotek Synergy 1 plate reader. Note, waaP ompA strains display a synthetic growth phenotype. Graphs represent mean OD600 +/- SEM. (B) Plate phenotype of the assembly mutants in the WT and waaP backgrounds. Strains were grown on LB agar at 37⁰ C overnight. Note, that the waaP mutation confers a RcsF-dependent mucoid phenotype. Introduction of a bamE mutation in waaP strain results in a loss of this phenotype. pZS21::rcsF complements waaP rcsF mutant but does not confer a gain-of-function phenotype in the WT background. pZS21::rcsF_A55Y failed to complement the mucoid phenotype.

https://doi.org/10.7554/eLife.15276.008
RcsF senses alteration in LPS structure.

(A) Structure and biosynthesis of E. coli K-12 LPS according to (Raetz and Whitfield, 2002). Non-essential enzymes are shown in bold. (B) The effect of mutations in non-essential genes in the LPS biosynthesis pathway on Rcs induction based on PrprA-lacZ expression. (C and D) OM permeability is not a physiological inducing signal for Rcs. (C) Mutations that cause defects in asymmetry (pldA mlaA) or LPS export (lptD4213 and lptE_R91D K136D) do not induce Rcs. RcsF is not generally inhibited in these strains because Rcs can still be induced by introducing the waaP mutation. (D) Mutations pldA mlaA, lptD4213 and lptE_R91D K136D confer OM permeability defects (see text for references) assayed by plating 10-fold serial dilutions of overnight cultures onto LB plates supplemented with antibiotics or detergents. Note, arabinose (Ara) is required for growth of lptE_R91D K136D mutant. (E) The addition of Mg2+ reduces Rcs signaling in the waaP background in a phoP-independent manner. (F) Lipid-truncated PMB derivative, PMBN, induces Rcs in concentration-dependent manner. Graphs B, C, E and F represent mean β-galactosidase activity +/- SEM, n=3

https://doi.org/10.7554/eLife.15276.009
Figure 5 with 2 supplements
Positive charge of the surface-exposed region of RcsF is required for LPS-sensing.

(A) Amino acid sequences and total charge of the surface exposed domain of RcsF_WT and the charge substitution mutant RcsF_A5. Positively charged residues (Lys and Arg) which were substituted by Ala are underlined. (B) Charge substitution mutations do not affect RcsF crosslinking to BamA and OmpA. (C) The rcsF_A5 mutant does not respond to PMB (upper panel) but does respond to Glb (lower panel) treatment based on expression of a PrprA-lacZ reporter analyzed by beta-galactosidase assay. (D) The rcsF_A5 mutant results in decreased PrprA-lacZ expression in the waaP background. Graphs C and D represent mean β-galactosidase activity +/- SEM, n=3. Corresponding OD600 graphs and untreated controls are shown in Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.15276.010
Figure 5—figure supplement 1
Kinetics of PrprA-lacZ expression in RcsF/OMP charge substitution strains upon treatment with PMB, Glb (same as Figure 5C) and untreated controls together with corresponding growth curves.

Graphs represent mean β-galactosidase activity or OD600 +/- SEM, n=3

https://doi.org/10.7554/eLife.15276.011
Figure 5—figure supplement 2
Plate phenotype of the charge substitution mutant in the WT and waaP background.

Strains were grown on LB agar at 37⁰C overnight. Note, that the waaP mutation confers a RcsF-dependent mucoid phenotype. pZS21::rcsF complements waaP rcsF mutant but does not confer gain-of-function phenotype in the WT background. pZS21::rcsF_A5 failed to complement mucoid phenotype.

https://doi.org/10.7554/eLife.15276.012

Additional files

Supplementary file 1

Strains used in this study.

https://doi.org/10.7554/eLife.15276.013

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