Outer membrane (OM) β-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli, the β-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a ‘top hat’-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel β1C–β6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the β16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique β16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA β1C–β6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts.

Introduction

Outer membrane proteins (OMPs) play important roles in importing nutrients, exporting wastes and transporting biological molecules in Gram-negative bacteria, mitochondria and chloroplasts [1,2]. Mitochondria and chloroplasts are eukaryotic organelles that derived from bacterial endosymbionts of α-proteobacteria and cyanobacteria [2], respectively. The OMPs are inserted and assembled into the outer membrane (OM) by conserved outer membrane protein of 85 kDa (OMP85) family proteins, which share the C-terminal 16 β-stranded barrel structure with different numbers of N-terminal polypeptide transport-associated (POTRA) domains [310]. In Gram-negative bacteria, BamA is the main OMP85 family protein for OMP insertion, while OMP85 family proteins sorting assembly machinery 50 kDa subunit (SAM50) and outer envelope protein of 80 kDa (OEP80) are proposed to be responsible for OMP insertion in mitochondria and chloroplasts [710], respectively, suggesting that the SAM50 and OEP80 may use a similar mechanism as BamA to insert OMPs.

In Escherichia coli, the unfolded OMPs are synthesized in the cytoplasm, and transported across the inner membrane (IM) by the SecYEG complex to the periplasm, where the unfolded OMPs are escorted by chaperon proteins Skp and SurA to the β-barrel assembly machinery (BAM) complex for insertion [11]. The E. coli BAM machinery complex contains an OMP BamA and four lipoproteins BamB, BamC, BamD and BamE, of which BamA and BamD are essential [12]. Individual crystal structures of BamA, BamB, BamC, BamD and BamE have been determined, as well as structures of BamCD complex, BamB in complex with POTRA3 and 4, and BamD in complex with POTRA4 and 5 [7,1328]. In addition, the OEP80 of chloroplasts is evolutionally from cyanobacterial Omp85 proteins, which are structurally characterized with three POTRA domains with a unique loop at POTRA domain 3. The loop is reported to regulate the barrel pore [3,4]. More recently, crystal structures of four-protein subcomplex BamACDE [29,30] and five-protein full complex BamABCDE [29,31] with functional assays and molecular dynamics simulations have been reported, as well as the cryo-EM structure of the BAM complex [32]; these revealed that the five POTRA domains of BamA and the BamB, BamC, BamD and BamE form a ring architecture in the periplasm, and the rotation of the ring structure may promote the scissor-like movement of the β1C–β6C of BamA barrel for OMP insertion [29]. The crystal structures showed that the BAM complex in two distinct conformations and functional assays have confirmed that the two states exist in E. coli [29]. In the BamABCDE complex, the β16C of BamA barrel coils into the lumen; however, whether the shift of the β16C is critical and how the rotation of the ring structure promotes the scissor-like movement are unknown. In contrast with the E. coli BAM complex, it has been reported that the Salmonella BamB can play a role independent of BAM machinery and BamD is not essential for Salmonella[33], suggesting that the BAM machinery subunits may be different from those of E. coli. Here, we report the crystal structure of BamA from S. enterica, which shows that the β16C partially coils into the barrel. We then show the importance of the last residue [tryptophan (Trp)]. Finally, we demonstrate that the periplasmic turns are essential, possibly involved in mediating the periplasmic ring rotation to the β1C–β6C scissor-like movement, while the extracellular loops 1–3 play a less important role for OMP insertion.

Materials and methods

Plasmid construction

Primers used in the present study are listed in Supplementary Table S1. The bamA and bamB genes were amplified by polymerase chain reaction (PCR) from genomic DNA of S. enterica Typhimurium str. LT2. The bamB was cloned into pACYCDuet-1 (Novagen) between NcoI and HindIII sites, and the bamA was sequentially cloned into NdeI and XhoI sites. A hexahistidine (His6) tag was introduced into BamA between Gly23 and Phe24 using site-directed mutagenesis [34], resulting in expression construct pACYC-SenBamAB. The nucleotide sequences were verified by Sanger sequencing. Plasmid pYG91 containing pelB-His10-EcoBamA was constructed previously [29] for E. coli BamA functional assays.

Protein expression and purification

The recombinant plasmid pACYC-SenBamAB was transformed into E. coli BL21(DE3) cells (Novagen) for overexpression. An overnight culture was inoculated in Miller's LB broth medium supplemented with 34 µg ml−1 chloramphenicol, and the cells were grown at 37°C to A600 nm of 0.3–0.4 and then shifted to 25°C. When A600 nm reached 0.5–0.7, expression of SenBamAB was induced with isopropyl β-d-1-thiogalactopyranoside at a final concentration of 100 µmol l−1 at 20°C overnight. Twelve litres of the cells were harvested by centrifugation and resuspended in a lysis buffer [20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 5 µg ml−1 DNase I and 100 µg ml−1 lysozyme], and then disrupted at 30 000 p.s.i. using a cell disruptor (Constant Systems Ltd). Cell debris and unbroken cells were removed by centrifugation, and the resulting supernatant was ultracentrifuged at 100 000 g for 1 h at 4°C. The membrane fraction was resuspended in 20 mM Tris–HCl (pH 8.0) and 150 mM NaCl supplemented with 1% (w/v) N-lauroylsarcosine sodium salt (Sigma–Aldrich) and incubated for 1 h at room temperature with moderate rocking, and then ultracentrifuged again. The pellet was resuspended in a solubilization buffer [20 mM Tris–HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole and 1% 3-(N,N-dimethylmyristylammonio)propanesulfonate (SB3-14) (Sigma–Aldrich)] and solubilized overnight at 4°C with moderate rocking. The solubilized membrane was ultracentrifuged, and the resulting supernatant was applied to a pre-equilibrated nickel–nitrilotriacetic acid superflow resin column (Qiagen). The His-tagged proteins were eluted with an elution buffer [20 mM Tris–HCl (pH 8.0), 300 mM NaCl, 300 mM imidazole, 0.023% (w/v) n-dodecyl-N,N-dimethylamine-N-oxide (LDAO; Anatrace) and 0.53% (w/v) n-octyl-β-d-glucopyranoside (OG; Anatrace)] after wash the resin with 20 column volumes of a wash buffer [20 mM Tris–HCl (pH 8.0), 300 mM NaCl, 30 mM imidazole, 0.023% (w/v) LDAO and 0.53% (w/v) OG]. The eluent was further purified by size-exclusion chromatography (HiLoad 16/600 Superdex 200 prep grade column; GE Healthcare) in a gel filtration buffer [20 mM Tris–HCl (pH 7.8), 100 mM NaCl, 0.023% (w/v) LDAO and 0.53% (w/v) OG]. Fractions containing SenBamAB were pooled and concentrated to 6 mg ml−1 for crystallization.

Crystallization, data collection and structure determination

The purified SenBamAB complex was crystallized using a sitting-drop vapour diffusion method at 22°C. Crystal trials were set up using various commercial crystallization screen conditions. A drop of reservoir solution (0.15 or 0.3 µl) was mixed with an equal amount of protein solution using a crystallization robot Gryphon (Art Robbins Instruments). Crystals were obtained in a buffer containing 0.1 M HEPES sodium (pH 7.5) and 1.0 M sodium citrate tribasic dehydrate. Crystals were harvested and cryoprotected in 20% glycerol supplemented with the precipitation solution, and then flash-cooled and stored in liquid nitrogen. X-ray diffraction data were collected at Diamond Light Source (U.K.) on beamline I04-1 at a wavelength of 0.9200 Å and indexed by iMosflm and further processed with CCP4 package [35]. Data collection and refinement statistics are summarized in Table 1. The structure was solved by the molecular replacement method with Phaser [36] in CCP4 suite [35] using an E. coli BamA β-barrel structure [Protein Data Bank (PDB) accession code 4N75] as the search model. The structure was built in Coot [37], and the structure was refined using REFMAC5 [38]. The final model was validated with Rwork/Rfree values of 0.305/0.348 (Table 1).

Table 1
Data collection and refinement statistics.
Data collectionBamA
Space group P62 2 2 
Cell dimensions 
a, b, c (Å) 124.03, 124.03, 131.48 
α, β, γ (°) 90.0, 90.0, 120.0 
Wavelength (Å) 0.9200 
Resolution (Å) 107.42–2.92 (3.024–2.92) 
I/σ 7.7 (2.5) 
Unique reflections 13 411(1311) 
Completeness 100% (100%) 
Redundancy 17.3 (18.2) 
Rmerge 0.30/1.52 
CC1/2 0.99 (0.5) 
Refinement 
 Resolution (Å) 62.02–2.92 (3.02–2.92) 
Rwork 0.305 (0.424) 
Rfree 0.348 (0.461) 
 RMSD (bonds, Å) 0.024 
 RMSD (angles, °) 2.60 
 Ramachandran favoured (%) 92 
 Ramachandran allowed (%) 6.1 
 PDB access code 5OR1 
Data collectionBamA
Space group P62 2 2 
Cell dimensions 
a, b, c (Å) 124.03, 124.03, 131.48 
α, β, γ (°) 90.0, 90.0, 120.0 
Wavelength (Å) 0.9200 
Resolution (Å) 107.42–2.92 (3.024–2.92) 
I/σ 7.7 (2.5) 
Unique reflections 13 411(1311) 
Completeness 100% (100%) 
Redundancy 17.3 (18.2) 
Rmerge 0.30/1.52 
CC1/2 0.99 (0.5) 
Refinement 
 Resolution (Å) 62.02–2.92 (3.02–2.92) 
Rwork 0.305 (0.424) 
Rfree 0.348 (0.461) 
 RMSD (bonds, Å) 0.024 
 RMSD (angles, °) 2.60 
 Ramachandran favoured (%) 92 
 Ramachandran allowed (%) 6.1 
 PDB access code 5OR1 

Site-directed mutagenesis

Primers used for site-directed mutagenesis of E. coli BamA are listed in Supplementary Table S1. A pRSFDuet-1-based construct, pYG91, containing coding sequences of an N-terminal pelB signal peptide followed by a decahistidine (His10) tag and the mature peptide of E. coli BamA starts from Glu22, was used as a template for PCR [29]. Site-directed mutagenesis was performed according to the protocol of Liu and Naismith [34], with minor modifications. All mutants were confirmed by Sanger sequencing.

Plate complementation assays

E. coli JCM166 cells [5], a BamA depletion strain, were used for functional assays. Plasmid pRSFDuet-1 (empty vector control), pYG91 (wild-type BamA) or BamA mutants were transformed into JCM166 competent cells, respectively. Single colony from LB agar plate containing 50 µg ml−1 kanamycin, 100 µg ml−1 carbenicillin and 0.05% l-(+)-arabinose was inoculated in 10 ml LB medium supplemented with antibiotics and l-(+)-arabinose, and cultured overnight at 37°C. The cells were collected by centrifugation and resuspended in fresh LB medium. After another centrifugation, the cell pellet was resuspended and diluted to A600 nm of ∼0.3. And then, the cells were streaked onto LB agar plates supplemented with antibiotics and incubated overnight at 37°C.

Western blotting

To assess the expression levels of BamA in cell membrane, recombinant cells were cultured in 50 ml LB medium supplemented with antibiotics and l-(+)-arabinose for overnight at 37°C. The cells were pelleted, resuspended in Tris-buffered saline [TBS, 20 mM Tris–HCl (pH 8.0) and 150 mM NaCl] and lysed by sonication. The lysate was clarified by centrifugation and the resultant supernatant was ultracentrifuged. The cell membrane was resuspended and solubilized in TBS supplemented with 1% SB3-14 for 30 min at room temperature. Membrane protein samples were mixed with 5× SDS–PAGE loading buffer and boiled. Proteins were resolved by SDS–PAGE on a 4–12% gradient gel (Invitrogen) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked overnight in Pierce™ Protein-Free (TBS) Blocking Buffer (Thermo Fisher Scientific) and then incubated with His·Tag Monoclonal Antibody (1 : 1000, Millipore) or anti-E. coli OmpA polyclonal antibody (Antibody Research Corporation). IRDye 800CW goat anti-mouse IgG (H + L) or IRDye 800CW donkey anti-rabbit IgG (H + L) (1 : 5000, LI-COR Biosciences) was used as a secondary antibody. The blot was scanned using the 800 nm channel of the Odyssey Infrared Imaging System (LI-COR Biosciences).

Urea extraction assays

To determine BamA insertion, urea extraction assays were performed [39]. Briefly, cell pellets were resuspended in 10 mM HEPES and sonicated; the unlysed cells and cell debris were removed and the cell membrane was isolated. The IM was solubilized with 2% (w/v) N-lauroyl sarcosine solution, and the OM was isolated by ultracentrifugation and resuspended in urea-containing (6 M urea) extraction solution. After incubation and ultracentrifugation, the pelleted membrane fractions were collected and resuspended in MilliQ water. Samples were analyzed by SDS–PAGE and western blotting.

Heat modifiability assays

BamA mutant was transformed into JCM166 cell and cultured in LB medium supplemented with antibiotics and l-(+)-arabinose. Cell membrane was isolated and solubilized, and the boiled or unboiled membrane protein samples were resolved by SDS–PAGE at 4°C. The proteins were transferred from the gel to the PVDF membrane and western blotting was performed as described above.

Results

BamA structure of S. enterica

The BamA and BamB of S. enterica were co-expressed, and the BamAB complex was purified (Figure 1A and Supplementary Figure S1). Crystallization trials of the BamAB complex were performed. The crystals belong to the space group P6222 with cell dimensions a = b = 124.03 Å, c = 131.48 Å, α = β = γ = 90°. The crystal structure was determined by molecular replacement to a resolution of 2.92 Å. Only the BamA barrel domain was found in the structure, indicating that the BamAB complex may be too flexible to be crystallized, while the BamA POTRA domains and BamB may be cleaved by contaminant proteases during the protein crystallization. There is one BamA molecule per asymmetric unit. The BamA barrel is formed by 16 β-strands with residues Asn427-Gly801 (Figure 1B and Supplementary Figure S2). Secondary-structure matching (SSM) analysis revealed that the BamA structure of S. enterica is very similar to that of the E. coli (PDB code 5D0O) with a root-mean-square deviation (RMSD) of 1.85 Å over 379 Ca atoms [40]. The obvious differences between the two-barrel structures are that the extracellular loops 4 and 6 of S. enterica are shorter than those of the E. coli by four and three residues, respectively (Figure 1A and Supplementary Figure S2). It is worth noting that the β16C of S. enterica BamA is very similar to that of the E. coli BamA of BamABCDE (Figure 1C), in which the β16C coils into the barrel, while β16C of E. coli BamA of BamACDE is further rotated into the barrel (Supplementary Figure S3). The significant conformational changes in the periplasm turns are observed with T1, T2 and T6 in particular (Supplementary Figure S4).

Structure of SenBamA β-barrel.

Figure 1.
Structure of SenBamA β-barrel.

(A) SDS–PAGE analysis of the purified SenBamAB protein complex. (B) Crystal structure of SenBamA β-barrel. A cartoon representation of SenBamA β-barrel in rainbow. The side view and the top view of the SenBamA. (C) The structure of SenBamA is similar to the EcoBamA barrel. SenBamA in rainbow is superimposed well with EcoBamA (PDB code 5D0O) in magenta.

Figure 1.
Structure of SenBamA β-barrel.

(A) SDS–PAGE analysis of the purified SenBamAB protein complex. (B) Crystal structure of SenBamA β-barrel. A cartoon representation of SenBamA β-barrel in rainbow. The side view and the top view of the SenBamA. (C) The structure of SenBamA is similar to the EcoBamA barrel. SenBamA in rainbow is superimposed well with EcoBamA (PDB code 5D0O) in magenta.

Trp810 of the BamA C-terminal residue is important for BamA insertion

It is reported that the last β-strand of bacterial OM barrel contains a β-signature sequence, which contains Phe (phenylalanine) or Trp at the C-terminus and hydrophobic residues at positions 3, 5, 7 and 9 from the C-terminus [41]. This motif is conserved in Gram-negative bacteria OM β-barrel proteins with the particular important C-terminal residue Phe or Trp. However, a recent study suggests that the E. coli BamA's β-signature sequence contains residues N765-P779, which forms the strand β14C rather than the last strand β16C [42]. For functional assays, we use the E. coli BamA depletion strain JCM166 and the plasmid pYG91 containing pelB-His10-EcoBamA (see Materials and Methods). The residue numbers correspond to E. coli BamA unless otherwise stated. To test whether the BamA's last residue W810 is critical for BamA's function, we generated W810F, W810Y, W810V, W810A, W810G and W810D substitutions; the W810F or W810Y substitutions do not slow bacterial growth, while Trp810 substitutions with valine (Val), alanine (Ala), Gly or aspartate (Asp) are lethal (Figure 2A). These data suggest that the C-terminal residue of BamA must be aromatic residues. To check whether the BamA variants can be inserted into the OM, the membrane fraction was collected and western blotting was performed, showing that BamA protein level of the Phe or tyrosine (Tyr) substitution in the membrane was similar to that of the wild-type BamA. BamA with Gly or Asp substitution was found located in the membrane fraction, but the protein level of Val or Ala substitution was extremely low in the membrane (Figure 2B). To further confirm that the BamA variants are inserted into the OM, the urea extraction was performed and BamA variants were detected. As expected, the BamA W810 Phe or Tyr substitution is the same as the wild type in the OM, while BamA mutants W810G and W810D have significantly less than those of the wild-type in the OM (Figure 2E). In contrast, no BamA was found in the OM for W810V and W810A mutants (Figure 2E). To check whether the BamA W810V and W810A mutants were degraded by proteinases or could not be inserted into the OM. Whole protein expressions of the Val and Ala substitutions were also examined at mid-logarithmic and late stationary phases, which showed that the protein level of BamA with Val and Ala substitutions was similar at the mid-logarithmic phase to the late stationary phase, but much less than the wild type (Figure 2C,D). These data may suggest that the BamA Val or Ala substitution can be expressed at very low level, but could not be inserted into the OM. The non-inserted BamA variants probably are cleaved by proteases in the periplasm. To further check whether the BamA mutants could be folded properly in the OM, heat modifiability experiments were performed, which showed that W810F, W810Y and W810G are folded properly, while BamA mutants W810D, W810V and W810A were not folded (Figure 2F).

C-terminal strand β16C terminal residue and periplasmic turn 7 are critical for BamA functionality.

Figure 2.
C-terminal strand β16C terminal residue and periplasmic turn 7 are critical for BamA functionality.

(A) Functional assays of BamA variants of the C-terminal strand and periplasmic turn 7. Numbers 1–13 represent BamA-depleted strain JCM166 harbouring wild-type BamA, empty vector, BamA variants W810F, W810Y, W810V, W810A, W810G, W810D, W810 + DDDD, ΔI806-W810, P779 + DDD, L780D and L780W, respectively. (B) Western blot analysis of protein expression levels of wild-type BamA and its variants in the cell membrane. (C) Western blot analysis of protein expression levels of wild-type BamA and its variants with total cell lysate of cells in the late stationary phase. (D) Western blot analysis of protein expression levels of wild-type BamA and its variants with total cell lysate of cells in the mid-logarithmic phase. (E) Urea extraction analysis of protein expression levels of wild-type BamA and its variants. (F) Heat modifiability analysis of protein expression levels of wild-type BamA and its variants. (G) Western blot analysis of protein expression levels of wild-type BamA and its variants with total cell lysate of cells in the late stationary phase. (H) Western blot analysis of protein expression levels of wild-type BamA and its variants with total cell lysate of cells in the mid-logarithmic phase.

Figure 2.
C-terminal strand β16C terminal residue and periplasmic turn 7 are critical for BamA functionality.

(A) Functional assays of BamA variants of the C-terminal strand and periplasmic turn 7. Numbers 1–13 represent BamA-depleted strain JCM166 harbouring wild-type BamA, empty vector, BamA variants W810F, W810Y, W810V, W810A, W810G, W810D, W810 + DDDD, ΔI806-W810, P779 + DDD, L780D and L780W, respectively. (B) Western blot analysis of protein expression levels of wild-type BamA and its variants in the cell membrane. (C) Western blot analysis of protein expression levels of wild-type BamA and its variants with total cell lysate of cells in the late stationary phase. (D) Western blot analysis of protein expression levels of wild-type BamA and its variants with total cell lysate of cells in the mid-logarithmic phase. (E) Urea extraction analysis of protein expression levels of wild-type BamA and its variants. (F) Heat modifiability analysis of protein expression levels of wild-type BamA and its variants. (G) Western blot analysis of protein expression levels of wild-type BamA and its variants with total cell lysate of cells in the late stationary phase. (H) Western blot analysis of protein expression levels of wild-type BamA and its variants with total cell lysate of cells in the mid-logarithmic phase.

The periplasmic turn 7 is located inside the membrane and is important for function

All the BamA structures suggest that the BamA periplasmic turn 7 is in the OM, based on the positioning of proteins in membrane (PPM) server's prediction (Figure 3A), while the periplasmic turn 7 of another OMP85 family protein TamA (PDB code 4C00), an autotransporter [43], is in the periplasm (Figure 3B), suggesting that BamA periplasmic turn 7 may play an important role for destabilization of the OM. To test this possibility, BamA periplasmic turn 7 variants L780D, L780W and insertion of three Asp residues between P779 and L780 were generated. Hydrophobic Trp is well known for being located inside of the membrane, whereas aspartic acid is a hydrophilic residue that is disfavoured in the membrane. We expected that the L780D mutant and the insertion of the triple Asp residues into the turn 7 would prevent the periplasmic turn 7 from moving inside of the OM, while L780W will help the periplasmic turn to remain in the OM. The functional assays showed that L780D mutant and the triple Asp insertion variant are lethal, but the L780W mutant has no impact on cell growth (Figure 2A). The BamA mutant L780D and the triple Asp insertion variant can be expressed (Figure 2B–D), but the urea extraction experiment indicates that the insertion efficiency was reduced (Figure 2E). It is worth noting that periplasmic turn T7 contains four residues (S778-G781), and deletion of these four residues causes bacteria death (Figure 4A), but it could be expressed and inserted into the OM (Figure 4B). The non-functional deletion BamA may be degraded by the proteinases as the mutant BamA level is similar to the wild type in the mid-logarithmic phase but much less than the wild type in the late-logarithmic phase (Figure 2G,H). These data strongly suggest that the periplasmic turn 7 is required to locate inside of the OM for BamA's function.

Periplasmic turns are important for the BamA's function.

Figure 3.
Periplasmic turns are important for the BamA's function.

(A) The PPM server predicts that the periplasmic turn 7 of EcoBamA in the OM. (B) The PPM server predicts that the periplasmic turn 7 of TamA in the periplasm. (C) The periplasmic turns adopt significant conformational changes from structure BamABCDE in red to BamACDE in green. (D) Functional assays of the periplasmic turn deletions. Numbers 1–14 represent BamA-depleted strain JCM166 harbouring wild-type BamA, empty vector, BamA mutations ΔG451-T452, ΔL450-G453, ΔW449-Y454, ΔT479-G482, ΔN475-S484, ΔN520-N523, ΔD582-D589, ΔD623-D624, ΔD622-H625, ΔP620-V628, ΔS726-Y729 and ΔT720-V733, respectively. (E) Western blot analysis of protein expression levels of wild-type BamA and its variants in the cell membrane.

Figure 3.
Periplasmic turns are important for the BamA's function.

(A) The PPM server predicts that the periplasmic turn 7 of EcoBamA in the OM. (B) The PPM server predicts that the periplasmic turn 7 of TamA in the periplasm. (C) The periplasmic turns adopt significant conformational changes from structure BamABCDE in red to BamACDE in green. (D) Functional assays of the periplasmic turn deletions. Numbers 1–14 represent BamA-depleted strain JCM166 harbouring wild-type BamA, empty vector, BamA mutations ΔG451-T452, ΔL450-G453, ΔW449-Y454, ΔT479-G482, ΔN475-S484, ΔN520-N523, ΔD582-D589, ΔD623-D624, ΔD622-H625, ΔP620-V628, ΔS726-Y729 and ΔT720-V733, respectively. (E) Western blot analysis of protein expression levels of wild-type BamA and its variants in the cell membrane.

Extracellular loops 1–3 play a less important role in OMP insertion.

Figure 4.
Extracellular loops 1–3 play a less important role in OMP insertion.

(A) Functional assays of BamA periplasmic turn 7 deletion and extracellular loop deletions. Numbers 1–8 represent BamA-depleted strain JCM166 harbouring wild-type BamA, empty vector, BamA mutations ΔS778-G781, ΔT434-G437, ΔY432-V438, ΔN463-Q466, ΔA499-S502 and ΔQ495-T505, respectively. (B) Western blot analysis of protein expression levels of wild-type BamA and its variants in the cell membrane.

Figure 4.
Extracellular loops 1–3 play a less important role in OMP insertion.

(A) Functional assays of BamA periplasmic turn 7 deletion and extracellular loop deletions. Numbers 1–8 represent BamA-depleted strain JCM166 harbouring wild-type BamA, empty vector, BamA mutations ΔS778-G781, ΔT434-G437, ΔY432-V438, ΔN463-Q466, ΔA499-S502 and ΔQ495-T505, respectively. (B) Western blot analysis of protein expression levels of wild-type BamA and its variants in the cell membrane.

Periplasmic turns are critical for protein insertion

Our structural work has revealed two conformations of the E. coli BAM machinery, indicating that the periplasmic ring rotates ∼30° promoting the scissor-like movement of the β1C–β6C of BamA barrel with ∼65° for unfolded OMP insertion [29]. We observed significant conformational changes in the periplasmic turns between the BamABCDE and BamACDE complexes; particularly, the turns 1 and 2 interact with POTRA 5 and turns 5 and 6 interact with POTRA 3 in the BamABCDE complex, while turns 1, 2, 3 and 4 interact with POTRA 5 in the BamACDE complex (Figure 3C). We therefore hypothesize that the movement of the barrel strands β1C–β6C may mediate through the periplasmic turns. To test this hypothesis, different BamA deletion mutants were generated in periplasmic turns 1, 2, 3, 4, 5, 6 and 7, and functional assays were performed (Figure 3D). The periplasmic turn T1 is formed by 10 residues Q446-A455, and a two-residue deletion ΔG451-T452 did not affect bacterial growth, while four and six residue deletions of ΔL450-G453 and ΔW449-Y454 resulted in the death of bacteria. T2 contains 10 residues N475-S484, and a four-residue deletion ΔT479-G482 has no impact on the bacterial growth, but a full T2 residue deletion ΔN475-S484 is lethal. T3 and T4 contain 6 residues (P518-N523) and 13 residues (N579-S591), respectively. Interestingly, a four-residue deletion ΔN520-N523 of T3 and eight-residue deletion ΔD582-D589 of T4 do not impair bacterial growth, suggesting that periplasmic turn T3 and T4 may not play an important role. T5 consists of nine residues (P620-V628). Two-residue deletion ΔD623-D624 of turn T5 did not slow bacterial growth. In contrast, both four-residue deletion ΔD622-H625 and nine-residue deletion ΔP620-V628 of turn T5 kill the bacteria. The western blot showed that the level of the BamA deletions ΔD622-H625 and ΔP620-V628 significantly lowers in the late-logarithmic phase than that of the mid-logarithmic phase, indicating that the BamA two deletions are degraded by the proteinases. T6 has 14 residues (T720-V733). A four-residue deletion ΔS726-Y729 of turn T6 has no affection on bacterial growth, whereas 14-residue deletion ΔT720-V733 is lethal. Similar to the turn T5 deletions ΔD622-H625 and ΔP620-V628, the BamA T6 turn ΔT720-V733 deletion was degraded (Figure 2G,H). These data may suggest that periplasmic turn deletions could express to the similar level of the wild type, but the lethal mutants are degraded by the OMP quality control proteinases.

Extracellular loops play a less important role in OMP insertion

BamA barrel β1C–β6C has scissor-like movement; the extracellular loops 1–3 have significant conformational changes between BamABCDE and BamACDE complexes, from pointing to the barrel to the outer side of the bacteria (Supplementary Figure S5). BamA deletion mutants were generated in extracellular loops 1, 2 and 3, and functional assays were performed (Figure 4A). L1 consists of four residues G433-S436 and L1 four-residue deletion ΔT434-G437 does not impair bacteria growth, but the deletion of Y432-V438 including residues of β1C and β2C causes bacterial death. L2 contains five residues K462-Q466; deletion of four residues ΔN463-Q466 slows the bacteria growth. L3 consists of 11 residues Q495-T505; deletion of four residues ΔA499-S502 do not show a growth defect, but deletion of the whole L3 impairs cell growth. All BamA variants with loop deletions are expressed into the membrane fraction (Figure 4B). These data suggest that the extracellular loops 1–3 play a less important or no role for OMP insertion.

Discussion

In Gram-negative bacteria, OM β-barrel proteins are inserted and folded into the OM by the BAM machinery. Our structural and functional studies of the BAM machinery suggest that the BAM machinery inserts OMP into the OM by periplasmic ring rotation that promotes the barrel conformational changes to insert OMPs without using ATP [29]. The SSM analysis showed that the BamA barrel structure of S. enterica is similar to that of E. coli (PDB code 5D0O [29]; PDB code 4C4V [17]) and Neisseria gonorrhoeae (PDB code 4K3B [14]) with RMSD of 1.84 Å over 290 aligned residues, 1.73 Å over 288 aligned residues and 2.418 Å over 249 aligned residues, respectively. In particular, these four barrels share the similar structural feature that all the C-termini of β16C coil into the lumen of the barrel. This may be involved in ‘thinning’ the OM and disturbing the OM for OMP insertion [14].

Bacterial OMP β-signature sequence is important for OMP insertion, and the β-signature sequences are at the C-terminal motif for most of the β-barrel protein with the last residue is normally Phe or Trp. More recently, the β-signature sequence of E. coli BamA is reported to be N765-P779, which forms the β14C and binds to BamD [42]. The Salmonella BamA structure showed that the C-terminal residues shift to the lumen, which is similar to those of the BamA structure from E. coli, and BamA C-terminus is Trp. E. coli BamA W810 substitutions revealed that the aromatic residue substitutions are functional, but other mutants are lethal. The BamA could not be inserted into the OM if Trp810 is mutated into the non-aromatic hydrophobic residues Val or Ala, even though the protein can be expressed. In contrast, BamA variants W810G or W810D can be inserted into the OM, but they are not functional. These data suggest that the aromatic residue Trp810 is not only for BamA insertion, but also for BamA's function for destabilizing OM, probably through movement inside the OM. The addition of four Asp residues after W810 prevents the Trp810 movement in the OM and kills the bacteria, which further confirmed this notion. The lipid heads of the inner leaflet of the OM prevent unfolded OMP insertion into the OM [44], and OM destabilization is required for the OMP insertion. The periplasmic turn deletion assays revealed that the partial deletion of the turns T1, T2 and T5 kills the bacteria, while the whole turn T6 deletion causes the death of the bacteria. In contrast, deletions of T3 and T4 have no effect on bacterial growth. These data are consistent with the structural findings. In the BamABCDE complex, T1 and T2 interact with POTRA5, while T5 and T6 interact with POTRA3; in this instance, the dimension of the central hole of periplasmic ring is ∼46 × 20 Å. However, the T1, T2, T3 and T4 interact with the POTRA5, with the T3 and T4 having fewer contacts with POTRA5 than T1 and T2, while T5 and T6 do not interact with POTRA3 in the BamACDE complex making the dimension of the central hole of the ring ∼34  × 33 Å. At the same time, the β1C–β6C adopts the significant conformational changes. These data may strongly suggest that the periplasmic turns T1, T2, T5 and T6 may play an important role in mediating the periplasmic ring rotation to the barrel strands β1C–β6C's movement. In addition, periplasmic turn 7 is located within the membrane, which may play a role in destabilizing the OM and helping OMP insertion.

The whole L1 deletion ΔT434-G437 shows no bacterial growth defect. Although deletion ΔY432-V438 causes bacterial death, this deletion not only deletes L1 loop residues, but also deletes residues of β1C and β2C. Deletion of individual loop 2 or 3 has some or no impact for bacterial growth. These data suggest that the extracellular loops 1–3 may play less important role in OMP insertion than those of the periplasmic turns, but play a role in sequestering the intact barrel from the environment.

Database Depositions

BamA co-ordinate and structure factor of S. enterica have been deposited in Protein Data Bank under access code 5OR1.

Abbreviations

     
  • Ala

    alanine

  •  
  • Asp

    aspartate

  •  
  • ATP

    adenosine triphosphate

  •  
  • BAM

    β-barrel assembly machinery

  •  
  • Gly

    glycine

  •  
  • IM

    inner membrane

  •  
  • LDAO

    n-dodecyl-N,N-dimethylamine-N-oxide

  •  
  • OEP80

    outer envelope protein of 80 kDa

  •  
  • OG

    n-octyl-β-d-glucopyranoside

  •  
  • OM

    outer membrane

  •  
  • OMP85

    outer membrane protein of 85 kDa

  •  
  • OMPs

    outer membrane proteins

  •  
  • PCR

    polymerase chain reaction

  •  
  • PDB

    protein data bank

  •  
  • Phe

    phenylalanine

  •  
  • POTRA

    polypeptide transport-associated

  •  
  • PPM

    positioning of proteins in membrane

  •  
  • PVDF

    polyvinylidene difluoride

  •  
  • RMSD

    root-mean-square deviation

  •  
  • SAM50

    sorting assembly machinery 50 kDa subunit

  •  
  • SB3-14

    3-(N,N-dimethylmyristylammonio)propanesulfonate

  •  
  • SSM

    secondary-structure matching

  •  
  • TBS

    Tris-buffered saline

  •  
  • Trp

    tryptophan

  •  
  • Tyr

    tyrosine

  •  
  • Val

    valine

Author Contribution

C.D. conceived and supervised the project; Y.G., Y.Z. and Z.W. performed the experiments and analyzed data; C.D. and Y.G. solved the structure and wrote the manuscript.

Funding

C.D. is a recipient of the Wellcome Trust investigator award (WT106121MA) and is supported by the Medical Research Council (G1100110/1).

Acknowledgments

We thank Prof. Thomas J. Silhavy for providing JCM166 cells. We thank Diamond Light Source for access to beamtime i04-1 (proposal mx9475) that contributed to the results presented here.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Knowles
,
T.J.
,
Scott-Tucker
,
A.
,
Overduin
,
M.
and
Henderson
,
I.R.
(
2009
)
Membrane protein architects: the role of the BAM complex in outer membrane protein assembly
.
Nat. Rev. Microbiol.
7
,
206
214
2
Walther
,
D.M.
,
Rapaport
,
D.
and
Tommassen
,
J.
(
2009
)
Biogenesis of β-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence
.
Cell. Mol. Life Sci.
66
,
2789
2804
3
Arnold
,
T.
,
Zeth
,
K.
and
Linke
,
D.
(
2010
)
Omp85 from the thermophilic cyanobacterium Thermosynechococcus elongatus differs from proteobacterial Omp85 in structure and domain composition
.
J. Biol. Chem.
285
,
18003
18015
4
Koenig
,
P.
,
Mirus
,
O.
,
Haarmann
,
R.
,
Sommer
,
M.S.
,
Sinning
,
I.
,
Schleiff
,
E.
et al
(
2010
)
Conserved properties of polypeptide transport-associated (POTRA) domains derived from cyanobacterial Omp85
.
J. Biol. Chem.
285
,
18016
18024
5
Wu
,
T.
,
Malinverni
,
J.
,
Ruiz
,
N.
,
Kim
,
S.
,
Silhavy
,
T.J.
and
Kahne
,
D.
(
2005
)
Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli
.
Cell
121
,
235
245
6
Heinz
,
E.
and
Lithgow
,
T.
(
2014
)
A comprehensive analysis of the Omp85/TpsB protein superfamily structural diversity, taxonomic occurrence, and evolution
.
Front. Microbiol.
5
,
370
7
Kim
,
S.
,
Malinverni
,
J.C.
,
Sliz
,
P.
,
Silhavy
,
T.J.
,
Harrison
,
S.C.
and
Kahne
,
D.
(
2007
)
Structure and function of an essential component of the outer membrane protein assembly machine
.
Science
317
,
961
964
8
Tommassen
,
J.
(
2010
)
Assembly of outer-membrane proteins in bacteria and mitochondria
.
Microbiology
156
,
2587
2596
9
Webb
,
C.T.
,
Heinz
,
E.
and
Lithgow
,
T.
(
2012
)
Evolution of the β-barrel assembly machinery
.
Trends Microbiol.
20
,
612
620
10
Day
,
P.M.
,
Potter
,
D.
and
Inoue
,
K.
(
2014
)
Evolution and targeting of Omp85 homologs in the chloroplast outer envelope membrane
.
Front. Plant Sci.
5
,
535
11
Rigel
,
N.W.
and
Silhavy
,
T.J.
(
2012
)
Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria
.
Curr. Opin. Microbiol.
15
,
189
193
12
Rossiter
,
A.E.
,
Leyton
,
D.L.
,
Tveen-Jensen
,
K.
,
Browning
,
D.F.
,
Sevastsyanovich
,
Y.
,
Knowles
,
T.J.
et al
(
2011
)
The essential β-barrel assembly machinery complex components BamD and BamA are required for autotransporter biogenesis
.
J. Bacteriol.
193
,
4250
4253
13
Noinaj
,
N.
,
Fairman
,
J.W.
and
Buchanan
,
S.K.
(
2011
)
The crystal structure of BamB suggests interactions with BamA and its role within the BAM complex
.
J. Mol. Biol.
407
,
248
260
14
Noinaj
,
N.
,
Kuszak
,
A.J.
,
Gumbart
,
J.C.
,
Lukacik
,
P.
,
Chang
,
H.
,
Easley
,
N.C.
et al
(
2013
)
Structural insight into the biogenesis of β-barrel membrane proteins
.
Nature
501
,
385
390
15
Zhang
,
H.
,
Gao
,
Z.-Q.
,
Hou
,
H.-F.
,
Xu
,
J.-H.
,
Li
,
L.-F.
,
Su
,
X.-D.
et al
(
2011
)
High-resolution structure of a new crystal form of BamA POTRA4–5 from Escherichia coli
.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
67
,
734
738
16
Ni
,
D.
,
Wang
,
Y.
,
Yang
,
X.
,
Zhou
,
H.
,
Hou
,
X.
,
Cao
,
B.
et al
(
2014
)
Structural and functional analysis of the β-barrel domain of BamA from Escherichia coli
.
FASEB J.
28
,
2677
2685
17
Albrecht
,
R.
,
Schütz
,
M.
,
Oberhettinger
,
P.
,
Faulstich
,
M.
,
Bermejo
,
I.
,
Rudel
,
T.
et al
(
2014
)
Structure of BamA, an essential factor in outer membrane protein biogenesis
.
Acta Crystallogr. D Biol. Crystallogr.
70
,
1779
1789
18
Albrecht
,
R.
and
Zeth
,
K.
(
2011
)
Structural basis of outer membrane protein biogenesis in bacteria
.
J. Biol. Chem.
286
,
27792
27803
19
Kim
,
K.H.
and
Paetzel
,
M.
(
2011
)
Crystal structure of Escherichia coli BamB, a lipoprotein component of the β-barrel assembly machinery complex
.
J. Mol. Biol.
406
,
667
678
20
Kim
,
K.H.
,
Aulakh
,
S.
and
Paetzel
,
M.
(
2011
)
Crystal structure of β-barrel assembly machinery BamCD protein complex
.
J. Biol. Chem.
286
,
39116
39121
21
Kim
,
K.H.
,
Aulakh
,
S.
,
Tan
,
W.
and
Paetzel
,
M.
(
2011
)
Crystallographic analysis of the C-terminal domain of the Escherichia coli lipoprotein BamC
.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
67
,
1350
1358
22
Kim
,
K.H.
,
Kang
,
H.-S.
,
Okon
,
M.
,
Escobar-Cabrera
,
E.
,
McIntosh
,
L.P.
and
Paetzel
,
M.
(
2011
)
Structural characterization of Escherichia coli BamE, a lipoprotein component of the β-barrel assembly machinery complex
.
Biochemistry
50
,
1081
1090
23
Warner
,
L.R.
,
Varga
,
K.
,
Lange
,
O.F.
,
Baker
,
S.L.
,
Baker
,
D.
,
Sousa
,
M.C.
et al
(
2011
)
Structure of the BamC two-domain protein obtained by Rosetta with a limited NMR data set
.
J. Mol. Biol.
411
,
83
95
24
Knowles
,
T.J.
,
Browning
,
D.F.
,
Jeeves
,
M.
,
Maderbocus
,
R.
,
Rajesh
,
S.
,
Sridhar
,
P.
et al
(
2011
)
Structure and function of BamE within the outer membrane and the β-barrel assembly machine
.
EMBO Rep.
12
,
123
128
25
Sandoval
,
C.M.
,
Baker
,
S.L.
,
Jansen
,
K.
,
Metzner
,
S.I.
and
Sousa
,
M.C.
(
2011
)
Crystal structure of BamD: An essential component of the β-barrel assembly machinery of Gram-negative bacteria
.
J. Mol. Biol.
409
,
348
357
26
Dong
,
C.
,
Hou
,
H.-F.
,
Yang
,
X.
,
Shen
,
Y.-Q.
and
Dong
,
Y.-H.
(
2012
)
Structure of Escherichia coli BamD and its functional implications in outer membrane protein assembly
.
Acta Crystallogr. D Biol. Crystallogr.
68
,
95
101
27
Jansen
,
K.B.
,
Baker
,
S.L.
and
Sousa
,
M.C.
(
2015
)
Crystal structure of BamB bound to a periplasmic domain fragment of BamA, the central component of the β-barrel assembly machine
.
J. Biol. Chem.
290
,
2126
2136
28
Bergal
,
H.T.
,
Hopkins
,
A.H.
,
Metzner
,
S.I.
and
Sousa
,
M.C.
(
2016
)
The structure of a BamA-BamD fusion illuminates the architecture of the β-barrel assembly machine core
.
Structure
24
,
243
251
29
Gu
,
Y.
,
Li
,
H.
,
Dong
,
H.
,
Zeng
,
Y.
,
Zhang
,
Z.
,
Paterson
,
N.G.
et al
(
2016
)
Structural basis of outer membrane protein insertion by the BAM complex
.
Nature
531
,
64
69
30
Bakelar
,
J.
,
Buchanan
,
S.K.
and
Noinaj
,
N.
(
2016
)
The structure of the β-barrel assembly machinery complex
.
Science
351
,
180
186
31
Han
,
L.
,
Zheng
,
J.
,
Wang
,
Y.
,
Yang
,
X.
,
Liu
,
Y.
,
Sun
,
C.
et al
(
2016
)
Structure of the BAM complex and its implications for biogenesis of outer-membrane proteins
.
Nat. Struct. Mol. Biol.
23
,
192
196
32
Iadanza
,
M.G.
,
Higgins
,
A.J.
,
Schiffrin
,
B.
,
Calabrese
,
A.N.
,
Brockwell
,
D.J.
,
Ashcroft
,
A.E.
et al
(
2016
)
Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM
.
Nat. Commun.
7
,
12865
33
Fardini
,
Y.
,
Trotereau
,
J.
,
Bottreau
,
E.
,
Souchard
,
C.
,
Velge
,
P.
and
Virlogeux-Payant
,
I.
(
2009
)
Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella
.
Microbiology
155
,
1613
1622
34
Liu
,
H.
and
Naismith
,
J.H.
(
2008
)
An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol
.
BMC Biotechnol.
8
,
91
35
Winn
,
M.D.
,
Ballard
,
C.C.
,
Cowtan
,
K.D.
,
Dodson
,
E.J.
,
Emsley
,
P.
,
Evans
,
P.R.
et al
(
2011
)
Overview of the CCP4 suite and current developments
.
Acta Crystallogr. D Biol. Crystallogr.
67
,
235
242
36
Mccoy
,
A.J.
,
Grosse-Kunstleve
,
R.W.
,
Adams
,
P.D.
,
Winn
,
M.D.
,
Storoni
,
L.C.
and
Read
,
R.J.
(
2007
)
Phaser crystallographic software
.
J. Appl. Crystallogr.
40
,
658
674
37
Emsley
,
P.
and
Cowtan
,
K.
(
2004
)
Coot: model-building tools for molecular graphics
.
Acta Crystallogr. D Biol. Crystallogr.
60
,
2126
2132
38
Murshudov
,
G.N.
,
Vagin
,
A.A.
and
Dodson
,
E.J.
(
1997
)
Refinement of macromolecular structures by the maximum-likelihood method
.
Acta Crystallogr. D Biol. Crystallogr.
53
,
240
255
39
Leo
,
J.C.
,
Oberhettinger
,
P.
and
Linke
,
D.
(
2015
)
Assessing the outer membrane insertion and folding of multimeric transmembrane β-barrel proteins
.
Methods Mol. Biol.
1329
,
157
167
40
Krissinel
,
E.
and
Henrick
,
K.
(
2004
)
Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions
.
Acta Crystallogr. D Biol. Crystallogr.
60
,
2256
2268
41
Lehr
,
U.
,
Schütz
,
M.
,
Oberhettinger
,
P.
,
Ruiz-Perez
,
F.
,
Donald
,
J.W.
,
Palmer
,
T.
et al
(
2010
)
C-terminal amino acid residues of the trimeric autotransporter adhesin YadA of Yersinia enterocolitica are decisive for its recognition and assembly by BamA
.
Mol. Microbiol.
78
,
932
946
42
Hagan
,
C.L.
,
Wzorek
,
J.S.
and
Kahne
,
D.
(
2015
)
Inhibition of the β-barrel assembly machine by a peptide that binds BamD
.
Proc. Natl Acad. Sci. U.S.A.
112
,
2011
2016
43
Gruss
,
F.
,
Zähringer
,
F.
,
Jakob
,
R.P.
,
Burmann
,
B.M.
,
Hiller
,
S.
and
Maier
,
T.
(
2013
)
The structural basis of autotransporter translocation by TamA
.
Nat. Struct. Mol. Biol.
20
,
1318
1320
44
Gessmann
,
D.
,
Chung
,
Y.H.
,
Danoff
,
E.J.
,
Plummer
,
A.M.
,
Sandlin
,
C.W.
,
Zaccai
,
N.R.
et al
(
2014
)
Outer membrane β-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA
.
Proc. Natl Acad. Sci. U.S.A.
111
,
5878
5883

Author notes

*

These authors contributed equally to this work.

Supplementary data