Dissection of β-barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli

Abstract

BamA of Escherichia coli is an essential component of the hetero-oligomeric machinery that mediates β-barrel outer membrane protein (OMP) assembly. The C- and N-termini of BamA fold into trans-membrane β-barrel and five soluble POTRA domains respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β-barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site-specific cross-linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β-barrel OMPs except for TolC, which folds into a unique soluble α-helical barrel and an OM-anchored β-barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalysed by the trans-membrane β-barrel domain of BamA.

Fig 1

Two-dimensional (2D) gel electrophoresis analyses of envelope fractions enriched for integral membrane proteins. (A and B) Coomassie blue stained gels containing protein samples from strains expressing bamA⁺ (A) and bamA66 (B) from pZS21. (C). Individual 2D gel images from A and B are overlaid to show relative protein abundance. Gel images containing protein samples from wild type and bamA66 strains are colored green and magenta, respectively.. Overlapping areas within a spot common in both wild type and mutant appear black; predominance of green indicates that the corresponding protein spot is more abundant in wild type than mutant, and predominance of magenta indicates the opposite. (D) A montage of individual Coomassie blue stained 2D gels containing envelope samples from wild type and mutant strains analyzed in (A and B). Areas outlined by green show a close-up view of gel regions encompassing BamA and LptD (Imp) spots. All numbered and labeled protein spots were identified using LC/MS/MS and are listed in (E). Isolation of envelopes and enrichment of integral membrane proteins were carried out as described in the Experimental procedures’ section.

Fig 2

Comparative analysis of selected envelope proteins from wild type, bamA66, and ΔsurA strains grown at 30°C. Wild type BamA and BamAΔR64 were expressed from pZS21 (low level constitutive expression), pBAD33 (induced with 0.1% arabinose), or from the native chromosomal location. Lanes 7 and 8 contain envelope samples prepared from surA⁺ and ΔsurA strains, respectively. Proteins were detected either by Western blot analysis using antibodies shown in the figure (A and C) or by SDS(urea)-PAGE analysis followed by Coomassie blue staining (B). AcrA serves as both an envelope preparation and gel loading control.

Fig 3

An examination of relative gene expression in the bamA66 (pZS21) and ΔsurA mutants using quantitative real-time PCR analysis. RNA was isolated from mid-log phase grown cultures at 30°C. Relative quantification of gene transcripts was performed in triplicate using the 2^−ΔΔCT method and both ndh and ftsL as reference genes. Relative fold-changes in gene expression and error bars representing standard deviation are shown (n=2).

Fig 4

The effects of ΔrseA and bamA66 (pZS21) on OmpF and LamB protein levels when expressed from the arabinose P_BAD promoter contained on the pBAD24 vector. (A) To determine ompF and lamB sensitivity to σ^E-mediated down-regulation, the levels of OmpF and LamB, expressed from either their native chromosomal location (left panel) or from the pBAD24 plasmid (middle and right panels, respectively), were evaluated in wild type (WT) and ΔrseA (Δ) backgrounds. The chromosomal status of ompF and lamB is shown below each panel. For Western blot analysis, 5 ml of LB containing ampicillin was inoculated with overnight grown cultures and grown at 37°C. Upon reaching an OD₆₀₀ of approximately 0.4, 0.001% arabinose (w/v, final) was added to all cultures for 30 min. After induction, cultures were chilled on ice and whole cell lysates were analyzed by SDS-PAGE. Proteins were detected by Western blots using appropriate polyclonal antibodies. (B and C) Expression of ompF (B) and lamB (C) from the chromosome or pBAD24 in backgrounds expressing wild type (WT) or mutant BamAΔR64 (ΔR64) from pZS21. Methods were followed as described in (A) with the exceptions that whole cell lysates were obtained from cultures grown at 30°C and inoculated with 0.001% or 0.005% arabinose for 45 or 60 minutes (B and C, respectively). The pBAD24-phoA construct was used as an induction control and GroEL as a sample loading control.

Fig 5

The effects of BamAΔR64 depletion on cell growth and TolC/LamB levels. (A) Cells deleted of chromosomal bamA and harboring pBAD33-bamA66 were grown in LB at 30°C with (circles) and without (squares) arabinose (0.1%, w/v). After the first dilution from overnight cultures, OD₆₀₀ was measured from samples taken every hour. (B) Western blot analysis to detect TolC, LamB, and AcrA from equal number of cells (based on OD₆₀₀) withdrawn at time points indicated by downward arrows in (A). An open downward arrow at the 3 h time point indicates that only the sample from cells grown without arabinose was analyzed. AcrA serves as a sample loading control.

Fig. 6

The effects of a BamA POTRA 1 deletion (ΔR36-K89) on LamB, TolC, and BamAΔR36-K89 levels in strains depleted of wild type BamA. (A to D) Strains used were deleted of chromosomal bamA and simultaneously expressed wild type BamA from pBAD33 (arabinose inducible promoter is inhibited by fucose) and wild type (A) or mutant BamA (B and C) from pZS21 (low level constitutive expression). Cultures were grown overnight at 30°C with appropriate antibiotics and 0.05% arabinose (final). Overnight cultures were subsequently diluted (first dilution) to an OD₆₀₀ of 0.025 in 5 ml of fresh medium containing appropriate antibiotics and 0.05% arabinose (Ara) or 0.05% D-fucose (Fuc). After five hours (average OD₆₀₀ 0.8), cultures were again diluted (second dilution) in fresh medium as before and growth was resumed. Samples were taken at hours 3, 4, and 5 (1^st dilution) and 8, 9 and 10 (2^nd dilution) every hour and prepared for Western blot analysis as described in Fig 6B. (D) Samples used in Fig 6C were further analyzed on a 7.5% polyacrylamide gel to separate the two BamA species; wild type BamA and the faster migrating BamA_ΔP1. Antibodies specific to the N-terminal of BamA were used to visualize protein levels. The ratios of BamA_ΔP1 levels when expression of wild type BamA was inhibited (fucose; Fuc) to the levels of BamA_ΔP1 when wild type was induced (arabinose; Ara) are shown along with standard deviation (n=2). Note that BamA N-terminal antibodies recognize BamA_ΔP1 better than wild type BamA. BamA_ΔP1 is short for BamAΔR36-K89.

Fig 7

BamA POTRA 1-SurA cross-linking using four BamA POTRA 1 cysteine variants. (A) A view of the BamA POTRA 1 structure (pdb 2qdf) with its N-terminus rotated in front. Residues R64 and R49 (red) with D28 and D74 (blue) represent the locations of the four BamA cysteine substitutions used for SPDP-mediated cross-linking with SurA. (B) For cross-linking, ΔbamA ΔbamB (pBAD24-BamB_6His) strains expressing wild type BamA or BamA cysteine variants from pBAD33 were grown at 30°C in LB plus arabinose (0.1%, w/v) and three equal culture aliquots were treated with DSP, SPDP or solvent. Immunoprecipitation was carried out as described in the Experimental procedures’ section. 6×His-tagged BamB was used as bait and the levels of BamB_6His and co-immunoprecipitated BamA and SurA from eluents were determined by Western blot analysis using Hisprobe-HRP, TolC/BamA antibodies, and SurA antibodies. SurA levels relative to BamB were quantified for each lane containing either DSP or SPDP cross-linker. The amounts of SurA for each cross-linker are shown as SPDP:DSP ratios ± standard deviation (n=3).

Fig 8

DSP-mediated cross-linking of BamAΔR64 and SurA. ΔbamA ΔbamB (pBAD24-BamB_6His) strains containing either pBAD33bamA (lanes 1–3) and pBAD33bamA66 (lanes 4–6) were grown in LB at 30°C and cross-linked using DSP as described in the Experimental procedures’ section. Eluents from DSP cross-linked cells (lanes 1 and 4) and uncross-linked cells (lanes 2 and 5) were analyzed by SDS-PAGE, followed by detection of protein by silver staining (A) or Western blots (B). BamB 6×His and SurA were visualized using Hisprobe-HRP and SurA antibodies, respectively. Lanes 3 and 6 contain control samples from cells receiving no cross-linker and His antibody.

Fig 9

A cartoon showing distinct pathways for assembly and insertion of integral outer membrane proteins (OMPs) in Escherichia coli. (A) Newly synthesized precursor OMPs traverse the inner membrane (IM) via the SecYEG complex and, upon cleavage of the signal sequence, enter the periplasmic compartment. Here, they are kept soluble/assembly-competent through interactions with chaperones, like SurA, or interact with factors that target them to the outer membrane (OM). The first two assembly pathways split at this step based on their SurA-dependence (LptD [Imp], LamB, and OmpF) or SurA-independence (BamA and TolC). TolC, being SurA-independent, also bypasses a requirement for the BamA POTRA 1 domain, which interacts with SurA. BamA on the other hand appears to require its own POTRA 1 domain for efficient assembly. The remaining two pathways rely on a specific OM-targeting factor, PulS (PulD) or Lol (Wza). Insertion of PulD into the OM has been shown to be independent on BamA, but may become dependent on the Lol system after PulD’s interactions with PulS, which is a lipoprotein. It is not known whether Wza gains entry into the OM exclusively through the Lol system or in addition requires the Bam complex. (B) Disruption of POTRA 1 negatively impacts the assembly of BamA and the SurA dependant OMPs, LptD, OmpF and LamB, leading to increased proteolysis and activation of envelope stress responses (σ^E and Cpx). The activated σ^E and Cpx systems attempt to reduce envelope stress by inhibiting synthesis of several OMPs, including LamB and OmpF, and elevating synthesis of factors that either degrade misfolded OMPs (DegP) or enhance OMP folding (SurA and Bam complex members).