Protease homolog BepA (YfgC) promotes assembly and degradation of β-barrel membrane proteins in Escherichia coli

Abstract

Gram-negative bacteria are equipped with quality-control systems for the outer membrane (OM) that sense and cope with defective biogenesis of its components. Accumulation of misfolded outer membrane proteins (OMPs) in Escherichia coli leads to activation of σ(E), an essential alternative σ factor that up-regulates transcription of multiple genes required to preserve OM structure and function. Disruption of bepA (formerly yfgC), a σ(E)-regulated gene encoding a putative periplasmic metalloprotease, sensitizes cells to multiple drugs, suggesting that it may be involved in maintaining OM integrity. However, the specific function of BepA remains unclear. Here, we show that BepA enhances biogenesis of LptD, an essential OMP involved in OM transport and assembly of lipopolysaccharide, by promoting rearrangement of intramolecular disulfide bonds of LptD. In addition, BepA possesses protease activity and is responsible for the degradation of incorrectly folded LptD. In the absence of periplasmic chaperone SurA, BepA also promotes degradation of BamA, the central OMP subunit of the β-barrel assembly machinery (BAM) complex. Interestingly, defective oxidative folding of LptD caused by bepA disruption was partially suppressed by expression of protease-active site mutants of BepA, suggesting that BepA functions independently of its protease activity. We also show that BepA has genetic and physical interaction with components of the BAM complex. These findings raised the possibility that BepA maintains the integrity of OM both by promoting assembly of OMPs and by proteolytically eliminating OMPs when their correct assembly was compromised.

Keywords: disulfide bond formation; extracytoplasmic function sigma factor; peptidase M48; protein quality control; tetratricopeptide repeat (TPR) motif.

Fig. 1.

Protease activity is required for BepA function. (A) Protease activity of BepA. α-casein (400 µg/mL) was incubated without (−) or with His₁₀-tagged wild-type BepA (WT) or its E137Q derivative (E137Q) (200 µg/mL) at 37 °C for 0 or 24 h. Proteins were separated by SDS/PAGE and visualized by staining with Coomassie brilliant blue R-250. Proteolytic product of α-casein is indicated by an open arrowhead. Migration positions of molecular weight markers are shown on the left. (B) Effects of metal chelators on the BepA protease activity. Protease activity of C-terminally His₁₀-tagged wild-type BepA was analyzed as in A except that the reaction mixture contained 50 μM ZnCl₂ (Zn), 250 μM 1,10-Phenanthroline (PT), or 250 μM EDTA. (C) Erythromycin sensitivity of cells expressing BepA or its derivative. The minimum inhibitory concentration (MIC) of erythromycin for the ΔbepA or wild-type strain transformed with pUC18 vector, pUC-bepA, pUC-bepA(H136R), or pUC-bepA(E137Q) encoding wild-type, H136R mutant, or E137Q mutant BepA, respectively, at 30 °C is shown. (D) Cellular levels of BepA. BepA levels in the ΔbepA cells used in C were determined by immunoblotting with anti-BepA antiserum. Maltose-binding protein (MBP) was also detected by immunoblotting with anti-MBP antibodies and was used as a loading control.

Fig. 2.

Overexpression of LptE suppresses the drug sensitivity of the ΔbepA strain. (A) Erythromycin sensitivity of the ΔbepA strain transformed with an ASKA clone carrying lptE. pCA24NΔNot was used as a vector control. For comparison, the MIC of the ΔbepA strain transformed with pTH-bepA-his₁₀ encoding wild-type BepA_His10 is shown. (B) LptE was overexpressed from pMAN-lptE carrying lptE under the control of the araBAD promoter. Expression of LptE was confirmed by SDS/PAGE of total cellular proteins followed by anti-LptE immunoblotting.

Fig. 3.

Defective oxidative folding of LptD in the absence of BepA. (A) Immunoblotting of LptD was carried out after reducing (+ME) or nonreducing (−ME) SDS/PAGE of total cellular proteins. Wild-type (+) or ΔbepA (−) cells were grown overnight at 30 °C. Anti-LptE immunoblotting was also carried out after reducing SDS/PAGE. The positions of reduced LptD (LptD^RED), properly oxidized LptD (LptD^NC), rapidly migrating LptD (LptD^C), LptE, and molecular weight markers are indicated. (B) Schematic representation of disulfide bonds in LptD^NC and LptD^C. Asterisk indicates a nonspecific band.

Fig. 4.

BepA facilitates disulfide isomerization in LptD. (A) Wild-type and ΔbepA cells were labeled with [³⁵S]-methionine for 1 min and chased for the indicated durations at 30 °C. Acid-precipitated proteins were subjected to immunoprecipitation with anti-LptD antiserum and were analyzed by nonreducing (−ME) or reducing (+ME) SDS/PAGE followed by phosphorimaging. Migration positions of molecular weight markers are shown on the left. (B) Oxidative folding of LptD was monitored as in A using ΔbepA cells transformed with empty vector or either of the plasmids encoding C-terminally His₁₀-tagged wild-type BepA or its H136R or E137Q derivative. LptD^NC (%) in Right was calculated by dividing the band intensity of LptD^NC by the sum of those of LptD^C and LptD^NC.

Fig. 5.

BepA destabilizes LptD in LptE-limiting conditions. (A) LptE-depleted cells carrying bepA (bepA⁺) or its disruptant were pulse-labeled and analyzed as described in the legend to Fig. 4A. (B and C) The stability of LptD was assessed as in A using the ΔbepA (B) or wild-type (C) cells transformed with empty vector or either of the plasmids encoding C-terminally His₁₀-tagged wild-type BepA or its H136R or E137Q derivative.

Fig. 6.

bamB/bepA and bamE/bepA double disruptants show elevated drug sensitivity. (A) MICs of erythromycin (ERM), rifampicin (RIF), vancomycin (VCN), and novobiocin (NOV) at 30 °C for strains lacking bepA, bamB, bamC, and bamE individually or in combinations as indicated. (B) Overnight cultures of strains lacking bepA, bamB, bamC, and bamE individually or in combinations as indicated were serially diluted with saline, spotted onto L agar with or without 0.5% SDS, and incubated at 30 °C for 24 h. (C) bamB/bepA and bamE/bepA double-knockout strains were transformed with a plasmid encoding wild-type BepA or its H136R or E137Q derivative and were grown on L agar containing 0.5% SDS for 24 h at 30 °C or 37 °C.

Fig. 7.

BepA interacts with the BAM complex. (A–G) BS³-mediated cross-linking. (A and B) Wild-type cells transformed with the pUC18 vector (−) or its derivative encoding the C-terminally His₁₀-tagged wild-type BepA (WT) or its H136R or E137Q derivative were subjected to cross-linking with BS³ as described in Materials and Methods. Membrane (M) and periplasmic (P) fractions were analyzed to SDS/PAGE followed by immunoblotting with anti-BepA (A) or anti-BamA (B) antisera. The cross-linked product of BepA and BamA is indicated (× BamA; also indicated by asterisks). (C–G) The ΔbepA strain (WT) or the ΔbepA strain also having deletions of bamB (ΔB), bamC (ΔC), or bamE (ΔE) were transformed with empty vector (–) or a plasmid encoding tag-free BepA(E137Q) and were subjected to BS³ cross-linking. Membrane fractions were analyzed by WIDE RANGE PAGE (Nacalai Tesque) (7.5% acrylamide), except for D, in which normal SDS/PAGE was used, followed by immunoblotting with anti-BepA (C), anti-BamA (D), anti-BamB (E), anti-BamC (F), or anti-BamD (G) antisera. (H) Photo–cross-linking of BepA and BamA. The ΔbepA cells transformed with pEVOL-pBpF (aminoacyl-tRNA synthetase/suppressor tRNA) and the plasmid encoding a BepA derivative with an amber codon at position 428 were grown with or without pBPA and were UV-irradiated for 10 min. Acid-precipitated proteins were analyzed by SDS/PAGE and immunoblotting with anti-BepA and anti-BamA antisera. The cross-linked product between BepA(Q428pBPA) and BamA is indicated. (I) Pull-down assay. Wild-type strain (WT) transformed with empty vector or the ΔbamB strain transformed with a plasmid encoding C-terminally His₆-tagged BamB (BamB_His6) was cotransformed with a plasmid encoding BepA(E137Q). Membrane proteins were solubilized and subjected to a pull-down assay using His₆ tag of BamB. Solubilized membrane (input) and affinity-purified proteins (eluate, a fivefold equivalent for Bam component immunoblotting and a 50-fold equivalent for BepA and OmpA immunoblotting) were analyzed by SDS/PAGE and immunoblotting.

Fig. 8.

BepA-dependent degradation of BamA in the ΔsurA strain. Total cellular protein was prepared from wild-type or mutant cells lacking bepA and/or surA (lanes 1–4) or ΔsurA/ΔbepA cells harboring empty vector or either of the plasmids encoding C-terminally His₁₀-tagged wild-type BepA or its H136R or E137Q derivative (lanes 5–8) and was subjected to SDS/PAGE followed by immunoblotting with anti-BamA or anti-BepA antisera. Putative BepA degradation products of BamA are indicated by asterisks.

Fig. 9.

A model for BepA function in the assembly/degradation of the OM LPS translocon. (Upper) BepA promotes disulfide rearrangement of LptD^C that is triggered by association of LptD^C with LptE. BepA may directly assist LptD-LptE interaction or act indirectly to facilitate formation of the productive LptD-LptE complex. Square brackets represent disulfide bonds (Lower). In the absence of LptE, BepA acts to proteolytically eliminate accumulated LptD^C.