Article Figures & Data
Figures
- FIG 1
NlpE and Cpx are required for tolerance of LolB depletion. (A) Overview of lipoprotein biogenesis and trafficking. Lipoproteins are secreted via the Sec translocon and are acylated at Cys+1 in the IM. Mature triacylated lipoproteins that are targeted for the OM enter LolCDE for extraction from the IM. LolA and LolB are part of an efficient trafficking pathway that is essential in wild-type cells. An alternate LolAB-independent pathway can also traffic lipoproteins but is insufficient in wild-type cells. Asp residues at +2 and +3 amino acids cause IM retention of lipoproteins. The targets of Glb and Cpd2 inhibitors are shown. (B) Strains tested for tolerance to LolB depletion. Expression of LolB was repressed by culturing in the absence of l-arabinose. Ten-fold serial dilutions of cultures are shown.
- FIG 2
The NlpE N-terminal domain is sufficient for tolerance of LolB depletion and activation of Cpx. (A) Schematic of NlpE structure in its extended conformation. The N-terminal domain (orange) is joined to the C-terminal domain (blue) via a linker region (black). Sites of truncations are marked with spheres; green spheres indicate truncations that are able to activate Cpx, red spheres indicate truncations that fail to activate Cpx, and gray spheres show the Cys residues in a putatively redox-sensitive CXXC motif. (B) nlpE mutants were tested for their ability to tolerate LolB depletion (− arabinose) in an lpp(ΔK58) ΔosmB background. (C) Relative LacZ levels in ΔnlpE cells harboring a PcpxP-lacZ transcriptional reporter and overproducing plasmid-borne NlpE variants targeted to the OM. (D) Relative LacZ levels in ΔnlpE cells encoding a PcpxP-lacZ transcriptional reporter and overproducing plasmid-borne NlpE(DD) variants targeted to the IM. Data are means ± standard deviations.
- FIG 3
Inhibitors of lipoprotein biogenesis Glb and Cpd2 activate Cpx through NlpE. Cells were treated with either Glb or Cpd2 lipoprotein trafficking inhibitors (or DMSO vehicle control) for 20 min. RNA was then extracted and subjected to qRT-PCR to quantitate levels of cpxP mRNA. Ksg-treated cells (+Ksg) were treated with a sub-MIC of Ksg for 15 min prior to Glb or Cpd2 treatment (see Materials and Methods). Data are means ± standard errors of the means.
- FIG 4
Cu impairs lipoprotein biogenesis and activates Cpx through NlpE. (A) Cultures were serially diluted on LB agar and LB agar supplemented with 4 mM CuCl2. (B) Cultures were grown in the presence of 3 mM CuSO4 to mid-log phase, and levels of cpxP mRNA were measured. Samples were prepared and analyzed together with samples presented in Fig. 3, the DMSO control presented is the same here as in Fig. 3 Data are means ± standard errors of the means. (C) Relative LacZ levels in ΔnlpE cells harboring a PcpxP-lacZ transcriptional reporter and overproducing plasmid-borne NlpE variants targeted to the OM. Data are means ± standard deviations. (D) Cultures were grown to mid-log phase in the presence (Cu +) or absence (Cu −) of 3 mM CuCl2. Lgt and Lnt replete (+) or deplete (−) samples were obtained by growing strains PAP9403 and KA472 in the presence or absence of arabinose; LspA activity was inhibited by treating cells with Glb (LspA −) in comparison to mock treatment (LspA +). Protein samples were taken and probed for Lpp by immunoblotting. Diacyl form Lpp is noted as +2. Lpp forms with signal peptides attached are noted as +SP. Peptidoglycan-bound Lpp forms are noted as * and **. See text for details.
Tables
- TABLE 1
Summary of NlpE constructs in this study
Name Description Membrane targeting Cpx activation NlpE Full-length wild-type NlpE OM trafficked Yes NlpE(DD) Full-length NlpE with N2D and N3D substitutions that cause avoidance of LolCDE IM retained Yes NlpE1–121 NlpE that lacks the C-terminal domain OM trafficked Yes NlpE(DD)1–121 Lacks the C-terminal domain; has the Lol avoidance signal IM retained Yes NlpE1–101 Lacks the C-terminal domain and the linker region OM trafficked Yes NlpE(DD)1–-101 Lacks the C-terminal domain and the linker region; has the Lol avoidance signal IM retained Yes NlpE1–94 Lacks the C-terminal domain, the linker region, and a portion of the N-terminal domain OM trafficked No NlpE(DD)1–94 Lacks the C-terminal domain, the linker region, and a portion of the N-terminal domain; has the Lol avoidance signal IM retained No NlpE1–82 Lacks the C-terminal domain, the linker region, and a portion of the N-terminal domain OM trafficked No NlpE(DD)1–82 Lacks the C-terminal domain, the linker region, and a portion of the N-terminal domain; has the Lol avoidance signal IM retained No NlpE(C31S C34S) Substitutions in N-terminal domain Cys residues proposed to form a disulfide bond OM trafficked Yes
FIG S1
CpxA is required for tolerance of LolB depletion. Strains were tested for tolerance to LolB depletion. Expression of LolB was repressed by culturing in the absence of l-arabinose. Ten-fold serial dilutions of cultures are shown. Download FIG S1, TIF file, 0.6 MB.
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FIG S2
NlpE activation of Cpx requires the IM CpxA sensor kinase. Relative LacZ levels in ΔnlpE cells harboring a PcpxP-lacZ transcriptional reporter and overproducing plasmid-borne NlpE variants targeted to the OM or NlpE(DD) variants targeted to the IM. Data are means ± standard deviations. Increased basal Cpx signaling in ΔcpxA::cam strains is due to low level phosphorylation of the CpxR response regulator by acetyl phosphate (1). Download FIG S2, TIF file, 0.5 MB.
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FIG S3
The lipoprotein biogenesis inhibitor Glb activates Cpx partially through NlpE but not Blc or YafY. Cells were treated with Glb (or DMSO vehicle control) for 20 min. RNA was then extracted and subjected to qRT-PCR to quantitate levels of cpxP mRNA. Data are means ± standard errors of the means. Download FIG S3, TIF file, 0.4 MB.
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FIG S4
The NlpE N-terminal domain is sufficient to confer resistance to Cu. Cultures were serially diluted, plated on LB agar and LB agar supplemented with 4 mM CuCl2, and incubated overnight at 37°C. Download FIG S4, TIF file, 0.3 MB.
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TABLE S1
Strains used in this study. Download Table S1, DOCX file, 0.1 MB.
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TABLE S2
Plasmids used in this study. Download Table S2, DOCX file, 0.1 MB.
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TABLE S3
Oligonucleotides used in this study. Download Table S3, DOCX file, 0.1 MB.
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TEXT S1
Supplemental references. Download Text S1, DOCX file, 0.1 MB.
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