Identification of residues critical for the membrane-binding activity of BacA in vivo.

(A) Schematic representation of the BacA-mVenus fusion protein used in this study. The proposed membranetargeting sequence is highlighted in red. The sequence at the botton shows that result of an amphipathic helix prediction for BacA using the AMPHIPASEEK software (Sapay et al., 2006). Residues predicted to be located in an unstructured, randomly coiled region are labeled with “c”. (B) Localization patterns of mutant BacA-mVenus variants. ΔbacAB cells producing BacA-mVenus or mutant variants thereof (LY84, LY89, LY90, LY97, LY111, LY112, LY113, LY119) were analyzed by phase contrast and fluorescence microscopy. The outlines of the cells are shown in the fluorescence images. Demographs summarizing the singlecell fluorescence profiles obtained from random subpopulations of cells are given next to the respective fluorescence images. The numbers of cells analyzed are: WT (130), Δ2-8 (292), F130R (156), F2Y (138), F2E (194), K4S-K7S (151), K4E-K7E (382), F2E-K4E-K7E (130). The vertical red line indicates the junction between cell body and the stalk. Scale bar: 1 μm. (C) Helical wheel diagram of the first eight amino acids of BacA. Residues are colored by properties: hydrophobic (gray), basic (blue), uncharged (yellow).

Verification of residues M1, F2 and K4/K7 as critical components of the BacA MTS.

(A) Mobility of the indicated BacA-mVenus fusion proteins. Shown is the average mean-squared displacement (MSD) (± SD) as a function of time, based on single-particle tracking analysis. (B) Cell fractionation experiment investigating the membrane-binding activity of BacA-mVenus (WT) or a mutant variant lacking the predicted MTS (Δ2-8). Whole-cell lysates (W) as well as the soluble (S) and pellet (P) fractions of cells producing the indicated proteins were subjected to immunoblot analysis with an anti-GFP antibody, detecting the BacA-mVenus fusion protein. As controls, the same samples were probed with antibodies raised against the soluble cell division regulator MipZ (Thanbichler and Shapiro, 2006) or the membranebound flagellar L-ring subunit FlgH (Mohr et al., 1996) from C. crescentus. (C) As in panel C, but for cells producing mutant BacA-mVenus variants with single or multiple amino-acid exchanges in the predicted MTS. Shown are representative images (n=3 independent replicates). The strains used are given in the legend to Figure 1B.

Co-sedimentation analysis of the association of various BacA variants with liposomes.

The indicated proteins (20 µM) were incubated without (-) or with (+) liposomes (0.4 mg/mL) prior to ultracentrifugation. The supernatant (S) and pellet (P) fractions of each mixture were analyzed by SDS gel electrophoresis. Shown are scans of representative gels and a quantification of the average amount of protein (± SD) contained in the different fractions (n=3 independent replicates).

Interdependence of BacA polymerization and membrane binding.

(A) Cell fractionation experiment investigating the membrane-binding activity of the polymerization-deficient F130R variant of BacA-mVenus in vivo (LY119). The analysis was performed as described for Figure 2B. (B) Co-sedimentation analysis of the association of BacA-F130R with liposomes in vitro, performed as described for Figure 3. (C) Role of polymerization in the membrane association of a BacA-mVenus variant carrying the membranetargeting sequence of E. coli MreB. Shown are phase contrast and fluorescence images of ΔbacAB mutants (LY103, LY123) producing either a BacA-mVenus variant in which the MTS is replaced by two tandem copies of the N-terminal amphiphilic helix of E. coli MreB (MreBWT) or a polymerization-deficient variant thereof (MreBF130A). Demographs summarizing the single-cell fluorescence profiles obtained from random subpopulations of cells are given next to the respective fluorescence images. The numbers of cells analyzed are: MreBWT (126), MreBF130A (169). The vertical red line indicates the junction between cell body and the stalk. Scale bar: 1 μm. (D) Mobility of the indicated BacA-mVenus fusion proteins. Shown is the average mean-squared displacement (MSD) (± SD) as a function of time, based on single-particle tracking analysis. (E) Cell fractionation experiment investigating the membrane-binding activity of MreBBacA-mVenus (MreBWT) and its polymerization-deficient F130R variant (MreBF130R) in vivo (LY103, LY123). The analysis was performed as described for Figure 2B.

Molecular dynamics simulation of the interaction between the BacA MTS and a model membrane.

(A) Snapshot of the molecular dynamics (MD) simulation system showing the 10-mer peptide MFSKQAKSNN (BacA1-10; red) after binding to the lipid bilayer. The water is shown in surface representation. K+ and Cl- counterions are not shown. (B) Close-up view of a representative snapshot from the MD simulation visualizing the binding mode of the peptide on the membrane surface. (C) Structural overlay of 40 snapshots from the MD simulation, taken after constant time intervals from the trajectory. (D) Density profiles of individual residues in the wild-type peptide along the membrane normal, i.e. the zcomponent of the distance vector from the center-of-mass (COM) of the bilayer, with the membrane midplane located at zero. The vertical dashed black line indicates the maximum of the density distribution of the lipid headgroup phosphates.

Contact numbers and interaction energies for different peptide-lipid bilayer interactions.

(A) The graph shows the total number of contacts between individual residues in the wild-type, K4S-K7S and F2Y peptides and the lipid bilayer as well as the number of contacts with PG lipids and GLY lipids. A contact between a peptide residue and a lipid was defined to exist if any two non-hydrogen atoms of the residue and a lipid molecule were within a distance of 0.5 nm to each other. Contacts were counted for each frame of the MD trajectories and averaged. Multiple contacts between a peptide and a lipid molecule were treated as a single contact, so that the number of contacts counted was either 1 or 0. The statistical errors plotted were obtained from the difference between the two different sets of 500-ns simulations, starting with peptides in an unfolded or α-helical conformation, respectively. (B) Energies of the interactions between individual residues in the wild-type, K4S-K7S and F2Y peptides and the lipid bilayer. The interaction energies plotted are the combined interaction energies of all Coulomb and van-der-Waals interactions in the force field averaged over the simulation trajectories.

Conservation of the N-terminal regions of bactofilin homologs in different bacterial phyla.

The pie chart in the middle shows the relative distribution of the 14337 unique bactofilin homologs analyzed among the indicated bacterial phyla. The sequence logos give the the most widespread N-terminal motifs obtained either by a global analysis of all 14337 bactofilin sequences (global consensus) or by an analysis of subsets of these sequences from specific phyla.

Interaction of BacA with its client protein PbpC.

(A) Localization patterns of different PbpC variants. ΔbacB ΔpbpC cells producing mVenus-PbpC (LY75) or mutant variants thereof lacking region C1 (LY76) or carrying an unstructured region from C. crescentus DipM in place of the unstructured region connecting region C1 and the transmembrane helix (LY77) were analyzed by phase contrast and fluorescence microscopy. The outlines of the cells are shown in the fluorescence images. Demographs summarizing the single-cell fluorescence profiles obtained from random subpopulations of cells are given next to the respective fluorescence images. The numbers of cells analyzed are: LY75 (158), LY76 (253), LY77 (119). Scale bar: 1 μm. (B) Biolayer interferometric analysis of the interaction between PbpC1-13aa and BacA. A synthetic peptide comprising the first 13 amino acids of PbpC (PbpCaa1-13) was immobilized on a biosensor and probed with increasing concentrations of BacA. After the association step, the sensor was transferred to a protein-free buffer to monitor the dissociation reaction. The graph shows a representative titration series (n=3 independent replicates). (C) Comparison of the interaction of PbpCaa1-13 with BacA and its polymerization deficient F130R variant, performed as described in panel B. (D) Mapping of the PbpC binding site on BacA by hydrogen-deuterium exchange (HDX) mass spectrometry. The plots show the extent of deuterium uptake by three representative peptides obtained after tryptic digestion of BacA protein (2.5 µM) that had been incubated in the absence or presence of PbpCaa1-13 peptide (10 µM) for the indicated time periods (see Data S1 for the full set of peptides). (E) Mapping of the the differences in deuterium uptake observed at t=1000 s onto the solid-state NMR structure of BacA (Shi et al., 2015).