Figure 1.
Mutational analysis of the BamA POTRA domains.
(A) The panel details the location of 5 amino acid insertions within the BamA POTRA domains. The BamA signal sequence (ss) and POTRA domains (PD1 to PD5) are aligned with the aa sequence number. The function of insertions was monitored by their ability to rescue BamA depletion and/or grow in the presence of vancomycin (i.e. 37.5, 75 and 150 µg ml−1). A severe growth defect (black lollipops) was defined as either an inability to rescue BamA depletion or to allow growth in the presence of vancomycin. Constructs producing a growth defect (grey lollipops) allowed growth on only 37.5 µg ml−1 vancomycin, whilst constructs which grew at 75 µg ml−1 vancomycin caused a minimal effect (white lollipops). Constructs which allowed growth at all vancomycin concentrations tested (arrows) had no effect. (B) Detection of BamA POTRA insertions. N-terminal 6His tags were introduced into the K89, F140, Y141, Q170, N181, L231, R237, T257, Y317 and Q384 insertion constructs, cloned into pET17b. Total membranes were prepared from JWD3 cells containing pET17b and the various pET17b/6hisbamA constructs, grown in the presence of arabinose. 1.6 µg of membrane protein was Western blotted with anti-6His antiserum (top) and 4 µg was analysed using SDS-PAGE and stained with Coomassie blue (bottom). (C) Deletion analysis of BamA POTRA domains. The panel shows the growth of JWD3 cells carrying pET17b, pET17b/bamA and pET17b containing bamA constructs with individual POTRA domains deleted (ΔP1 to ΔP5). Cells were grown in Lennox broth in the presence of arabinose or fructose (+Ara or +Fru). After 300 minutes cultures were sampled and subcultured into fresh medium. (D) A Western blot of normalised total cellular protein samples from JWD3 cells after 300 minutes of growth. Blots were probed with anti-BamA POTRA antiserum.
Figure 2.
Topology model of the BamA β-barrel.
(A) The panel shows a model of BamA, displaying the five POTRA domains and β-barrel of BamA. The model was generated by combining the available crystal structures of BamA POTRA1 to POTRA4 (3ECF) [8] and POTRA4 and POTRA5 (3OG5) [10] with the model of the BamA β-barrel generated in this study using Coot [33] and is visualised using PyMol [35]. POTRA domains are indicated (PD1 to PD5), as are β-strands β1 and β16. The tip of loop L6 has been placed within the pore of the β-barrel lumen and has been modelled as a β-hairpin. (B) The panel shows a topology model of the BamA β-barrel (N422 to W810) derived from bioinformatics predictions. Amino acids within β-strand regions are shown as blue squares and those in external loops and periplasmic turns are shown as pink circles. β-strands β1 to β16, extracellular loops L1 to L8 and periplasmic turns T1 to T7 are indicated. The tip region of L6 is predicted to form a β-turn and the RGF motif important in BamA function is starred [14], [15]. The figure also details the position of 5 amino acid insertions isolated by linker scanning mutagenesis (Table S3). Insertions that either failed to rescue BamA depletion or did not allow growth in the presence of vancomycin (severe mutations) are coloured red, whilst insertion constructs which allowed growth on vancomycin concentration of 37.5, 75 and 150 µg ml−1 are coloured orange, yellow and white, respectively. The location of HA epitopes inserted within the β-barrel is also indicated using triangles (Table S5). The severity of the insertion is colour coded as for the linker scanning mutations.
Figure 3.
Mutational Analysis of the BamA β-barrel.
Detection of BamA proteins carrying insertions within the β-barrel domain. N-terminal 6His tags were introduced into the Q441, Q466, D503, Y509, W546, Y574, L613, Q664, N666, A714, A770, Q789 and A799 insertion constructs cloned into pET17b (see Fig. 2). Total membranes were prepared from JWD3 cells, containing pET17b, pET17b/6hisbamA or pET17b carrying 6His insertion mutants, grown in the presence of arabinose. 1.6 µg of total membrane protein was subjected to Western blotting with anti-6His antiserum (top panel) and 4 µg of protein was analysed using SDS-PAGE and stained with Coomassie blue (bottom panel). The location of 6HisBamA proteins, OmpF, OmpC and OmpA are indicated. The location of each insertion with respect to the secondary structure detailed in Fig. 2 is also given.
Figure 4.
Analysis of HA epitopes within the BamA β-barrel.
The figure shows the detection of BamA proteins carrying HA insertions within the β-strands and external loops of the BamA β-barrel. HA epitopes were introduced into β-strands (β1 to β16) and loops (L2 to L8) (see Fig. 2) and bamA insertion constructs were cloned into pET17b. Outer membranes (OM) were prepared from JWD3 cells containing each construct and normalised protein samples were subjected to Western blotting with anti-HA antiserum. (A) shows a Western blot analysis of BamA proteins carrying HA epitopes in β-strands β1 to β16 and (B), in loops L2 to L8. Panel (B) also shows the urea insoluble fraction (UI) obtained after outer membrane preparations were washed with urea. All BamA proteins carrying HA epitopes in their loop domains were localised within the urea insoluble fraction, indicating that they are correctly folded within the membrane. (C) Immunofluorescence analysis of BamA constructs carrying HA epitopes within external loops. JWD3 cells, containing pET17b, pET17b/bamA or pET17b carrying HA insertions in L2 to L8, were fixed, probed with anti-HA and Alexa Fluor® 488 antibody and visualized using phase contrast (shown inverted) and fluorescence microscopy.
Figure 5.
Deletion of BamA extracellular loops.
(A) The location of external loop deletions within the BamA β-barrel. The panel shows the location of loop deletions (yellow) introduced into BamA based on the homology model detailed in Fig. 2. For deletions ΔL3, ΔL4, ΔL7 and ΔL8 the deleted sequence was replaced by three glycine residues to maintain flexibility between β-strands. (B) Detection of BamA proteins carrying loop deletions. Total cellular membranes were prepared from JWD3 cells carrying pET17b, pET17b/6hisbamA and pET17b carrying 6HisBamA loop deletion constructs (see panel A). 1.6 µg of total membrane protein was subjected to Western blotting with anti-6His antiserum (top panel) and 4 µg of protein was analysed using SDS-PAGE and strained with Coomassie blue (bottom panel). The location of 6HisBamA proteins, OmpF, OmpC and OmpA are indicated. (C) Urea washing of membrane-localised BamA proteins containing loop deletions. Total cellular membranes from JWD3 cells carrying pET17b/6hisbamA or pET17b containing 6HisBamA loop deletion constructs were washed with urea and the insoluble (UI) and soluble (US) fractions subjected to Western blotting with anti-6His antiserum. All 6HisBamA proteins were localised within the urea insoluble fraction indicting that they are full folded within the membrane.