Biochemical analysis of B. subtilis MinD ATPase cycle.

(A) Phosphate release plotted against different MinD concentrations, fitted with a simple linear regression (R² = 0.97). Phosphate release was measured using the EnzChek™ phosphate assay kit. Samples contained 0.2 mg ml-1 liposomes and were pre-incubated for 10 minutes before addition of 2 mM Mg2+-ATP; n ≥ 3. (B) Specific activity of the MinD ATPase from (A) measured as a function of MinD concentration. (C) Specific activity of MinD measured as a function of ATP concentration, determined as in (B) with fixed MinD concentration of 10 µM. Fitting the Michaelis–Menten equation (black lines) gives kcat = 36.27 h−1, Vmax = 19.50 nmol mg-1 min-1 and KM = 0.173 mM. (D) Specific activity of MinD measured as a function of liposome concentration, determined as in (B) with fixed MinD concentration of 6 µM. Fitting the Michaelis–Menten equation (black lines) gives kcat = 41.01 h−1, Vmax = 22.05 nmol mg-1 min-1 and KM = 0.0437 mg ml-1.

Biochemical analysis of effect of His-MinC or His-PDZ on MinD ATPase activity.

(A) Left: Specific activity of MinD measured as a function of ATP concentration. Phosphate release was measured using the EnzChek™ phosphate assay kit. Samples contained 6 µM His-MinD, 6 µM His-MinC and 0.2 mg ml-1 liposomes and were pre-incubated for 10 minutes before addition of 2mM Mg2+-ATP; n ≥ 3. Fitting the Michaelis–Menten equation (black lines) gives kcat = 15.77 h−1, Vmax = 8.30 nmol mg-1 min-1 and KM = 0.10 mM. Right: Relative activity of 6 µM His-MinD in the presence of 6 µM His-MinC (blue) compared to an internal control of 6 µM His-MinD (black, relative activity = 1). Whiskers represent SD. Specific activity of only His-MinD was 8.61 [nmol mg-1 min-1] (SD = 0.03, n=2). Relative activity of 6 µM His-MinD in the presence of 6 µM His-MinC was 0.95 with SD = 0.071 (n = 3). Significance was assessed by performing Welch’s t-test on the raw data. p < 0.05. (B) Left: Specific activity of MinD measured as a function of ATP concentration. Phosphate release was measured using the EnzChek™ phosphate assay kit. Samples contained 6 µM His-MinD, 5 µM His-PDZ and 0.2 mg ml-1 liposomes and were pre-incubated for 10 minutes before addition of 2mM Mg2+-ATP n ≥ 3. Fitting the Michaelis–Menten equation (black lines) gives kcat = 12.07 h−1, Vmax = 6.35 nmol mg-1 min-1 and KM = 0.233 mM. Right: Relative activity of 6 μM His-MinD in the presence of different concentrations of His-PDZ (blue) compared to only 6 μM His-MinD (black, relative activity = 1). Whiskers represent SD. Specific activity of an internal control of His-MinD was 4.60 nmol mg-1 min-1 (SD = 0.03, n=2). Relative activities of His-MinD in the presence of 1 µM, 3 µM and 5 µM His-PDZ were 1.22, 1.37 and 1.25 with SD = 0.07, 0.13 and 0.03, respectively (n = 3 each). Significance was assessed by performing Welch’s t-test on raw data of all samples versus control. p < 0.05 for all PDZ concentrations.

Cartoon of MinD AlphaFold model with highlighted key amino acids and the effects of their mutagenesis.

(A) Left: Rendering of MinD AlphaFold model with ATP binding region highlighted in light blue, membrane targeting sequence (MTS) in turquoise and key residues in orange; Box: explanation of mutagenesis effects. Right: Close-up on hydrophobic region of amphipathic helix.(B) Representative wide-field microscopy images of B. subtilis expressing indicated Halo-MinD variants as the only MinD copy from the native locus, stained with TMR ligand. Left: Phase contrast; scale bar 5 µm. Right: fluorescence.

His-MinD mutants are catalytically inactive in ATP hydrolysis assay.

(A) Phosphate release plotted against different His-MinD-I260E concentrations and His-MinD as control, fitted with a simple linear regression. Phosphate release was measured using the EnzChek™ phosphate assay kit. Samples contain 0.2 mg ml-1 liposomes and were pre-incubated for 10 minutes before addition of 2 mM Mg2+-ATP. (B) Same as (A) but with different y-axis scaling for better visualization of His-MinD-I260E baseline activity. (C) Same as (B) but comparing His-MinD mutants G12V, K16A and D40A to His-MinD at 6 µM concentrations, respectively.

Measurements of cell length and minicell formation of B. subtilis strains.

BLI analysis of His-MinD and different mutants.

(A) Left: Cartoon representation of the different BLI steps, starting with (1) establishing a baseline in protein buffer, (2) binding of liposomes through the biotinylated DSPE-PEG(2000) phospholipid, (3) establishing a new baseline, (4) binding of MinD and finally (5) dissociation of MinD. Right: Exemplary graph resulting from sensor readouts of steps (1) - (5). (B) MinD binding plotted against time through association and dissociation phase (steps 4-5) at different protein concentrations, including the His-MinD-I260E mutant. (C) – (E) Same as (B) using the indicated respective mutant of His-MinD (G12V, K16A and D40A).

Kinetic constants of His-MinD and variants obtained via BLI.

Kinetic constants of His-MinD in combination with His-MinC or His-PDZ.

Single-molecule localization microscopy analysis of Halo-MinD and mutants expressed in B. subtilis.

Exponentially growing B. subtilis cells expressing Halo-MinD and variants (n ≥ 48 cells, respectively) were stained with TMR ligand and subsequently imaged. Individual protein trajectories were recorded using SMLM and analyzed with Zen blue (Zeiss), Trackmate, the SMTracker 2.0 software package and manual scripts in R. Minimum track-length 4 frames of 24 ms, with at least 2596 trajectories per strain. (A) Heat map representation of intracellular localization of individual molecules of Halo-MinD and variants, respectively, plotted on normalized cells. Brighter colors indicate higher abundance. (B) Barplot of stationary localization analysis (SLA), comparing different track types within the protein population. Tracks were considered static when not leaving a circular area of 97 nm diameter within 5+ frames. Mobile populations were further divided into free and mixed tracks, where mixed tracks displayed a switch between free and confined movement. (C) Plot of the mean-squared displacement of Halo-MinD and variants over time, fitted with a linear fit excluding the last timelag. (D) Bubble plot displaying single-molecule diffusion rates of the indicated MinD fusions. Populations were determined by fitting the probability distributions of the frame-to-frame displacement (jump distance) data of all respective tracks to a three-component model (fast mobile, slow mobile and confined protein populations). (E) Probability distributions of jump distances of Halo-MinD and variants. Data was fit with a three-component model, indicating confined, slow and fast tracks.

Diffusion coefficients obtained from non-comparative SQD analysis of Halo-MinD and variants fitted with three populations.

Single-molecule localization microscopy analysis of Halo-MinD expressed in B. subtilis wild type and mutant backgrounds.

Exponentially growing B. subtilis cells expressing Halo-MinD in the indicated genetic backgrounds (n ≥ 115 cells, respectively) were stained with TMR ligand and subsequently imaged. Individual protein trajectories were recorded using SMLM and analyzed with Zen blue (Zeiss), Trackmate, the SMTracker 2.0 software package and manual scripts in R. Minimum track-length 4 frames of 24 ms, with at least 2688 trajectories per strain. (A) Heat map representation of intracellular localization of individual molecules of Halo-MinD in the indicated genetic background, respectively, plotted on normalized cells. Brighter colors indicate higher abundance. (B) Barplot of stationary localization analysis (SLA), comparing different track types within the protein population. Tracks were considered static when not leaving a circular area of 97 nm diameter within 5+ frames. Mobile populations were further divided into free and mixed tracks, where mixed tracks displayed a switch between free and confined movement. (C) Bubble plot displaying single-molecule diffusion rates of Halo-MinD in different genetic backgrounds. Populations were determined by fitting the probability distributions of the frame-to-frame displacement (jump distance) data of all respective tracks to a three components model (fast mobile, slow mobile and confined protein populations). (D) Probability distributions of jump distances of Halo-MinD in different genetic backgrounds. Data was fit with a three-component model, indicating confined, slow and fast tracks.

Diffusion coefficients obtained from non-comparative SQD analysis of Halo-MinD in different genetic backgrounds fitted with three populations.

His-MinD purification analysis.

(A) SDS-PAGE gel analysis of purification products of His-MinD (31.7 kDa), stained with Coomassie blue stain. From left to right: Ladder, pellet after lysate, supernatant, Ni-NTA peaks A2, A7, C4 (Main peak), concentrated sample, gel filtration peak. Ladder = Color Prestained Protein Standard, Broad Range (New England Biolabs). (B) Western blot of replicate gel from (A) using Penta-His antibody (QIAGEN).

Gel filtration chromatograms of His-MinD and variants.

Gel filtration analysis of purified His-MinD and indicated mutants. Curves were aligned according to the respective void volume after sample injection. Green and red dotted lines mark the calibrated elution volumes corresponding to proteins of 31.69 kDa (His-MinD monomer) and 63.38 kDa (His-MinD dimer), as determined from molecular-weight standards.

His-MinC purification analysis.

(A) SDS-PAGE gel analysis of purification products of His-MinC (27.3 kDa), stained with Coomassie blue stain. From left to right: Ladder, Ni-NTA peak, SEC peak. Ladder = Color Prestained Protein Standard, Broad Range (New England Biolabs). (B) Western blot of replicate gels from (A) using Penta-His antibody (QIAGEN).

His-PDZ purification analysis and rendering of MinJ AlphaFold model.

(A) SDS-PAGE gel analysis of purification products of His-PDZ (17.6 kDa), stained with Coomassie blue stain. From left to right: Ladder (L), Ni-NTA main peak. Ladder = Color Prestained Protein Standard, Broad Range (New England Biolabs). (B) Western blot of replicate gel from (A) using Penta-His antibody (QIAGEN). (C) Rendering of AlphaFold 3 model of B. subtilis MinJ (grey) (83). The truncation expressed with pET-28a-PDZ, which includes the PDZ domain (blue), is highlighted in pink.

His-MinD-I260E purification analysis.

(A) SDS-PAGE gel analysis of purification products of His-MinD-I260E (31.7 kDa), stained with Coomassie blue stain. From left to right: Ladder, supernatant after lysis, pellet, Ni-NTA flowthrough, Ni-NTA peaks A10 and C4 (Main peak), concentrated sample, gel filtration peak. Ladder = Color Prestained Protein Standard, Broad Range (New England Biolabs). (B) Western blot of replicate gel from (A) using Penta-His antibody (QIAGEN).

His-MinD-G12V purification analysis.

(A) SDS-PAGE gel analysis of purification products of His-MinD-G12V (31.7 kDa), stained with Coomassie blue stain. From left to right: Ladder, pellet after lysate, supernatant, Ni-NTA flowthrough, Ni-NTA peaks A2, A10, C4 (Main peak), concentrated sample, gel filtration peak. Ladder = Color Prestained Protein Standard, Broad Range (New England Biolabs). (B) Western blot of replicate gel from (A) using Penta-His antibody (QIAGEN).

His-MinD-K16A purification analysis.

A) SDS-PAGE gel analysis of purification products of His-MinD-K16A (31.7 kDa), stained with Coomassie blue stain. From left to right: Ladder, pellet after lysate, supernatant, Ni-NTA flowthrough, Ni-NTA peaks A2, A10, C4 (Main peak), concentrated sample, gel filtration peak. Ladder = Color Prestained Protein Standard, Broad Range (New England Biolabs). (B) Western blot of replicate gel from (A) using Penta-His antibody (QIAGEN).

His-MinD-D40A purification analysis.

(A) SDS-PAGE gel analysis of purification products of His-MinD-D40A (31.7 kDa), stained with Coomassie blue stain. From left to right: Ladder, supernatant after lysis, pellet, Ni-NTA flowthrough, Ni-NTA peaks A2 and C4 (Main peak), concentrated sample, gel filtration peak. Ladder = Color Prestained Protein Standard, Broad Range (New England Biolabs). (B) Western blot of replicate gel from (A) using Penta-His antibody (QIAGEN).

BLI analysis of interactions between His-MinD and His-MinC or His-PDZ.

(A) MinD binding (see Figure 5 A) plotted against time through association and dissociation phase (steps 4-5) at different indicated His-MinC concentrations, including His-MinD and His-MinC only controls, all in presence of freshly added ATP [2 mM]. (B) Same as (A) using the indicated respective concentrations of His-PDZ.

BLI analysis of His-MinD without addition of ATP.

His-MinD binding to liposome associated BLI sensor (see Figure 5), plotted against time through association and dissociation phase at different protein concentrations without the addition of Mg2+-ATP.

Oligonucleotides used in this study.

Plasmids used in this study.

Strains used in this study.

BLI Advanced Kinetics Protocol.