A modular BAM complex in the outer membrane of the alpha-proteobacterium Caulobacter crescentus

Abstract

Mitochondria are organelles derived from an intracellular alpha-proteobacterium. The biogenesis of mitochondria relies on the assembly of beta-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an alpha-proteobacterium, and the BAM (beta-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A approximately 150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane.

Figure 1. BamA in Caulobacter crescentus.

(A) The domain structure of BamA. The 5 predicted POTRA domains (P1–P5) are shown in white, with the secondary structure β-α-α-β-β indicated – and the predicted β-barrel domain in grey. The defining “Omp85” motif is shaded a darker grey. The mitochondrial Sam50 proteins from yeast (S. cerevisiae) and humans (H. sapiens) are shown for comparison. (B) Membranes from wild-type C. crescentus were fractionated on sucrose gradient and analysed by SDS-PAGE. Coomassie Brilliant Blue staining (upper panel) reveals separation of the membrane protein profiles and immunoblotting (lower panel) for the inner membrane protein TimA and the outer membrane protein BamA. Mass spectrometry of the major protein bands indicated revealed identities of fifteen proteins (CC0288, CC0925, etc) annotated as TonB-dependent receptors .

Figure 2. The BAM complex is modular.

(A) Outer membrane vesicles (100 µg protein) were solubilised with 0.38% (w/v) dodecyl-maltoside and loaded for analysis by blue native-PAGE. The Coomassie Brilliant Blue-stained gel shows the major outer membrane protein complexes. (B) Outer membrane vesicles (100 µg protein per lane) solubilised with the indicated concentrations of dodecyl-maltoside, separated by blue native-PAGE and analysed by immuno-staining with an antiserum to BamA. The migration positions of the molecular weight markers are shown. Arrows indicate three modular forms of the BAM complex.

Figure 3. Subunit composition of the BAM complex in C. crescentus.

(A) Outer membrane vesicles (800 µg protein) were solubilised with 0.75% (w/v) dodecyl-maltoside and the BAM complex immunoprecipitated with an antiserum recognizing the BamA subunit (lane 3) or preimmune serum (lane 4). Samples corresponding to 8 µg protein of total outer membranes (Lane 1), 8 µg protein of unbound fraction (Lane 2) and all of the material immunoprecipitated (Lane 3, 4) are shown. Asterisks indicate the IgG heavy and light chains, with the migration positions of the molecular markers shown in kDa. (B) Mass spectrometry data summarizing the identification of immunoprecipitated proteins (see Methods). In addition to the number of high confidence peptides identified by MS/MS data and sequence coverage; a MOWSE score is included, which is a probabilistic score that indicates the match of the experimental peptide precursor masses (peptide mass fingerprint) to the sequence of a candidate parental protein. Typically a MOWSE score >75 is considered as significant .

Figure 4. Pal is an essential outer membrane protein, associated with the BAM complex.

(A) BAM complex was immunoprecipitated using BamA antiserum added to outer membrane vesicles that were solubilised with 0.75% (w/v) (Lane 1) and 2.25% (w/v) (Lane 2) dodecyl-maltoside. Immunoprecipitate obtained with preimmune serum was loaded in Lane 3. Asterisks indicate the IgG heavy and light chains, with the migration positions of the molecular markers shown in kDa. (B) Cells with the pal gene under the control of a xylose-inducible promoter (↓ pal) were grown in the presence (right montage) and absence (left montage) of xylose (0.3% [w/v]) for 10 hrs. Outer membrane blebs that form predominantly from the division site or cell poles are evident only in the Pal-depleted cells. Scale bars (white) represent 1 micrometer. (C) Membranes were fractionated on sucrose gradient and analysed by SDS-PAGE. Coomassie Brilliant Blue staining (upper panel) reveals separation of the membrane protein profiles and immunoblotting (lower panel) for the inner membrane protein TimA and the outer membrane protein BamA, and the mCherry epitope to determine the location of Pal.

Figure 5. Pal anchors the BAM complex to the peptidoglycan layer of the cell wall.

(A) Photobleaching analysis of Pal-mCherry dynamics required pre-treatment with chloramphenicol to block protein synthesis, and cells expressing Pal-mCherry were immobilized on an agarose-padded slide containing chloramphenicol. The left panel shows a representative cell in which the circle indicates the region that will be photobleached. The right panel shows a kymograph representation of Pal-mCherry fluorescence intensity along the cell axis before (−1 min) and following photobleaching. (B) Fluorescence micrographs showing a cell expressing Pal-mCherry before photobleaching (left), right after being completely photobleached (middle) and after 126 min of growth (right). (C) Outer membrane vesicles (100 µg protein per lane) from cells expressing Pal-mCherry were solubilised with dodecyl-maltoside and analysed by blue native-PAGE and immuno-staining with antisera recognizing either Pal or BamA. The migration position of molecular markers (kDa) are shown, arrow indicates the 300 kDa form of the BAM complex. Scale bars (white) represent 1 micrometer.

Figure 6. Structural homology model for CC3229 as the Pal from C. crescentus.

The homology model is based on the known structures of Pal (PDB code 2AIZ) and YiaD (PDB code 2K1S) from E. coli, and RmpM (PDB code 1R1M) from N. meningitidis. Note that YiaD, CcPal and RmpM have N-terminal extensions that are not resolved in the crystal structures. Residues that show at least 50% conservation in structure are colour coded according to their properties. Below the sequence alignment is the conservation plot, which is based on a 1–10 scale with completely conserved residues highlighted with yellow bars and asterisks.