Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry
- National University of Singapore, Singapore
Figures
Figure 1 with 4 supplements
MlaF, MlaE, MlaD and MlaB form a stable complex.
(A) Co-TALON affinity purification using WT and indicated mutant strains harboring empty vector (pET23/42) or pET23/42His-mlaE (pHis-mlaE). Samples (heated or non-heated) were subjected to SDS-PAGE (12% Tris.HCl gel), and visualized by silver staining and immunoblot analyses using antibodies against the pentahistidine tag. (B) SEC profile of MlaF(His-E)DB complex purified from cells over-expressing MlaF(His-E)DCB. The peak fraction (heated or non-heated) was subjected to SDS-PAGE (4–20% Tris.HCl gel) followed by Coomassie Blue (CB) staining and immunoblot analysis. His-MlaE can only be detected on immunoblots when samples are not heated. Under the same conditions, MlaD migrates as a high molecular weight species. Positions of relevant molecular weight markers are indicated in kDa.
Figure 1—figure supplement 1
His-tagged Mla proteins are able to rescue SDS/EDTA sensitivity in the respective mla mutant strains.
Serial dilutions of cultures of wild-type (WT), ∆mlaF, ∆mlaE, ∆mlaD and ∆mlaB strains harboring pET23/42 empty vector, pET23/42mlaF-His (pmlaF-His), pET23/42His-mlaE (pHis-mlaE), pET23/42mlaD-His (pmlaD-His) or pET23/42His-mlaB (pHis-mlaB), respectively, were spotted on LB agar plates containing 200 μg/mL ampicillin, supplemented with or without 0.50% SDS and 0.60 mM EDTA as indicated, and incubated overnight at 37°C.
Figure 1—figure supplement 2
MlaF, MlaE, MlaD and MlaB form a stable complex.
Co-TALON affinity purification experiments using wild-type (WT) and indicated mutant strains harboring (A) pmlaF-His, pmlaD-His, or (B) pHis-mlaB. Samples were heated and subjected to SDS-PAGE (12% Tris.HCl gel), and visualized by silver staining and immunoblot analyses using antibodies against the pentahistidine tag. Positions of relevant molecular weight markers are indicated in kDa.
Figure 1—figure supplement 3
MlaF, MlaD and MlaB co-purify with His-tagged MlaE following overexpression and affinity purification.
Tandem MS/MS sequencing confirmed the identities of protein bands corresponding to MlaF, MlaD and MlaB in a preparation of the purified MlaFEDB complex. The top three most abundant proteins in each band are shown. Positions of relevant molecular weight markers are indicated in kDa.
Figure 1—figure supplement 4
SEC-MALS analysis of the MlaF(His-E)DB complex.
Molecular mass: 256 kDa (predicted, MlaF2E2D6B2), 285 ( ± 0.5%) kDa (observed). Molecular masses of DDM fraction in complex and free micelles are 35 ( ± 4.4%) kDa and 65 ( ± 2.1%) kDa, respectively. Numbers stated after ± show statistical consistency of analysis.
Figure 2
MlaD forms SDS-resistant hexamers via its soluble domain.
(A) Domain organization of MlaD. (B) SEC profile of purified soluble domain of MlaD (sMlaD-His). Elution volumes of standard globular proteins (aldolase 158 kDa, conalbumin 75 kDa and ovalbumin 44 kDa) are indicated. The peak fraction (heated or non-heated) was subjected to SDS-PAGE (4–20% Tris.HCl gel) followed by CB staining. Positions of relevant molecular weight markers are indicated in kDa. (C) SEC-MALS analysis of sMlaD-His. Hexamer molecular mass: 107 kDa (predicted), 110.7 ( ± 0.5%) kDa (observed). Numbers stated after ± show statistical consistency of the analysis.
Figure 3 with 2 supplements
sMlaD co-purifies with endogenous PLs.
(A) TLC analysis of PLs extracted from BL21(λDE3) cells, purified dLolB-His and sMlaD-His. (B) 31P NMR analysis of PL extracts from BL21(λDE3) cells and purified sMlaD-His in 5% Triton X-100. Compositions of bound PLs were obtained via integration of peak areas, and normalized to the number of phosphorus atoms per PL molecule (i.e. one for PE/PG and two for CL). Unknown peaks that cannot be assigned to any PL species in E. coli (see Figure 3—figure supplement 1) are annotated with asterisks (*). (C) Positive mode, non-denaturing electrospray ionization (ESI) mass spectrum of sMlaD-His. Under native conditions (20 mM ammonium acetate, pH 6.9), sMlaD hexamers with charge states centred around +24 could be detected. After deconvolution, the molecular weight of the native hexamers was ~110 kDa, indicating the presence of at least four bound PLs (i.e. P6L4,assuming an average mass of 750 Da per PL).
Figure 3—figure supplement 1
31P NMR analysis of E. coli PLs. 31P chemical shifts (ppm) of phosphatidic acid (14:0) (PA), phosphatidylserine (PS), cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) are given in the table.
PS were extracted from E. coli EH150 strain, which accumulates PS when grown at 42°C (Hawrot et al., 1975).
Figure 3—figure supplement 2
MS analyses of sMlaD-His.
(A) Positive-mode ESI-MS spectrum of sMlaD-His under denaturing conditions (50% acetonitrile 0.2% formic acid), revealing monomeric sMlaD-His at various charge states. Inset: Deconvoluted spectrum of monomeric sMlaD-His(experimental 17828.32 Da, theoretical 17827.77 Da). (B) Positive mode ESI-MS spectrum of sMlaD-His under non-denaturing conditions (20 mM ammonium acetate, pH 6.9) with increasing collision energies (0 V (black), 80 V (green), 100 V (pink) and 150 V (blue)), progressively revealing hexameric sMlaD-His with four to zero bound PL molecules.
Figure 4 with 2 supplements
MlaB is required for the stability and/or assembly of the canonical ABC transporter.
SEC profiles of (A) His-MlaE purified from cells over-expressing MlaF(His-E), (B) (His-MlaE)D purified from cells over-expressing MlaF(His-E)D, and (C) MlaF(His-MlaE)B purified from cells over-expressing MlaF(His-E)B. The respective peak fractions (non-heated) were subjected to SDS-PAGE (4–20% Tris.HCl gel) followed by CB staining. (D) Co-TALON affinity purification using WT and ∆mlaB strains harboring empty vector (pET23/42) or pET23/42His-mlaE (pHis-mlaE). Samples (heated or non-heated) were subjected to SDS-PAGE (12% Tris.HCl gel), and visualized by silver staining and immunoblot analyses using antibodies against the pentahistidine tag. Positions of relevant molecular weight markers are indicated in kDa.
Figure 4—figure supplement 1
MlaF is produced at high levels in strains over-expressing full and sub-complexes of the IM ABC transporter.
SDS-PAGE (12% Tris.HCl gel) analysis of cell lysates isolated from strains over-expressing MlaF(His-E), MlaF(His-E)D, MlaF(His-E)B and MlaF(His-E)DCB from indicated vectors, with or without IPTG induction. Positions of relevant molecular weight markers are indicated in kDa.
Figure 4—figure supplement 2
MlaD is not co-purified with MlaF-His in the absence of MlaB.
Co-TALON affinity purification using wild-type (WT), ∆mlaB and ∆mlaE strains harboring an empty pET23/42 vector or pmlaF-His. Samples were heated and subjected to SDS-PAGE (12% Tris.HCl gel), and visualized by silver staining and immunoblot analyses using antibodies against the pentahistidine tag. Positions of relevant molecular weight markers are indicated in kDa.
Figure 5 with 1 supplement
MlaD and MlaB modulate ATP hydrolytic activity of the IM ABC transporter.
(A) Enzyme-coupled ATPase assays of indicated complexes (0.1 μM) performed in detergent micelles (0.05% DDM). Average ATP hydrolysis rates (obtained from triplicate experiments, see Figure 5—source data 1) were plotted against ATP concentrations, and fitted to an expanded Michaelis-Menten equation that includes a term for Hill coefficient (n); MlaFEDB (kcat = Vmax/[complex] = 0.6 ± 0.3 μmol ATP s-1/μmol complex, Km = 181.1 ± 203.6 μM, n = 1.0 ± 0.6) and MlaFEB (kcat = 1.8 ± 0.5 μmol ATP s-1/μmol complex, Km = 161.4 ± 75.57 μM, n = 1.5 ± 0.5). SDS-PAGE analysis of the complexes (non-heated) used for these assays is shown on the right. Error bars in the graph and numbers stated after ± are standard deviations of triplicate data. (B) A proposed model for how the MlaFEDB complex functions to drive PL transport from the OM to the IM.
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Figure 5—source data 1
Source data for ATPase assay.
- https://doi.org/10.7554/eLife.19042.016
Figure 5—figure supplement 1
mlaFK47R and mlaBT52A are non-functional alleles.
Serial dilutions of cultures of wild-type (WT) and ∆mlaF strains harboring pET22/42 empty vector, pET22/42mlaF-His or pET22/42mlaFK47R-His, or WT and ∆mlaB strains harboring pCDF empty vector, pCDFmlaB or pCDFmlaBT52A were spotted on LB agar plates containing appropriate antibiotics, supplemented with or without 0.50% SDS, 0.65/0.70 mM EDTA as indicated, and incubated overnight at 37°C. The strains used here do not express T7 polymerase. IPTG (1 mM) was added to allow de-repression and thus low-level transcription (by endogenous polymerases) from the T7-lacO promoter in these plasmids.
Additional files
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Supplementary file 1
Bacteria strains used in this study.
- https://doi.org/10.7554/eLife.19042.018
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Supplementary file 2
Plasmids used in this study.
- https://doi.org/10.7554/eLife.19042.019
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Supplementary file 3
Primers used in this study.
- https://doi.org/10.7554/eLife.19042.020
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Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry
eLife 5:e19042.
https://doi.org/10.7554/eLife.19042
