The role of FtsZ-associated proteins in the regulation of the assembly dynamics of Mycobacterium smegmatis FtsZ is not clear. In this work, we examined the effect of M. smegmatis SepF on the assembly and stability of M. smegmatis FtsZ polymers. We discovered a single dominant point mutation in SepF (G51D or G51R) that renders the protein inactive. SepF promoted the polymerization of FtsZ, induced the bundling of FtsZ filaments, stabilized FtsZ filaments and reduced the GTPase activity of FtsZ. Surprisingly, both G51D-SepF and G51R-SepF neither stabilized FtsZ filaments nor showed a discernable effect on the GTPase activity of FtsZ. The binding affinity of SepF to FtsZ was found to be stronger than the binding affinity of G51R/D-SepF to FtsZ. Interestingly, the binding affinity of SepF to G51R-SepF was determined to be 45 times stronger than FtsZ. In addition, the interaction of SepF with G51R-SepF was found to be 2.6 times stronger than SepF–SepF interaction. Furthermore, G51R-SepF impaired the ability of SepF to promote the assembly of FtsZ. In addition, the overexpression of G51R-SepF in M. smegmatis mc2 155 cells retarded the proliferation of these cells and increased the average length of the cells. The results indicated that SepF positively regulates the assembly of M. smegmatis FtsZ and the G51 residue has an important role in the functioning of SepF.

Introduction

FtsZ, a bacterial cytoskeletal protein, plays an important role in the division of bacterial cells. FtsZ monomers assemble to form a Z-ring at the mid-cell position, and the disassembly of the ring is considered to engineer the cell membrane constriction to form two daughter cells [14]. The mechanism by which the Z-ring regulates the septum constriction is still elusive. It was suggested that FtsZ could generate force for the membrane constriction [5]. Recently, it has been observed that FtsZ needs other peptidoglycan-synthesizing proteins to constrict the membrane [6]. The treadmilling of FtsZ has been shown to play an important role in the septum synthesis [7]. Furthermore, FtsZ forms discrete membrane-associated filaments with the help of one of its accessory proteins, FtsA, at the potential division site. These filaments move around the division plane directionally by treadmilling of FtsZ filaments at the potential division site forming the Z-ring. The ring eventually recruits peptidoglycan synthesis enzymes and helps the inward synthesis of nascent peptidoglycan leading to the membrane constriction [7]. The Z-ring is highly dynamic in nature [8,9], and the dynamics of the Z-ring is controlled by the combined actions of several regulatory proteins [10] such as ZipA [3,11], FtsA [12], ZapA-D [1317], and SepF [1820]. These accessory proteins either stabilize and destabilize the Z-ring, and thus regulate the ring formation spatially as well as temporally [21].

Interestingly, different bacterial species have different divisome proteins with similar functions. For example, SepF is considered to stabilize the Z-ring in Bacillus subtilis, while ZipA performs the similar function in Escherichia coli [4,22,23]. SepF is conserved in all Gram-positive bacteria [19] and it regulates the assembly of FtsZ in bacteria [20]. It is encoded by the gene ylmF in B. subtilis. The presence of SepF was first identified during the isolation of FtsZ-associated cell division complex in vivo [19]. The function of SepF overlaps with FtsA in the formation of a functional Z-ring [19]. A disruption of SepF produced slightly elongated Bacillus subtilis cells, whereas the disruption of both SepF and FtsA inhibited cell division due to the formation of inefficient Z-rings [19]. In vitro, SepF was found to promote the assembly of B. subtilis FtsZ (BsFtsZ) and to suppress the disassembly of FtsZ polymers [18].

B. subtilis SepF polymerizes itself to form a ring-like polymer 50–60 nm in diameter and facilitate the lateral association between the FtsZ protofilaments [24]. SepF binds to the C-terminal tail of BsFtsZ like many other FtsZ-associated proteins [18,25] and has a membrane-binding motif on its N-domain [22]. The presence of SepF in Mycobacterium sp. was reported in 2015 [26]. SepF was found to interact with FtsZ and localize at the division site in an FtsZ-dependent manner in vivo [26]. The overexpression or the deletion of SepF was found to inhibit the division of Mycobacterium smegmatis [26]. Furthermore, SepF was shown to interact with the peptidoglycan-synthesizing enzyme MurG, indicating its additional role in cell wall synthesis in M. smegmatis [27]. A recent study suggested that the overexpression of SepF perturbs membrane invagination by interfering with the recruitment of the late divisome proteins such as Pbp2B, FtsW, and FtsL [28]. The studies indicated that SepF plays dual roles in cell division as well as in peptidoglycan synthesis. However, the role of SepF in the assembly of M. smegmatis FtsZ remains elusive.

In the present study, we sought to elucidate the effect of M. smegmatis SepF on the polymerization of M. smegmatis FtsZ. To achieve the objective, SepF was cloned from the genomic DNA of M. smegmatis mc2 155 cells. During the cloning, a serendipitous mutation (G51D) occurred in the retrieved SepF construct. Surprisingly, G51D-SepF could not promote the assembly of FtsZ. Then, we prepared the wild-type form of SepF by correcting the mutation. SepF promoted the polymerization of FtsZ and stabilized the preformed FtsZ polymers against dilution in vitro. To determine the effect of the charge of the amino acid at the 51st position on the function of SepF, another mutation (G51R) in SepF was introduced. SepF and G51D/R-SepF displayed similar secondary structural contents, indicating that the mutation did not alter the secondary structure of the protein. Like G51D-SepF, G51R-SepF also could not promote the polymerization of FtsZ. G51D/R-SepF were found to bind to FtsZ with a weaker affinity than SepF. Interestingly, G51R/D-SepF inhibited the polymerization-inducing effect of SepF on FtsZ. The overexpression of G51R-SepF in M. smegmatis mc2 155 strain increased the mean length of the cells. Together, the results suggested that SepF acts as a positive regulator of FtsZ assembly and the G51 residue in M. smegmatis SepF may play an important role in its ability to promote FtsZ assembly.

Experimental methods

Extraction of genomic DNA from M. smegmatis mc2 155 cells

M. smegmatis mc2 155 cells were grown in Middlebrook 7H9 medium with Tween 20 and glycerol and incubated at 37°C and 200 rpm. The cells were harvested and the cell pellet was dissolved in TE buffer containing glass beads and lysozyme. Furthermore, 10% SDS and proteinase K were added to the lysate and incubated for 15 min at 65°C. Pre-warmed NaCl (5 M) and CTAB (12%) were added to the lysate and incubated for 10 min at 65°C. A mixture of phenol : chloroform : isoamyl alcohol (25 : 24 : 1) was added to it and the mixture was centrifuged at room temperature for 15 min at 10 000 rpm. The aqueous supernatant was transferred to a microcentrifuge tube and mixed with chloroform and isoamyl alcohol (24 : 1). Then, the vial was centrifuged at room temperature for 10 min at 10 000 rpm. The nucleic acids were precipitated by incubating the supernatant with chilled isopropanol for 30 min at −20°C and the pellet was collected by centrifuging at 10 000 rpm for 10 min. The pellet was washed with 75% ethanol and dissolved in TE buffer.

Cloning of M. smegmatis SepF

M. smegmatis SepF was cloned into pET-28a vector between XhoI and BamHI restriction sites. The genomic DNA isolated from M. smegmatis mc2 155 was used as a template for PCR using forward (5′-CGCGGATCCATGAGCACACTGCATAA-3′) and reverse (5′-CCGCTCGAGCTAACGGTAGGAGTAGA-3′) primers. The PCR product was gel-extracted and digested with the XhoI and BamHI restriction enzymes at 37°C for 30 min. The digested PCR product was then ligated to the digested pET-28a vector in the presence of T4 DNA ligase at 16°C for 4 h. The ligated product was transformed into XL1-B competent cells. Positive clones, confirmed by sequencing, were transformed into BL21 competent cells for overexpression. Colonies positive for SepF expression were selected.

Site-directed mutagenesis of M. smegmatis SepF

Site-directed mutations at the 51st amino acid position in M. smegmatis SepF were done using primers designed by the QuikChange primer design tool. Forward and reverse primers were used for D51G (Fwd 5′-GTGGCCCTCGTAGCCGTAGGCCTCTTC-3′ and Rev 5′ GAAGAGGCCTACGGCTACGAGGGCCAC-3′) and D51R (Fwd 5′-CGTGGCCCTCGTAGCGGTAGGCCTCTTCCT-3′ Rev 5′-AGGAAGAGGCCTACCGCTACGAGGGCCACG-3′) amplification. The PCR products were subjected to DpnI digestion by incubating at 37°C for 1.30 h. The digested products were ligated using T4 DNA ligase at 25°C for 1 h. The final products were transformed into XL1-B competent cells. Colonies were confirmed for the mutation by sequencing. The positive clone was transformed into BL21 competent cells.

Purification of M. smegmatis FtsZ and M. smegmatis L66W-FtsZ

M. smegmatis FtsZ was overexpressed in E. coli BL21 (DE3) cells using the pET-15b-ftsZ construct and the protein was purified using Ni2+-NTA affinity chromatography as described recently [29]. The 6×-histidine tag of FtsZ was removed from the protein using thrombin as described recently [29]. All experiments were performed using FtsZ without the histidine tag.

The QuikChange site-directed mutagenesis kit from Stratagene was used to introduce a single tryptophan mutation in M. smegmatis FtsZ at the 66th position. L66W-FtsZ was overexpressed in E. coli BL21 (DE3) cells, by inducing with 1 mM IPTG, and the protein was purified using a Ni2+-NTA column [29]. The eluted 6×-His-tagged FtsZ fractions were dialyzed for 12 h in 50 mM PIPES-NaOH buffer containing 50 mM KCl and 5 mM MgCl2, pH 7.2. The dialyzed proteins were concentrated and stored in aliquots at −80°C.

Purification of M. smegmatis SepF

SepF, G51D-SepF, and G51R-SepF were cloned in pET-28a (+) and overexpressed as the 6×His-tagged protein in E. coli BL21 (DE3) cells. The proteins were purified using the following protocol. Briefly, E. coli BL21 (DE3) cells having different recombinant SepF constructs were induced with 0.5 mM IPTG for 4 h at 30°C when the OD600 reached 0.2. The cells were harvested by centrifugation and lysed in buffer A (0.5 M NaCl, 20 mM NaH2 PO4) containing 8 M urea. The lysed cells were homogenized on ice for 30 min and then sonicated till the lysate became clear. The soluble fraction was separated from the cell debris by centrifugation at 33 902 g for 45 min at 30°C. The soluble lysate was incubated with Ni2+-NTA beads for 3 h at room temperature and the beads were then washed with buffer A (0.5 M NaCl, 20 mM NaH2 PO4) containing 6, 4, and 2 M urea separately. Subsequently, the beads were washed with buffer A (0.5 M NaCl and 20 mM NaH2 PO4) and kept at 4°C for 2 h to allow the protein to fold. After the refolding, the column was washed with buffer A containing 10, 50, and 100 mM imidazole, respectively. The histidine-tagged SepF was eluted using elution buffer containing 0.5 M NaCl, 20 mM NaH2 PO4, and 500 mM imidazole. The eluted fractions were collected together and desalted using 25 mM HEPES–NaOH buffer containing 50 mM KCl, 2% glycine, and 10% glycerol (pH 7.2). The His-tag was removed from SepF using thrombin (Novagen, San Diego, CA) and then thrombin was removed using the streptavidin-agarose beads [29]. The eluted fractions were concentrated and stored at −80°C.

Light scattering

The polymerization kinetics of the FtsZ was monitored by 90° light scattering using a JASCO FP-8500 fluorescence spectrophotometer (Tokyo, Japan). Purified FtsZ (6 µM) was incubated in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl and 5 mM MgCl2 in the absence and in the presence of 1, 2, and 3 µM SepF, G51D-SepF, and G51R-SepF separately for 10 min on ice. GTP (1 mM) was added to the reaction mixtures and the assembly kinetics was monitored for 600 s at 37°C [30]. The light scattering of 1, 2, and 3 µM SepF, G51D-SepF, and G51R-SepF was also recorded. Similarly, the effects of SepF and G51R/D-SepF on the assembly of FtsZ were monitored at pH 7.5.

Furthermore, the effect of SepF on the polymerization of FtsZ in the presence of G51R/D-SepF was monitored by light scattering. Briefly, FtsZ (6 µM) was incubated with SepF (3 µM) in 25 mM HEPES (pH 6.8) containing 100 mM KCl and 5 mM MgCl2 on ice for 5 min. Then, either G51R-SepF (0.25, 0.5, and 1 µM) or G51D-SepF (3 µM) was added to the reaction mixture and incubated for an additional 5 min on ice. The polymerization kinetics was recorded at 37°C for 10 min immediately after the addition of 1 mM GTP.

Sedimentation assay

FtsZ (6 µM) in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl and 5 mM MgCl2 was incubated without and with different concentrations (2, 4, 6, and 9 µM) of either SepF, G51D-SepF, or G51R-SepF on ice for 10 min. Then, the samples were incubated in the presence of 1 mM GTP for 10 min at 37°C. FtsZ polymers were retrieved by centrifugation at 32 047 g for 30 min at 30°C. The polymers were dissolved, analyzed by SDS–PAGE, and stained with Coomassie brilliant blue. The band intensity was quantified using the ImageJ software. Similarly, the effects of SepF, G51R-SepF, and G51D-SepF on the amount of polymerized FtsZ were examined at pH 7.5.

In a separate experiment, FtsZ (6 µM) was incubated in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl and 5 mM MgCl2 with SepF (4 µM) for 5 min and then G51R-SepF (1 and 2 µM) was added to the reaction milieu. The samples were further kept on ice for 5 min. Then, 1 mM GTP was added and the samples were polymerized at 37°C for 10 min. The polymers were collected and the pellets were loaded in 12% SDS–PAGE. Similarly, the polymerization kinetics of 1, 2, 4, 6, and 8 µM SepF, G51D-SepF and G51R-SepF were observed using the above-mentioned protocol. Furthermore, the assembly properties of L66W-FtsZ and FtsZ (6, 8, 10, and 12 µM) were compared under similar conditions.

Transmission electron microscopy

FtsZ (3 µM) was incubated in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl and 5 mM MgCl2 in the absence and in the presence of either 3 µM SepF or G51D-SepF and G51R-SepF independently on ice for 10 min. The samples were polymerized in the presence of 1 mM GTP at 37°C for 10 min. An aliquot of the polymers was transferred to a glow-discharged formvar/carbon-coated copper grid (300 mesh) and stained with 2% uranyl acetate. The grids were analyzed using JEOL JEM 1220 HRTEM (200 kV) [31].

Separately, FtsZ (3 µM) was incubated with SepF (2 µM) in polymerizing buffer for 5 min on ice and then G51R-SepF (2 µM) was added. The samples were further incubated for an additional 5 min and then polymerized with 1 mM GTP for 10 min at 37°C. The polymers formed were analyzed under the electron microscope.

Measurement of the GTPase activity of FtsZ

The effect of SepF on the GTPase activity of FtsZ was determined using the ammonium molybdate assay [32]. Briefly, FtsZ (4 µM) was incubated in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl and 5 mM MgCl2 without and with different concentrations (2, 4, 6 and 9 µM) of SepF, G51D-SepF, and G51R-SepF on ice for 10 min. Then, 1 mM GTP was added to the samples and the mixtures were incubated for 15 min at 37°C. The reaction was stopped by the addition of 10% (v/v) perchloric acid and the moles of Pi released per mole of FtsZ were determined by taking the absorbance of the reaction mixture at 650 nm.

Dilution-induced disassembly assay

FtsZ (15 µM) was polymerized in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl, 5 mM MgCl2, and 1 mM GTP at 37°C for 10 min. Then, the FtsZ polymers were diluted 20 times in warm 25 mM HEPES buffer with and without 1, 2, 3, and 4 µM SepF, G51D-SepF, and G51R-SepF. The final concentration of FtsZ after dilution was 0.75 µM. The diluted reaction milieu was incubated for an additional 5 min and was subjected to centrifugation at 32 047 g for 30 min at 30°C. The pelleted polymers were dissolved in SDS loading buffer and loaded in 12% SDS–PAGE. The intensities of the protein bands were estimated from the SDS–PAGE gel stained with Coomassie brilliant blue R using the ImageJ software.

Circular dichroism study

The secondary structures of SepF, G51D-SepF, and G51R-SepF were determined using circular dichroism. To achieve that, 4 µM SepF, G51D-SepF, and G51R-SepF were incubated in 5 mM HEPES buffer (pH 6.8). The secondary structures were monitored by measuring the far-UV CD spectra (200–260 nm) using a cuvette of 0.1 cm path length. CD experiment was performed in a Jasco CD spectrometer (J-1500) [33].

Size-exclusion chromatography

The interactions between FtsZ, SepF, and mutant SepF constructs were detected by size-exclusion chromatography using AKTA Pure (GE Healthcare) liquid chromatography [29]. The superdex™ G 75 column (GE Healthcare) was used for the experiment and the column was equilibrated with 25 mM HEPES (pH 6.8). Bovine serum albumin (BSA) and lysozyme (20 µM) were used to calibrate the column at 4°C. FtsZ (20 µM) was incubated with SepF, G51D-SepF, and G51R-SepF individually (20 µM) in 25 mM HEPES buffer (pH 6.8) on ice for 30 min and the samples were loaded onto the column at 4°C. The flow rate was adjusted to 0.5 ml/min. The elution profiles of FtsZ, SepF, G51D-SepF, G51R-SepF, and FtsZ-SepF complexes were monitored individually by taking the absorbance at 280 nm. The peak fractions were loaded in 12% SDS–PAGE.

Labeling of SepF

SepF (20 µM) was incubated with 100 µM FITC in 50 mM phosphate buffer (pH 8.0) for 4 h on ice. The reaction was terminated by adding 5 mM Tris–HCl (pH 8.0). The free FITC and WT-SepF-bound FITC were separated using a P4 column (Bio-Rad, CA, USA) [33]. The stoichiometry of labelling of FITC per SepF was determined to be 0.5.

Fluorescence spectroscopy

Determination of dissociation constant using tryptophan fluorescence

The binding affinity of SepF, G51D-SepF, and G51R-SepF for FtsZ was determined using tryptophan fluorescence of L66W-FtsZ. Briefly, L66W-FtsZ (1 µM) was incubated without and with different concentrations (100–8000 nM) of SepF, G51D-SepF, and G51R-SepF for 10 min in 25 mM HEPES buffer (pH 6.8) at 25°C. The fluorescence spectra (300–400 nm) were monitored using 295 nm as the excitation wavelength. The increase in the tryptophan fluorescence intensity (at 345 nm) upon binding to SepF was used to determine the dissociation constant (Kd) of the interaction of FtsZ and SepF using a formula:

 
formula

ΔF is the change in the fluorescence intensities of L66W-FtsZ upon binding to SepF; ΔFmax is the maximum change in the fluorescence intensity of FtsZ upon binding to SepF. L is the concentration of SepF used. ΔF was obtained by subtracting the fluorescence intensity of L66W-FtsZ in the presence of SepF from the fluorescence intensity of only L66W-FtsZ. The data were analyzed using a GraphPad prism5 software (Graph Pad Software, La Jolla, CA, USA).

Determination of the dissociation constant of the interaction of FtsZ and SepF using FITC-SepF

To check the specificity of binding of SepF to FtsZ, FITC-SepF (50 nM) was incubated with either 500 nM BSA, alcohol dehydrogenase or tubulin in 25 mM HEPES (pH 6.8) for 10 min at 25°C. The fluorescence spectra of FITC-SepF (510–600 nm) were monitored by exciting the reaction mixtures at 495 nm.

Furthermore, the dissociation constant of the binding interaction of SepF to itself, FtsZ, or G51R-SepF was determined by monitoring the change in the fluorescence intensity of FITC-SepF. FITC-SepF (50 nM) was incubated with different concentrations of either FtsZ (50–5000 nM) or unlabeled SepF/G51R-SepF (2–500 nM) for 10 min in 25 mM HEPES buffer (pH 6.8) at 25°C. The fluorescence spectra (510–600 nm) of FITC-SepF were monitored by exciting the samples at 495 nm. The changes in the fluorescence intensities of FITC-SepF in the presence of different concentrations of either FtsZ or unlabeled SepF/G51R-SepF were plotted against concentrations of FtsZ, unlabeled SepF, and G51R-SepF used. The dissociation constants of binding were determined by fitting the values in a binding equation as mentioned previously.

Effect of potassium chloride on the binding of FtsZ and SepF

L66W-FtsZ (1 µM) was incubated in the presence of SepF (6 µM) for 10 min in 25 mM HEPES buffer (pH 6.8) at 25°C. The reaction mixtures were further incubated for 10 min without or with different concentrations (50, 100, 200, and 500 mM) of KCl. Separately, L66W-FtsZ (1 µM) was incubated with different concentrations (50, 100, 200, and 500 mM) of KCl for 10 min in 25 mM HEPES buffer (pH 6.8). The fluorescence spectra were monitored using 295 nm as an excitation wavelength.

Preparation of recombinant M. smegmatis mc2 155 cells expressing SepF constructs

SepF and G51R-SepF were cloned in Mycobacterium shuttle vector pSC300 (Addgene) at BamHI and EcoRV restriction sites using the forward (5′-CGCGGATCCATGAGCACACTG-3′) and the reverse (5′-CCGGATATCCTAACGGTAGGAG-3′) primers. These constructs were screened on LB Agar plates containing 100 µg/ml hygromycin. The confirmed clones were transformed into M. smegmatis mc2 155 by electroporation (Bio-Rad gene pulsar).

Briefly, 5 µg of pSC300-SepF and pSC300-G51R-SepF plasmids were added to 200 µl of mc2 155 competent cells in a pre-chilled 0.2 cm electroporation cuvette (Bio-Rad). The electroporation was performed at 2.5 kV, 800 Ω, and 25 µF capacitance. The time constant for the pulses was ranged from 8.5 to 15.5 ms. Electroporated cells were immediately diluted with 5 ml of Middlebrook 7H9 media and kept in a shaker at 37°C for 2 h. The transformed M. smegmatis mc2 155 cells were plated on LB Agar containing hygromycin (50 µg/ml). Positive mc2 155 colonies were further cultured in 7H9 media containing 50 µg/ml hygromycin.

Effects of expression of SepF and G51R-SepF on the growth and length of M. smegmatis mc2 155 cells

The wild-type and the recombinant M. smegmatis mc2 155 cells (harboring pSC300-SepF and pSC300-G51R-SepF) were inoculated in Middlebrook 7H9 media containing 0 and 50 µg/ml hygromycin, respectively, so that the initial OD600 reached 0.1. The cultures were grown at 37 °C for 35 h under shaking condition. The cells were collected every 3 h and OD600 was plotted against time.

To determine the average cell length, the cells were harvested at 23 h. The cells were suspended in PBS containing 0.1% Tween 80 and then fixed with 2.8% formaldehyde and 0.4% glutaraldehyde at 25°C. The fixed cells were visualized under a microscope (FV500 laser scanning confocal microscope, Olympus, Tokyo, Japan). The bacterial cell length was measured using Image-Pro Plus (Media Cybernetics, Silver Spring, MD).

Membrane staining with FM4–64

M. smegmatis mc2 155 cells were stained with a lipophilic fluorescent dye FM 4–64, which has been widely used to image outer membranes as well as the septal membrane of bacterial cells [34,35]. M. smegmatis mc2 155 harboring SepF or G51R-SepF were grown for 30 h. The cells were incubated with 1.5 µg/ml FM 4–64 for 1 h at 37°C under shaking condition. The cells were washed with PBS before mounting on poly l-lysine-coated glass slides to visualize under an FV500 laser scanning confocal microscope (Olympus, Tokyo, Japan).

Dynamic light-scattering spectroscopy

The size distribution of SepF and mutant SepF was determined using dynamic light scattering (DLS). SepF (2 µM) and mutant SepF (2 µM) in 25 mM HEPES (pH 6.8) were subjected to DLS. Size distribution profiles of the samples were determined at 25°C using the Malvern zeta sizer DLS instrument.

Results

Three SepF constructs showed similar secondary structures

The secondary structures of SepF, G51D-SepF, and G51R-SepF were analyzed by the far-UV CD spectra of the purified protein (Supplementary Figure S1). The α-helix, β-sheet, and random coil contents of the SepF were estimated to be 44 ± 3, 11 ± 3, and 45 ± 2%, respectively, indicating that SepF contains a significant level of α helices and random coil structures. Furthermore, G51D-SepF and G51R-SepF showed secondary structures similar to that of the native SepF.

SepF, but not G51R-SepF and G51D-SepF, promoted the polymerization of M. smegmatis FtsZ

The effect of SepF, G51D-SepF, and G51R-SepF on the assembly kinetics of M. smegmatis FtsZ was monitored using 90° light scattering at pH 6.8 (Figure 1A–C) and at pH 7.5 (Supplementary Figure S2). The light-scattering signal of FtsZ in the presence of 2 µM SepF was found to increase by 98 ± 7 and 125 ± 10% at pH 6.8 and 7.5, respectively (Figure 1A and Supplementary Figure S2A). In contrast with the polymerization-promoting ability of SepF, G51D-SepF and G51R-SepF reduced the light-scattering signal of the assembly of FtsZ. For example, 3 µM G51D-SepF and G51R-SepF reduced the scattering of FtsZ by 61 ± 10 and 78 ± 8% at pH 6.8 (Figure 1B,C) and 50 ± 11 and 60 ± 6% at pH 7.5, respectively (Supplementary Figure S2B,C). The results indicated that the mutant SepF proteins could not promote the assembly of FtsZ and these mutants displayed negative effects on the assembly of FtsZ.

Effects of SepF, G51R-SepF, and G51D-SepF on the polymerization kinetics of FtsZ at pH 6.8.

Figure 1.
Effects of SepF, G51R-SepF, and G51D-SepF on the polymerization kinetics of FtsZ at pH 6.8.

FtsZ (6 µM) was polymerized in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl, 5 mM MgCl2, and 1 mM GTP in the absence (□) and presence of 1 (○), 2 (Δ), and 3 (◊) µM (A) SepF, (B) G51D-SepF, and (C) G51R-SepF. The light scattering of only 1 (●), 2 (▴), and 3 (♦) µM SepF, G51D-SepF, and G51R-SepF and buffer (▪) was monitored under similar conditions. Each experiment was repeated three times (n = 3). Furthermore, FtsZ (6 µM) was polymerized in the absence and presence of different concentrations of (D) SepF, (E) G51D-SepF, and (F) G51R-SepF at 37°C in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl, 5 mM MgCl2, and 1 mM GTP. The polymers were collected by centrifugation and the amount of polymerized FtsZ was estimated by Coomassie blue staining of the pelleted proteins. Lanes 1–5 in the gels show the FtsZ pelleted in the absence and presence of 2, 4, 6, and 9 µM (D) SepF, (E) G51D-SepF, and (F) G51R-SepF, respectively. The experiments were performed three times (n = 3).

Figure 1.
Effects of SepF, G51R-SepF, and G51D-SepF on the polymerization kinetics of FtsZ at pH 6.8.

FtsZ (6 µM) was polymerized in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl, 5 mM MgCl2, and 1 mM GTP in the absence (□) and presence of 1 (○), 2 (Δ), and 3 (◊) µM (A) SepF, (B) G51D-SepF, and (C) G51R-SepF. The light scattering of only 1 (●), 2 (▴), and 3 (♦) µM SepF, G51D-SepF, and G51R-SepF and buffer (▪) was monitored under similar conditions. Each experiment was repeated three times (n = 3). Furthermore, FtsZ (6 µM) was polymerized in the absence and presence of different concentrations of (D) SepF, (E) G51D-SepF, and (F) G51R-SepF at 37°C in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl, 5 mM MgCl2, and 1 mM GTP. The polymers were collected by centrifugation and the amount of polymerized FtsZ was estimated by Coomassie blue staining of the pelleted proteins. Lanes 1–5 in the gels show the FtsZ pelleted in the absence and presence of 2, 4, 6, and 9 µM (D) SepF, (E) G51D-SepF, and (F) G51R-SepF, respectively. The experiments were performed three times (n = 3).

The effects of SepF, G51D-SepF, and G51R-SepF on the assembly of FtsZ were further determined using the sedimentation assay at pH 6.8 and 7.5. SepF enhanced the assembly of FtsZ polymers in a concentration-dependent manner at pH 6.8 (Figure 1D). On the other hand, G51D-SepF and G51R-SepF decreased the amount of FtsZ polymerized compared with the control at pH 6.8 (Figure 1E,F). For example, 9 µM SepF increased the amount of polymerized FtsZ by 2.5-fold, while 9 µM G51R-SepF or G51D-SepF reduced the amount of polymerized FtsZ by ∼44 and 37%, respectively (Supplementary Figure S3). The effects of SepF and mutant SepF on the assembly of FtsZ at pH 7.5 were found to be similar to those at pH 6.8 (Supplementary Figure S4). For example, 9 µM SepF increased the amount of polymerized FtsZ by 1.7-fold, while 9 µM G51R-SepF or G51D-SepF reduced the amount of polymerized FtsZ by ∼49 and 28%, respectively. The results indicated that the mutation at the G51 position impaired the ability of SepF to enhance FtsZ assembly.

SepF induced the bundling of FtsZ polymers, but G51D and G51R-SepF reduced the formation of FtsZ filaments

Under the condition used, FtsZ formed thin polymers in the absence of SepF (Supplementary Figures S5A and S6A). SepF increased the thickness of the FtsZ filaments at both pH 6.8 and pH 7.5 (Supplementary Figures S5 and S6). The thickness of the polymers formed in the absence and in the presence of 3 µM SepF was determined to be, respectively, 13 ± 7 and 29 ± 5 nm (n = 150 polymers) at pH 6.8, suggesting that SepF induced the bundling of the FtsZ polymers (Supplementary Figures S5A,B). In the presence of 3 µM G51D-SepF and G51R-SepF, few small polymers of FtsZ could be seen per field of microscopic view at pH 6.8 (Supplementary Figure S5C,D). Furthermore, the thickness of the FtsZ polymers was increased from 10 ± 5 to 33 ± 2 nm in the presence of SepF at pH 7.5 (Supplementary Figure S6A,B). However, very few small polymers of FtsZ could be seen in the presence of 3 µM G51D and G51R-SepF at pH 7.5 (Supplementary Figure S6C,D).

SepF stabilized FtsZ polymers against dilution and reduced the GTPase activity of FtsZ

When diluted below the critical concentration, FtsZ polymers are reported to disassemble [18]. The preformed FtsZ polymers were diluted 20 times without and with different concentrations of SepF, G51R-SepF, and G51D-SepF. The polymers were recovered by centrifugation and the amount of FtsZ pelleted was estimated by Coomassie blue staining of the SDS–PAGE (Figure 2A–C). The amount of polymerized FtsZ was found to increase with increasing concentration of SepF (Figure 2A). For example, the intensity of FtsZ bands was estimated to be 1.7 ± 0.6, 4.8 ± 0.7, and 5.9 ± 0.3 (arbitrary units) in the absence and presence of 3 and 4 µM SepF, respectively. However, the amount of pelleted FtsZ did not increase detectably when the preformed FtsZ polymers were diluted in the presence of either G51R-SepF or G51D-SepF when compared with the control (Figure 2B,C). The results indicated that SepF suppressed the disassembly of FtsZ polymers upon dilution, whereas G51D-SepF and G51R-SepF could not prevent the disassembly of the FtsZ polymers.

Effects of SepF, G51R-SepF, and G51D-SepF on the dilution-induced disassembly of preformed FtsZ polymers and the GTPase activity of FtsZ.

Figure 2.
Effects of SepF, G51R-SepF, and G51D-SepF on the dilution-induced disassembly of preformed FtsZ polymers and the GTPase activity of FtsZ.

FtsZ (15 µM) was polymerized for 10 min. The preformed FtsZ polymers were diluted 20 times in warm 25 mM HEPES buffer (pH 6.8) in the absence and presence of different concentrations of native and mutant SepFs and incubated for an additional 5 min at 37°C. Lanes 1–5 show the amount of FtsZ polymers recovered in the absence and in the presence of 1, 2, 3, and 4 µM (A) SepF, (B) G51R-SepF, and (C) G51D-SepF, respectively. (D) The moles of Pi released per mole of FtsZ without and with different concentrations of SepF (▪), G51D-SepF (●), and G51R-SepF (□) were calculated. Each experiment was performed three times (n = 3).

Figure 2.
Effects of SepF, G51R-SepF, and G51D-SepF on the dilution-induced disassembly of preformed FtsZ polymers and the GTPase activity of FtsZ.

FtsZ (15 µM) was polymerized for 10 min. The preformed FtsZ polymers were diluted 20 times in warm 25 mM HEPES buffer (pH 6.8) in the absence and presence of different concentrations of native and mutant SepFs and incubated for an additional 5 min at 37°C. Lanes 1–5 show the amount of FtsZ polymers recovered in the absence and in the presence of 1, 2, 3, and 4 µM (A) SepF, (B) G51R-SepF, and (C) G51D-SepF, respectively. (D) The moles of Pi released per mole of FtsZ without and with different concentrations of SepF (▪), G51D-SepF (●), and G51R-SepF (□) were calculated. Each experiment was performed three times (n = 3).

FtsZ was polymerized without and with different concentrations of SepF, G51D-SepF, and G51R-SepF for 10 min, and the effects of SepF, G51D-SepF, and G51R-SepF on the GTPase activity of FtsZ were determined [32]. After 10 min of assembly, 15 ± 2 moles of inorganic phosphate per mole of FtsZ were released in the absence of SepF (Figure 2D). The moles of inorganic phosphate released per mole of FtsZ decreased with increasing concentrations of SepF (Figure 2D). In contrast, G51D-SepF and G51R-SepF did not produce any significant effect on the GTPase activity of FtsZ. SepF alone did not hydrolyze GTP.

SepF interacts with FtsZ with strong affinity in vitro

The interaction between FtsZ and SepF was first characterized using gel filtration chromatography (Supplementary Figure S7). Initially, BSA (66 kDa) and lysozyme (16 kDa) were found to be eluted at elution volumes 9 and 22 ml, respectively (Supplementary Figure S7A). Subsequently, FtsZ and three different SepF constructs were eluted individually. FtsZ was eluted at a 15 ml elution volume when loaded individually on the column (Supplementary Figure S7B). SepF was found to elute in two fractions (elution volumes 10–15 and 20 ml) (Supplementary Figure S7C). The 20 ml elution volume indicated that a fraction of SepF was eluted as monomers, whereas the elution volumes 10–15 ml indicated that SepF formed dimers and higher-order oligomers (Supplementary Figure S7C). G51D-SepF and G51R-SepF displayed elution patterns similar to that of SepF (Supplementary Figure S7C). Finally, the mixture of FtsZ and SepF was loaded on the column and the proteins were found to be eluted with peak fractions at 10 and 20 ml (Supplementary Figure S7B). The peak fractions of the elutions were loaded in 12% SDS–PAGE (Supplementary Figure S7D,E). The eluted fraction at 10 ml was found to contain both FtsZ and SepF, whereas the elution fractions at 15 and 20 ml contained only FtsZ and only SepF, respectively (Supplementary Figure S7B,D,E). The results indicated that SepF binds to FtsZ in vitro.

Furthermore, the binding of FtsZ and SepF was determined by monitoring the fluorescence of FITC-labeled SepF (FITC-SepF). A fixed concentration of FITC-SepF (50 nM) was incubated without or with either 500 nM bovine serum albumin (BSA), tubulin, alcohol dehydrogenase, or FtsZ. The fluorescence intensity of FITC-SepF did not alter significantly in the presence of 500 nM BSA, alcohol dehydrogenase, and tubulin (Figure 3A). However, the fluorescence intensity of FITC-SepF increased significantly in the presence of FtsZ, indicating that SepF specifically interacts with FtsZ. Furthermore, FITC-SepF (50 nM) was incubated with different concentrations (50–5000 nM) of FtsZ. The fluorescence of FITC-SepF increased with increasing concentrations of FtsZ (Figure 3B). Using the change in the fluorescence of FITC-SepF in the presence of FtsZ, the Kd for the interaction of SepF with FtsZ was determined to be 443 ± 53 nM (Figure 3B, inset).

SepF bound to FtsZ with high affinity.

Figure 3.
SepF bound to FtsZ with high affinity.

(A) FITC-SepF (50 nM) was incubated without (▪) and with 500 nM of bovine serum albumin (Δ), alcohol dehydrogenase (▴), tubulin (●), and FtsZ (□). The experiment was performed four times (n = 4). (B) FITC-SepF (50 nM) was incubated without (▪) or with 50 (□), 100 (▴), 200 (Δ), 400 (●), 600 (○), 800 (∇), 1000 (▾), 2000 (⋆), 4000 (◊), and 5000 (♦) nM FtsZ. The increase in the fluorescence intensity of FITC-SepF upon binding to FtsZ is shown. The inset shows the increase in the fluorescence intensities of FITC-SepF with increasing concentrations of FtsZ. The experiment was performed four times (n = 4).

Figure 3.
SepF bound to FtsZ with high affinity.

(A) FITC-SepF (50 nM) was incubated without (▪) and with 500 nM of bovine serum albumin (Δ), alcohol dehydrogenase (▴), tubulin (●), and FtsZ (□). The experiment was performed four times (n = 4). (B) FITC-SepF (50 nM) was incubated without (▪) or with 50 (□), 100 (▴), 200 (Δ), 400 (●), 600 (○), 800 (∇), 1000 (▾), 2000 (⋆), 4000 (◊), and 5000 (♦) nM FtsZ. The increase in the fluorescence intensity of FITC-SepF upon binding to FtsZ is shown. The inset shows the increase in the fluorescence intensities of FITC-SepF with increasing concentrations of FtsZ. The experiment was performed four times (n = 4).

SepF bound to FtsZ with stronger affinity than the mutant SepF constructs

The binding affinities of SepFs to FtsZ were also compared using a tryptophan mutant of FtsZ (L66W-FtsZ). The assembly of L66W-FtsZ was found to be comparable with that of FtsZ as determined by the sedimentation assay. Under similar assembly conditions, 26 ± 9, 42 ± 5, 52 ± 8, and 66 ± 10% of the total L66W-FtsZ were pelleted when 6, 8, 10, and 12 µM L66W-FtsZ were polymerized, while 35 ± 9, 47 ± 5, 66 ± 6, and 68 ± 10% of the WT-FtsZ were pelleted, respectively. The results indicated that the mutation (L66W) had a minimal inhibitory effect on the assembly of FtsZ; therefore, the tryptophan fluorescence of L66W-FtsZ was used to monitor its interaction with SepF.

The fluorescence intensity of L66W-FtsZ was increased when incubated with increasing concentrations of SepF, G51D-SepF, and G51R-SepF (Figure 4A,C,E). A dissociation constant (Kd) of the interaction between L66W-FtsZ and SepF was estimated to be 1.1 ± 0.4 µM (Figure 4B). The dissociation constants of the interactions of L66W-FtsZ with G51D-SepF and G51R-SepF were determined to be 3.8 ± 0.1 and 3.1 ± 0.9 µM, respectively, indicating that SepF binds to FtsZ with a stronger affinity than G51D-SepF and G51R-SepF (Figure 4D,F).

SepF binds to FtsZ with a higher affinity than G51D-SepF and G51R-SepF.

Figure 4.
SepF binds to FtsZ with a higher affinity than G51D-SepF and G51R-SepF.

The binding of L66W-FtsZ with (A and B) SepF, (C and D) G51D-SepF, and (E and F) G51R-SepF was monitored by tryptophan fluorescence of L66W-FtsZ. L66W-FtsZ (1 µM) was incubated without (▪) and with different concentrations of 100 (□), 200 (●), 400 (○), 600 (▴), 800 (Δ), 1000 (♦), 2000 (◊), 4000 (◄), 6000 (◅) and 8000 nM (⋆) (A) SepF, (C) G51D-SepF, and (E) G51R-SepF. The increase in the fluorescence intensity of L66W-FtsZ upon binding to SepF, G51D-SepF, and G51R-SepF is shown. The increase in the fluorescence intensities of L66W-FtsZ upon interaction with native and mutant SepF was plotted against different concentrations of (B) SepF, (D) G51D-SepF, and (F) G51R-SepF used. The experiments were performed three times (n = 3).

Figure 4.
SepF binds to FtsZ with a higher affinity than G51D-SepF and G51R-SepF.

The binding of L66W-FtsZ with (A and B) SepF, (C and D) G51D-SepF, and (E and F) G51R-SepF was monitored by tryptophan fluorescence of L66W-FtsZ. L66W-FtsZ (1 µM) was incubated without (▪) and with different concentrations of 100 (□), 200 (●), 400 (○), 600 (▴), 800 (Δ), 1000 (♦), 2000 (◊), 4000 (◄), 6000 (◅) and 8000 nM (⋆) (A) SepF, (C) G51D-SepF, and (E) G51R-SepF. The increase in the fluorescence intensity of L66W-FtsZ upon binding to SepF, G51D-SepF, and G51R-SepF is shown. The increase in the fluorescence intensities of L66W-FtsZ upon interaction with native and mutant SepF was plotted against different concentrations of (B) SepF, (D) G51D-SepF, and (F) G51R-SepF used. The experiments were performed three times (n = 3).

The interaction between FtsZ and SepF was ionic in nature

The interaction between L66W-FtsZ and SepF was monitored in the presence of different concentrations of KCl. The fluorescence intensity of only L66W-FtsZ did not change in the presence of KCl. The fluorescence intensity of L66W-FtsZ and SepF reduced with increasing concentrations of KCl (Supplementary Figure S8A). For example, the fluorescence intensity of L66W-FtsZ in the presence of 6 µM SepF decreased by 42 ± 9 and 64 ± 8% when incubated with 200 and 500 mM KCl, respectively (Supplementary Figure S8B). The results indicated that the interaction between FtsZ and SepF was perturbed by KCl and that the interaction of FtsZ and SepF is electrostatic in nature.

G51R/D-SepF poisoned the activity of SepF to promote the assembly of FtsZ

The addition of G51R-SepF to SepF impaired the ability of SepF to promote the assembly of FtsZ as indicated by light scattering (Figure 5A), sedimentation (Figure 5B), and transmission electron microscopy (Figure 5C). The polymerization of FtsZ in the presence of SepF was found to decrease with increasing concentrations of G51R-SepF (Figure 5A). For example, the light scattering of the assembly of FtsZ in the presence of SepF was reduced by 30 ± 5, 65 ± 10, and 77 ± 8% when incubated with 0.25, 0.5, and 1 µM G51R-SepF, respectively. Similar to the result obtained from the light-scattering assay, the amount of sedimentable FtsZ polymers was found to reduce with increasing concentrations of G51R-SepF. For example, the addition of 2 µM G51R-SepF reduced the amount of polymerized FtsZ by 27 ± 5% in the presence of 4 µM SepF (Figure 5B). Electron microscopy analysis also revealed that 2 µM G51R-SepF reduced the polymerization of FtsZ in the presence of 2 µM SepF (Figure 5Ciii). Similarly, the extent of FtsZ assembly in the presence of SepF was found to reduce by 78 ± 4% when 3 µM G51D-SepF was added to the reaction mixture, indicating that G51D-SepF could also impair the SepF-induced polymerization of FtsZ (Figure 5D).

G51R-SepF impaired the polymerization-enhancing effect of SepF on FtsZ assembly.

Figure 5.
G51R-SepF impaired the polymerization-enhancing effect of SepF on FtsZ assembly.

(A) FtsZ (6 µM) was polymerized in the absence (□) or presence of SepF (3 µM) (◊) in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl, 5 mM MgCl2, and 1 mM GTP. Separately, FtsZ (6 µM) was incubated with SepF (3 µM) in the polymerizing buffer for 5 min and then G51R-SepF was added. The samples were further incubated for 5 min and the scatterings of FtsZ with increasing concentrations of G51R-SepF [0.25 (▴), 0.5 (Δ), and 1 µM (●)] were recorded. The assembly kinetics of FtsZ was monitored for 600 s. (B) A sedimentation assay was performed to quantify the amount of FtsZ polymers under similar conditions as described in A. The retrieved polymers were run in 12% SDS–PAGE. Lanes 1–2 show the pelleted FtsZ when polymerized alone and with SepF (4 µM). Lanes 3–4 show the FtsZ pellet when polymerized with SepF (4 µM) followed by the addition of G51R-SepF (1 and 2 µM), respectively. Lanes 5–8 indicate the supernatant fractions of the same reaction milieu. (C) The TEM images of polymers of FtsZ (3 µM) when polymerized (i) alone or with (ii) SepF (2 µM). (iii) Furthermore, FtsZ was incubated first with SepF (2 µM) for 5 min and then with G51R-SepF (2 µM) for an additional 5 min. The polymers obtained were observed under TEM. The scale bar for each panel is 200 nm. (D) The polymerization of FtsZ (6 µM) without (□) and with SepF (◊) and G51D-SepF (▴) (3 µM each) was monitored for 600 s. Furthermore, G51D-SepF (3 µM) was found to reduce the SepF-induced assembly of FtsZ (Δ). Each experiment was repeated three times (n = 3).

Figure 5.
G51R-SepF impaired the polymerization-enhancing effect of SepF on FtsZ assembly.

(A) FtsZ (6 µM) was polymerized in the absence (□) or presence of SepF (3 µM) (◊) in 25 mM HEPES buffer (pH 6.8) containing 100 mM KCl, 5 mM MgCl2, and 1 mM GTP. Separately, FtsZ (6 µM) was incubated with SepF (3 µM) in the polymerizing buffer for 5 min and then G51R-SepF was added. The samples were further incubated for 5 min and the scatterings of FtsZ with increasing concentrations of G51R-SepF [0.25 (▴), 0.5 (Δ), and 1 µM (●)] were recorded. The assembly kinetics of FtsZ was monitored for 600 s. (B) A sedimentation assay was performed to quantify the amount of FtsZ polymers under similar conditions as described in A. The retrieved polymers were run in 12% SDS–PAGE. Lanes 1–2 show the pelleted FtsZ when polymerized alone and with SepF (4 µM). Lanes 3–4 show the FtsZ pellet when polymerized with SepF (4 µM) followed by the addition of G51R-SepF (1 and 2 µM), respectively. Lanes 5–8 indicate the supernatant fractions of the same reaction milieu. (C) The TEM images of polymers of FtsZ (3 µM) when polymerized (i) alone or with (ii) SepF (2 µM). (iii) Furthermore, FtsZ was incubated first with SepF (2 µM) for 5 min and then with G51R-SepF (2 µM) for an additional 5 min. The polymers obtained were observed under TEM. The scale bar for each panel is 200 nm. (D) The polymerization of FtsZ (6 µM) without (□) and with SepF (◊) and G51D-SepF (▴) (3 µM each) was monitored for 600 s. Furthermore, G51D-SepF (3 µM) was found to reduce the SepF-induced assembly of FtsZ (Δ). Each experiment was repeated three times (n = 3).

The interaction of SepF with G51R-SepF was stronger than SepF–SepF interaction

The binding affinity of SepF to itself or to G51R-SepF was determined by incubating a fixed concentration of FITC-SepF with increasing concentrations of either unlabeled SepF (Figure 6A) or unlabeled G51R-SepF (Figure 6B). The fluorescence intensity of FITC-SepF was found to decrease with increasing concentrations of either SepF or G51R-SepF (Figure 6A,B). The Kd of the binding of SepF to itself and G51R-SepF was determined to be 26 ± 1.7 and 10 ± 1.8 nM, respectively (Figure 6A,B, inset). The results indicated that SepF binds to G51R-SepF more strongly than either to itself or FtsZ.

The binding interaction of FITC-SepF with SepF or with G51R-SepF.

Figure 6.
The binding interaction of FITC-SepF with SepF or with G51R-SepF.

FITC-SepF (50 nM) (▪) was incubated with different concentrations of 2 (□), 3 (▴), 5 (Δ), 10 (●), 20 (○), 30 (♦), 50 (◊), 80 (∇), 100 (▾), 200 (⋆), and 500 (⋆) nM either (A) wild-type SepF or (B) G51R-SepF. The decrease in the fluorescence intensity of FITC-SepF upon binding to either wild-type SepF or G51R-SepF is shown. One of the three independent experiments is shown (n = 3). The changes in the fluorescence intensities of FITC-SepF were plotted against different concentrations of wild-type SepF and G51R-SepF and are shown in the inset. The experiment was performed three times (n = 3).

Figure 6.
The binding interaction of FITC-SepF with SepF or with G51R-SepF.

FITC-SepF (50 nM) (▪) was incubated with different concentrations of 2 (□), 3 (▴), 5 (Δ), 10 (●), 20 (○), 30 (♦), 50 (◊), 80 (∇), 100 (▾), 200 (⋆), and 500 (⋆) nM either (A) wild-type SepF or (B) G51R-SepF. The decrease in the fluorescence intensity of FITC-SepF upon binding to either wild-type SepF or G51R-SepF is shown. One of the three independent experiments is shown (n = 3). The changes in the fluorescence intensities of FITC-SepF were plotted against different concentrations of wild-type SepF and G51R-SepF and are shown in the inset. The experiment was performed three times (n = 3).

Overexpression of G51R-SepF in M. smegmatis mc2 155 cells reduced cell viability and increased the length of the cells

The kinetics of growth of recombinant M. smegmatis mc2 155 cells containing either sepf or G51R-sepf was monitored for 35 h in the M7H9 medium. M. smegmatis mc2 155 strain was used as the control for the experiment. Cells overexpressing SepF showed a similar growth pattern as that of the wild-type strain (Figure 7A). Interestingly, the cells overexpressing G51R-SepF showed delayed growth compared with the wild-type strain. Furthermore, the DIC images of the cells were recorded after 23 h of growth (Figure 7B). The cell lengths of M. smegmatis mc2 155 cells and cells overexpressing SepF were determined to be 5.6 ± 1 and 5.5 ± 1 µm, respectively (Figure 7Ba,b,d). Overexpression of G51R-SepF increased the mean length of M. smegmatis mc2 155 cells to 9 ± 2 µm (Figure 7Bc,d). Using FM 4–64 dye [34,35], 85 and 88% of wild-type and SepF-overexpressing M. smegmatis mc2 155 cells exhibited single septal membrane staining, respectively. In contrast, only 20% of G51R-SepF-expressing cells displayed single septal membrane staining. Nearly 80% of the G51R-sepF-expressing cells exhibited more intense FM4–64 staining at multiple sites throughout the cell length, suggesting the presence of multiple septa (Supplementary Figure S9). The result indicated that the overexpression of G51R-SepF reduced the growth and increased the cell length of M. smegmatis mc2 155 cells even in the presence of the genomic SepF. The finding suggests that G51R mutation in SepF is a dominant-negative mutation.

The overexpression of G51R-SepF in M. smegmatis mc2 155 cells retarded the cell growth and increased the mean length of the cells.

Figure 7.
The overexpression of G51R-SepF in M. smegmatis mc2 155 cells retarded the cell growth and increased the mean length of the cells.

(A) Growth curves of M. smegmatis mc2 155 strains grown in M7H9 media in the absence of any plasmid (▪), with SepF overexpressed (□), with G51R-SepF overexpressed (●) are shown. Growth was monitored for 35 h by measuring OD600. (B) (a) Control M. smegmatis mc2 155 cells, (b) M. smegmatis mc2 155 cells with SepF overexpressed, and (c) M. smegmatis mc2 155 cells with G51R-SepF overexpressed were collected after 23 h of growth and the DIC images were captured to determine the cell length. The scale bar is 10 µm. (d) The overexpression of G51R-SepF increased the length of bacterial cells (P < 0.005). Three hundred cells were monitored to determine the cell length (n = 300 cells).

Figure 7.
The overexpression of G51R-SepF in M. smegmatis mc2 155 cells retarded the cell growth and increased the mean length of the cells.

(A) Growth curves of M. smegmatis mc2 155 strains grown in M7H9 media in the absence of any plasmid (▪), with SepF overexpressed (□), with G51R-SepF overexpressed (●) are shown. Growth was monitored for 35 h by measuring OD600. (B) (a) Control M. smegmatis mc2 155 cells, (b) M. smegmatis mc2 155 cells with SepF overexpressed, and (c) M. smegmatis mc2 155 cells with G51R-SepF overexpressed were collected after 23 h of growth and the DIC images were captured to determine the cell length. The scale bar is 10 µm. (d) The overexpression of G51R-SepF increased the length of bacterial cells (P < 0.005). Three hundred cells were monitored to determine the cell length (n = 300 cells).

SepF forms oligomers

The assembly of SepF constructs was monitored by sedimentation (Supplementary Figure S10). The amount of polymerized SepF was found to increase in a concentration-dependent manner and three SepF constructs were found to yield a similar amount of polymers. The oligomers formed by SepF, G51D-SepF, and G51R-SepF were further observed under AFM (Supplementary Figure S11). In addition, the size distribution of the SepF oligomers was determined using DLS (Supplementary Figure S12). Large oligomers of SepF were detected by DLS. For example, the average size of the 82, 9, and 8% population of the SepF oligomers was estimated to be 413, 97, and 1 nm, respectively (Supplementary Figure S12A). The size distribution of G51D-SepF in solution was found to be similar compared with the SepF (Supplementary Figure S12B). Interestingly, G51R-SepF predominantly formed higher-order oligomers. For example, a population of 74 and 24% of the G51R-SepF in the solution formed oligomers of size 675 and 146 nm, respectively (Supplementary Figure S12C). The result indicated that the substitution of the G51 residue in SepF by arginine increased the propensity of forming large oligomers.

Discussion

In the present study, M. smegmatis SepF was found to promote the polymerization and bundling of M. smegmatis FtsZ and also stabilized the FtsZ filaments. The polymerization-enhancing ability of M. smegmatis SepF has been found to be comparable with several other assembly-promoting proteins such as ZipA in E. coli and SepF in B. subtilis [3,11,18]. Interestingly, a point mutation (G51D or G51R) at the G51 residue in SepF impaired the FtsZ assembly-promoting activity of SepF. Furthermore, the G51R/D-SepF also impaired the assembly-promoting activity of the native SepF. The G51 residue has been found to be conserved in most of the nonpathogenic Mycobacterium sp. such as M. smegmatis, M. fortuitum, M. mageritense, and M. phlei, indicating that the residue may be important for the functioning of SepF in nonpathogenic Mycobacterium strains. The dissociation constant of the interaction between SepF and FtsZ was determined to be 443 nM, which was found to be comparable with the binding affinity of ZipA, ZapC, and SulA for FtsZ [11,17,36]. G51D/R-SepF bound to FtsZ with a lower affinity than the native SepF. It was reported that a mutation of the G109 residue in BsSepF hindered its dimerization as well as its interaction with FtsZ by perturbing the close-packed conformation of SepF [22,24]. Similarly, the G51D/R mutation of SepF may have altered the compact conformation of the protein and diminished its ability to polymerize FtsZ. Furthermore, the interaction between FtsZ and SepF was found to be electrostatic in nature as it was perturbed by the addition of KCl. Though the effects of M. smegmatis SepF on the assembly of FtsZ were found to be similar with B. subtilis SepF; the B. subtilis SepF was reported to form ring polymers of 50–60 nm diameter [24], while M. smegmatis SepF was found to form non-structured oligomers.

G51R mutation is a dominant-negative mutation

In vitro, G51R-SepF inhibited the FtsZ assembly-promoting activity of native SepF. To correlate with the in vitro observation, G51R-SepF was overexpressed in M. smegmatis mc2 155 cells without deleting its genomic SepF. The overexpression of wild-type SepF had no visible effect on the septal membrane in the M. smegmatis mc2 155 strain. However, the overexpression of G51R-SepF in the M. smegmatis mc2 155 strain perturbed septal formation, slowed down the division of the cells, and increased their mean cell length. Using high-resolution imaging, it has been recently reported that the overexpression of native SepF in B. subtilis cells can also produce similar structures, which are not septa but membrane invaginations [28]. A plausible explanation could be that G51R-SepF impaired the activity of the native SepF in M. smegmatis mc2 155 cells. The results together suggested that G51R-SepF acquired a dominant-negative characteristic and the mutation of the G51 residue in SepF perturbed the cell division by affecting the FtsZ assembly.

How did SepF promote the bundling of FtsZ?

SepF increased light-scattering signal of FtsZ assembly and also increased the amount of polymerized FtsZ, suggesting that SepF promoted the assembly of FtsZ. The instantaneous increase in the scattering of FtsZ in the presence of SepF suggested that SepF facilitated the nucleation of the FtsZ monomers. SepF could form small oligomers with FtsZ. These small oligomers of FtsZ-SepF could add to the end of FtsZ polymers and increased the rate of FtsZ assembly. Electron microscopy images showed that SepF strongly increased the bundling of FtsZ polymers. The incorporation of SepF into the FtsZ filaments could increase the lateral interaction between the FtsZ protofilaments and promoted the bundling of filaments (Figure 8A).

Model depicting a possible mechanism of action of G51R-SepF on FtsZ.

Figure 8.
Model depicting a possible mechanism of action of G51R-SepF on FtsZ.

(A) FtsZ monomers (green circles) start to assemble in the presence of SepF (yellow oval circles). SepF aids the lateral interactions between the FtsZ protofilaments and induces bundling. (B) FtsZ (green circles) forms small oligomers upon interaction with G51R/D-SepF (red oval circles). G51R/D-SepF sequesters FtsZ monomers and reduces FtsZ assembly. (C) G51R/D-SepF (red oval circles) interacts with SepF (yellow oval circles) to form inactive oligomers (yellow and red stars) and impairs the activity of SepF. Hence, FtsZ forms short and thin filaments instead of forming bundles when polymerized in the presence of a mixture of SepF and mutant SepF.

Figure 8.
Model depicting a possible mechanism of action of G51R-SepF on FtsZ.

(A) FtsZ monomers (green circles) start to assemble in the presence of SepF (yellow oval circles). SepF aids the lateral interactions between the FtsZ protofilaments and induces bundling. (B) FtsZ (green circles) forms small oligomers upon interaction with G51R/D-SepF (red oval circles). G51R/D-SepF sequesters FtsZ monomers and reduces FtsZ assembly. (C) G51R/D-SepF (red oval circles) interacts with SepF (yellow oval circles) to form inactive oligomers (yellow and red stars) and impairs the activity of SepF. Hence, FtsZ forms short and thin filaments instead of forming bundles when polymerized in the presence of a mixture of SepF and mutant SepF.

How did G51D/R-SepF reduce the assembly of FtsZ polymers?

Both G51D/R-SepF reduced the polymerization of FtsZ and did not stabilize the preformed FtsZ polymers. The decrease in the light-scattering intensity of FtsZ in the presence of G51D/R-SepF could be due to the sequestration of the FtsZ monomers by G51D/R-SepF as done by other negative regulatory proteins of FtsZ assembly such as SulA [36] and EzrA [37]. FtsZ could produce small oligomers when incubated with G51D/R-SepF, which were unable to form large size polymers, thereby reducing the light-scattering signal and the amount of FtsZ polymers (Figure 8B). Alternatively, G51D/R-SepF might have bound to the FtsZ subunits and the presence of a charged residue at the 51st position of SepF hindered the interaction between FtsZ protofilaments.

G51R-SepF bound to SepF with a high affinity and impaired the ability of SepF to enhance the assembly of FtsZ

G51R-SepF inhibited SepF-induced polymerization of FtsZ. A possible mechanism of the impairing activity could be that G51R-SepF formed a complex with SepF and produced inactive SepF oligomers containing both wild-type SepF and G51R-SepF. The wild-type SepF was found to bind to G51R-SepF with 45 times higher affinity than FtsZ. Furthermore, the binding affinity of SepF to G51R-SepF was determined to be 2.6-fold higher than its affinity for itself. Thus, the interaction could remove the native SepF from the reaction mixture and, thereby, effectively reduced the amount of SepF available to enhance the assembly of FtsZ monomers. In addition, the inactive oligomers of SepF and mutant SepF might not be able to increase the interaction between the FtsZ polymers and, therefore, failed to increase the bundling of FtsZ filaments (Figure 8C). Since the binding affinities of G51R-SepF and G51D-SepF to FtsZ are only ∼2.8 and 3.4 times lower than the binding affinity of wild-type SepF to FtsZ, the mutant SepF could sequester a fraction of FtsZ monomers by forming an inactive complex with FtsZ and thereby inhibit the nucleation of FtsZ monomers even in the presence of wild-type SepF (Figure 8C).

Conclusion

In the present study, SepF was found to act as a positive regulator of the assembly of M. smegmatis FtsZ. Interestingly, a mutation of the G51 residue in M. smegmatis SepF rendered the protein inactive. Furthermore, G51D/R-SepF poisoned the ability of SepF to enhance the assembly and stability of FtsZ filaments. In addition, the mutation of the G51 residue in SepF suppressed the rate of division of M. smegmatis mc2 155 cells, indicating that the mutation is a dominant-negative mutation.

Abbreviations

     
  • B. subtilis

    Bacillus subtilis

  •  
  • DIC

    differential interference contrast

  •  
  • FITC

    fluorescein isothiocyanate

  •  
  • FtsZ

    filamentous temperature sensitive Z

  •  
  • GTP

    guanosine-5′-triphosphate

  •  
  • IPTG

    isopropyl-β-d-1-thiogalactopyranoside

  •  
  • SDS–PAGE

    sodium dodecyl sulfate–polyacrylamide gel electrophoresis

  •  
  • SepF

    septum-forming protein

Author Contribution

D.B. and K.S. designed and performed the experiments, and analyzed the data, while D.P. designed the experiments and analyzed the data. D.B. and D.P. wrote the manuscript.

Funding

This work was supported by the Council of Scientific and Industrial Research (CSIR), Government of India to D.P.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Supplementary data