High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function

Abstract

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the β-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF's partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.

Keywords: Rcs phosphorelay; envelope biogenesis; envelope stress response; surface-exposed lipoproteins.

Fig. 1.

Proposed mechanistic models for the Rcs stress response. Rcs components (orange) are shown in the context of the envelope structure and biogenesis pathways. The sensory lipoprotein RcsF and the negative regulator IgaA are central to the regulation of RcsCDB phosphorelay. RcsF is exported to the OM by the Lol pathway; the Bam complex assembles RcsF with partner OMPs, leading to a partially surface-exposed topology. Red arrows represent proposed signaling events in response to stress (red stars) that are not yet fully understood. (A) Proposed model for the OM/LPS sensing by RcsF. Cell surface localization of RcsF NTD enables RcsF to monitor the integrity of the outer leaflet. Upon LPS stress (e.g., PMB treatment), the signal is transduced to the periplasmic CTD through the conformational change in the RcsF/OMP complex stimulating downstream signaling. (B) Proposed model for the Bam complex sensing function of RcsF. Envelope stress by an unknown mechanism inhibits the Bam complex function; as a result, RcsF/BamA interaction is prevented, and RcsF is accumulated in the periplasmic-facing orientation stimulating downstream signaling.

Fig. 2.

A pattern of RcsF-pBPA site-specific cross-linking to BamA in bamE+ or ΔbamE backgrounds. Cells expressing RcsF^pBPA variants at the indicated codons or an RcsF^WT (no pBPA negative control indicated by “−“) were subjected to UV cross-linking, and cell lysates were analyzed by immunoblotting with an α-BamA antibody. Size shifts indicate RcsF/BamA complexes. RcsF sites of BamA cross-link in both bamE+ or ΔbamE backgrounds are highlighted in red. RcsF sites of BamA cross-link specific to ΔbamE are highlighted in blue.

Fig. 3.

RcsF mutant library performance. (Left) Log2[FC] of individual RcsF variants plotted against the RcsF residue number. Nonsense variants (green squares), synonymous variants (red circles), and RcsF^SUP variants characterized in detail (blue diamonds) are highlighted. (Right) Violin plot of log2[FC] distribution within RcsF variant groups. The solid horizontal line represents the median. Statistical analysis was performed using one-way ANOVA (multiple comparison). (A) A total of 11 generations of outgrowth in an rcsB+ background; (B) A total of 18 generations of outgrowth in an rcsB+ background; (C) A total of 18 generations of outgrowth in a ΔrcsB background. The complete data for all detected RcsF variants are presented in Dataset S1. n.s. = P ≥ 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 4.

Phenotype of RcsF^SUP variants in different genetic backgrounds. (A) Chromosomal rcsF^SUP mutants express RcsF at a WT level. (B) Immunoblot analysis of in vivo formaldehyde cross-linked samples probed with α-BamA (Top) or α-RcsF antibodies (Bottom). Immunoblot quantification can be found in SI Appendix, Fig. S5. (C) Rcs activity as measured by a β-galactosidase assay using a PrprA-lacZ transcriptional reporter. Strains grown in LB and treated with 0.75 μg/mL PMB, where indicated, for 40 min. Graphs represent mean β-galactosidase activity normalized to OD₆₀₀ ± SEM. Statistical analysis was performed, using the two-way ANOVA (multiple comparison), between RcsF^SUP variants and a WT RcsF control under the same conditions. n.s. = P ≥ 0.05, ***P < 0.001, ****P < 0.0001.

Fig. 5.

Phenotype of BamA^SUP variants in different genetic backgrounds. (A) Immunoblot analysis of in vivo formaldehyde cross-linked samples probed with α-BamA (Top) and α-RcsF antibodies. Immunoblot quantification can be found in SI Appendix, Fig. S11. (B) Rcs activity measured as described in Fig. 4. Statistical analysis was performed using two-way ANOVA (multiple comparison) between the BamA^S715R variant and a WT BamA control under the same conditions. (C) Strep-Tactin purification of RcsF/BamA^SUP and RcsF^SUP/BamA complexes in the absence of cross-linking. Immunoblots were probed with α-BamA and α-RcsF antibodies. n.s. = P ≥ 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

In vitro pull-down of variant RcsF/BamA complexes. Purified RcsF-Strep and His-BamA, and their variants, were incubated in equimolar amounts before being subjected to Ni-NTA purification. Equal volume of the first elution fractions was resolved on an SDS-PAGE gel and visualized using Imperial Protein Stain. All other fractions are shown in SI Appendix, Fig. S12. (A) Interactions between RcsF variants and full-length BamA (BamA_FL). (B) Interactions between RcsF variants and the BamA β-barrel domain (BamA_β). (C) Interactions between BamA_β variants and RcsF^WT.

Fig. 7.

Phenotypes of RcsF and BamA variants in a BamA+++ background. (A) The structure of the RcsF/BamA complex (Protein Data Bank 6T1W); RcsF is shown in orange, and BamA is shown in green (33). The BamA Potra domains are omitted for clarity. RcsF^SUP residues are shown as yellow spheres. BamA^SUP residues described previously (38) and in this study are shown as purple and magenta spheres, respectively (B and D). Immunoblot analysis of in vivo formaldehyde cross-linked samples probed with α-BamA (Top) and α-RcsF antibodies. Immunoblot quantification is shown in SI Appendix, Fig. S13. (C) Rcs activity measured as described in Fig. 4. Statistical analysis was performed, using two-way ANOVA (multiple comparison), in comparison with the untreated WT with EV. n.s. = P ≥ 0.05, **P < 0.01, ****P < 0.0001.

Fig. 8.

The RcsF/BamA complex that forms in the absence of BamE is a dead-end product. The AK-1478 strain was grown until midlog in the presence of tetracycline to allow expression of RcsF, and the sample was taken (time point 0). Remaining cells were washed and resuspended in fresh media supplemented with tetracycline and arabinose to promote RcsF and BamE expression, as indicated, and grown for 30 min. Immunoblot analysis of total cell pellet (Top) and in vivo formaldehyde cross-linked samples (Middle) probed with α-BamE and α-RcsF antibodies. Quantification of RcsFxOmpA band (Bottom) as a percentage of total RcsF based on the three biological replicates. Statistical analysis was performed using one-way ANOVA in comparison with the time point 0. ***P < 0.0001.

Fig. 9.

Disruption of the RcsF/BamA complex does not induce Rcs signaling. (A) Immunoblot analysis of in vivo formaldehyde cross-linked samples probed with α-BamA (Top) and α-RcsF antibodies. Immunoblot quantification is shown in SI Appendix, Fig. S14. (B) Rcs activity measured as described in Fig. 4. Statistical analysis was performed, using two-way ANOVA (multiple comparison), in comparison with a WT control. (C) RcsF and BamA variant sensitivity to detergent or vancomycin. Overnight cultures were serially diluted and plated on LB agar supplemented with 0.5% SDS/0.5 mM EDTA or 75 µg/mL vancomycin. n.s. = P ≥ 0.05, ***P < 0.001.

Fig. 10.

Rcs activation upon Bam complex inhibition. (A and B) Rcs activity of strains grown in LB or LB supplemented with 10 mM MgSO₄ or CaCl₂ were measured as described in Fig. 4. Statistical analysis was performed using one-way ANOVA and indicated as follows: P > 0.05 (ns), **P < 0.01. (C) Activity of fluorescent Rcs (P_rprA-GFP) and σE (P_micA-GFP) reporters expressed as relative fluorescence units (RFU) normalized to OD₆₀₀. Strains were grown in LB or LB supplemented with 10 mM MgSO₄ or CaCl₂, as indicated. After 90 min, cells were treated with 32 μg/mL MRL-494, 1 μg/mL darobactin, or a vehicle control (DMSO or H₂O, respectively) for 3 h. Statistical analysis was performed, using one-way ANOVA, in comparison with the vehicle control. n.s. = P ≥ 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.