Differentiated roles for MreB-actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis

Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin-like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins.


Supplementary Experimental procedures Supplementary Figures:
. Phenotypes associated with ftsX and cwlO mutant strains. Figure S2. LytE and CwlO mutants are synthetically lethal. Figure S3. CwlO localization in different genetic backgrounds. Figure S4. FtsX and CwlO interact within the same protein complex at the cell membrane. Figure S5. LytE is synthetically lethal with Mbl Supplementary Tables: Table S1. E-values and identity homologies for FtsEX and the CW hydrolases CwlO and LytE Table S2. Bacterial strains Table S3. Plasmids Table S4. Primers Supplementary References transform Bs168CA, with selection for spectinomycin resistance, to generate the strain PDC534, in which the gfp sf -fused to ftsX is expressed from the xylose inducible promoter P xyl at the amyE locus on the B. subtilis chromosome. Disruption of amyE was confirmed using a starch plate assay, and the correct integration of the inserts at the amyE locus was confirmed by PCR.

CwlO-Flag
To construct pMUTin-'cwlO-flag a fragment containing the last 550 bp of the cwlO orf was amplified by PCR with the primers cwlOCterFHindIIIFlag and cwlOrevKpnIFlag from the wildtype strain 168 genomic DNA, digested with HindIII and KpnI, and inserted into the corresponding HindIII-KpnI sites of pMUTin-flag plasmid. The resulting plasmid was used to transform PDC538, with selection for erythromycin resistance, to generate the strain PDC609, in which the flag-fused to cwlO is expressed from the native promoter at the native cwlO locus on the B. subtilis chromosome. The newly generated strain showed a wt phenotype, indicating that the flag fusion is fully functional. The correct integration of the plasmid at the native locus was confirmed by PCR.

Inducible expression strains amyE::P xyl -cwlO
cwlO was amplified by PCR from the wild-type strain 168 genomic DNA using primers CwlO-FXhoI and cwlORev-EcoRI, then cloned between the XhoI and EcoRI sites of plasmid pSG1728, creating pSG1728-cwlO. The resulting plasmid was used to transform Bs168CA, with selection for spectinomycin resistance, to generate the strain PDC567, in which cwlO is expressed from the xylose inducible promoter P xyl at the amyE locus on the B. subtilis chromosome.
aprE::P spac -lytE lytE was amplified by PCR from the wild-type strain 168 genomic DNA using primers LytEFXmaI and LytERevEcoRI, then cloned between the XmaI and EcoRI sites of plasmid pAPNC213-erm, creating pAPNerm-P spac -lytE. The resulting plasmid was used to transform Bs168CA, with selection for erythromycin resistance, to generate the strain PDC620, in which lytE is expressed from the IPTG inducible promoter P spac at the aprE locus on the B. subtilis chromosome. The correct integration of the inserts at the aprE locus was confirmed by PCR. aprE::P xyl -cwlO, aprE::P xyl -ftsEX and aprE::P xyl -lytE cwlO, ftsEX and lytE orfs were amplified by PCR from the corresponding plasmids pSG-P xyl-cwlO, pSG-P xyl -ftsEX-gfp sf and pSG-P xyl -P wt -LytEmcherry, respectively, using primers P xyl -FsphI and amyEtoAprERevBamHI (or LytE-revSacI, in the case of LytE), then cloned between the SphI and BamHI (or SacI, in the case of LytE) sites of plasmid pAPNC213-ery, creating the plasmids pAPNC-P xyl -cwlO, pAPNC-P xyl -ftsEX and pAPNC-P xyl -Pwt-LytE. The resulting plasmids were used to transform the corresponding deletion mutant strains, with selection for erythromycin resistance, to generate strains PDC639, PDC635 and PDC702 respectively, in which the different orfs are expressed from the xylose inducible promoter P xyl at the aprE locus on the B. subtilis chromosome. The correct integration of the inserts at the aprE locus was confirmed by PCR.

Microscopic imaging
For fluorescence microscopy, cells were grown to mid-exponential phase at 30°C or 37°C and mounted on microscope slides covered with a thin film of 1.2% agarose. See figure legends for specific growth conditions employed for each experiment. Fluorescence microscopy was carried out using Zeiss Axiovert 200M, Nikon Eclipse Ti-U, spinning disk confocal microscope. The images were acquired with Metamorph 6 (Molecular Devices, Inc) and FRAP-AI 7 (MAG Biosystems) software, and analyzed using ImageJ v.1.44o (National Institutes of Health). Images from a single focal plane were deconvolved using the 'No Neighbours' algorithm from the Metamorph software package. When required, cells were incubated in the presence of the membrane dye FM5-95 (90 µg ml -1 , Molecular Probes) prior to microscopic examination.

Sample preparation for microscopy
For sample preparation, overnight pre-cultures of B. subtilis were grown in CH medium supplemented with 20 mM MgSO 4 (CH-Mg) and appropriate antibiotic selection, from freshly isolated colonies on plates. Day cultures were performed by diluting pre-culture to an OD600 of 0.02 in CH-Mg and grown at 30°C. Expression of fluorescent CwlO-GFP fusion was induced by addition of xylose to 0.3%. Samples for microscopic observation were taken at mid-exponential phase and immobilized on 1.2% agarose-coated microscope slides.

Protoplast preparation for microscopy
Cells of strains PDC528 (wt, CwlO-GFP sf ) and PDC560 (ΔftsX::neo, CwlO-GFP sf ) were grown in CH media in the presence of 0.5% xylose. Cells were harvested and re-suspended in CH-MSM media in the presence of 0.5% xylose. Cells were protoplasted by incubation with 0.5 mg ml -1 lysozyme during 30 min at 30°C. After CW removal, the protoplasts suspensions were split in two. One half was treated with proteinase K (10 µg ml -1 ) for 30 min.

Cell measurements
Cells from strains included in table I, constitutively expressing soluble/cytosolic GFP protein (aprE::P rpsD -gfp) were grown in LB media at 37°C and samples were taken at different time points along the growth curve. Cells were imaged by epifluorescence microscopy using an Axiovert M200 microscope (Zeiss, Oberkochen, Germany) with a 300 W lambda light source (Sutter Instrument Company, California, USA) and a Zeiss x 100 plan-neofluar oil immersion objective lens (1.3 numerical aperture). Images were captured on a 1395 x 1040 pixel CoolSNAP HQ camera (Photometrics, Ottobrunn, Germany) controlled by Metamorph software version 6.1r3 (Universal Imaging Corporation, Marlow, UK). Image analysis was performed using the open source Cell Profiler software and consisted of the following two successive steps: (i) identification of cell contour by a segmentation pipeline of fluorescence images; (ii) automatic measurement of several cell characteristics: cell length and width, perimeter and area of cells. For each image, ~100-300 cells were identified and analyzed. For each strain and time point >1000 cells were analyzed.

Cell fractionation and immunoblotting
The generous gift from K. Devine's laboratory of a polyclonal antibody raised against the native CwlO protein allowed us to detect it in cell fractionation experiments. In order to be able to perform pull-down experiments we constructed an epitope-tagged version of CwlO fused to the Flag tag. The CwlO-Flag fusion was expressed from the native chromosomal cwlO locus (strain PDC609). Flag epitope was fused to the carboxyl-terminal part of CwlO. To increase the stability of the CwlO-Flag bait, all pull down experiments were performed in a wprA epr double mutant background. The growth rates and cell shapes of these strains were indistinguishable from that of the wild type indicating that the fusion protein is functional. When cells reached mid-exponential phase, cultures (50 ml) were collected by centrifugation (8,000 × g for 10 min at 25°C). Culture supernatants' protein content (S) was recovered by cold-acetone precipitation. Five volumes of cold acetone were added to 5 ml of culture supernatant and incubated at -20°C for 1 h. Then, samples were collected by centrifugation (10,000 x g for 20 min at 4°C). Pellets were washed with 70 % cold-ethanol and air dried, before re-suspending the protein pellet in 0.5 ml of Tris buffer (100 mM Tris-HCl pH 7.5, 1x complete protease inhibitor). Culture pellets were re-suspended in 4 mL 1× SMM buffer [0.5 M sucrose, 20 mM MgCl 2 , 20 mM maleic acid), pH 7]; 250 μL 10 mg ml -1 lysozyme (Sigma), and 50 μL complete protease inhibitor (EDTA-free, Roche) were added to cell suspensions and incubated at 37°C for 1 h with gentle shaking. Then cultures were split into two (2x 2 ml). First half constituted the total fraction (T). Protoplasts from the second half were collected by centrifugation. Supernatants (2 ml) were collected to constitute the CW fraction (CW). Cell membranes and cytoplasmic fractions were obtained from the protoplasts' pellets. Pellets were re-suspended in 2 ml of Tris-buffer (100 mM Tris-HCl, pH 7.5, 1x complete protease inhibitor) and sonicated until a clear solution was obtained. Membrane fraction pellets (M) were collected by centrifugation (50,000 x g for 40 min at 4°C) and supernatants were also kept as cytoplasmic fractions (C). Membrane pellets were re-suspended in 2 ml of the Tris buffer. 10 μg of total protein from each extract was separated on a 4-12% SDS-PAGE gradient gel (Novex, Life technologies). Proteins were transferred to a PVDF membrane (Amersham Hybond-P) and the membrane was blocked with 5% milk in PBST (PBS, 0.1% Tween-20) for 3 h. The membrane was incubated with appropriate antibodies (anti-Flag or anti-CwlO antibodies (1:10,000 or 1:3000 respectively in PBST)) o-n at 4°C temperature. The membrane was washed three times with PBST for 10 minutes. Following the wash, the membrane was incubated with rabbit antimouse or goat anti-rabbit antibodies conjugated with HRP (Sigma, A9044) (1:10,000 in 5% milk in PBST) for 1 hour at room temperature. Finally, the membrane was washed three times as above and developed using the Pierce ECL 2 Western Blotting substrate reagent. Chemiluminescence was detected using an ImageQuant LAS4000mini GE Healthcare system.

Formaldehyde Cross-Linking and Pull Down of CwlO Complexes
Cross-Linking and Pull Down experiments were performed with some modifications as described by Sham et al 2011. Briefly, cultures (400 mL) of strains PDC612 (Bs168CA ΔwprA::hyg Δepr::tet ΩcwlO-FLAG amyE::P xyl -ftsEX-gfp) and PDC613 (Bs168CA ΔwprA::hyg Δepr::tet amyE::P xyl -ftsEX-gfp parent negative control) were grown exponentially to OD600 ∼ 0.5. Cells were collected by centrifugation (8,000 × g for 10 min at 25°C). Cell pellets were washed with 18 mL 1× PBS at 25°C, and cells were collected again by centrifugation (8,000 × g for 5 min at 4°C). Residual supernatants were removed. Washed pellets were suspended in 19 mL 1× PBS, to which 1200 μL 37% of formaldehyde solution (Sigma) were added. Mixtures were incubated at 37 °C for 1 h. Cross-linking reactions were quenched by the addition of 4 mL 1.0 M glycine followed by incubation for 10 min at 25°C. Cells were collected by centrifugation (8,000 × g for 10 min at 4°C), washed with 20 mL 1× PBS at 25°C, and centrifuged again. Residual supernatants were removed using a fine pipette tip. Pellets of cross-linked cells were re-suspended in 5 mL 1× SMM buffer [0.5 M sucrose, 20 mM MgCl 2 , 20 mM maleic acid), pH 7]; 250 μL 10 mg ml -1 lysozyme (Sigma), and 50 μL complete protease inhibitor were added to cell suspensions and incubated at 37°C for 1 h with gentle shaking. Protoplast formation was monitored by phase-contrast microscopy. Protoplasts were collected by centrifugation (8,000 × g for 10 min at 4°C). Pellets were then suspended in 5 mL buffer H (20 mM Hepes, pH 8, 200 mM NaCl, 1 mM DTT, 1 x complete protease inhibitor) at 4°C. After mixing, 5 μL 0.1 M MgCl 2 , 5 μL 0.1 M CaCl2, 10 μL 5 mg ml -1 DNase (D4527; Sigma), and 10 μL 10 mg ml -1 RNase (R5500; Sigma) were added, and mixtures were incubated for 20 min on ice. Cells were then disrupted by sonication (five pulses of 40 µm amplitude for 10 s), and membrane fraction pellets were collected by centrifugation (16,000 × g for 30 min at 4°C). Membranes were dissolved in 2 mL room temperature CoIP lysis buffer [50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton X-100], chilled, and incubated for 30 min at 4°C; 80 μL anti-FLAG M2 affinity gel (A2220; Sigma) were added. The gel was washed before use five times with 0.5 mL 1× wash buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) at room temperature as described in the manufacturer's protocol. The mixture of dissolved membranes and washed gel was added to a 2-mL gravity-flow column (Pierce) and incubated at 4°C overnight with gentle rotation. Lysate was removed, and the resin was washed three times with 1 mL CoIP lysis buffer at 25°C. FLAG-tagged protein was eluted from the column by incubation with 200 μL FLAG elution buffer (1× wash buffer containing 150 ng 3× FLAG peptide/μL) (F4799; Sigma) for 30 min at 4°C. Residual FLAG-tagged protein was eluted from the column by washing two times with 200 μL 1× wash buffer at 25°C. Eluates were filtered and concentrated (to ∼40 μL) through 100-kDa cut-off Microcon columns (Millipore) by centrifugation (10,000 × g at RT). Concentrated samples were split evenly into two parts, and each one was mixed with 20 μL 2 × Laemmli sample buffer containing 5% (vol/vol) β-mercaptoethanol. One half was heated for 1 h at 95°C to remove crosslinks, while the other was kept intact. Samples were separated on 4-12% gradient SDS-PAGE gels in MES buffer and blotted into PVDF membranes, ready for immunoblotting with different antibodies (monoclonal anti-Flag and polyclonals anti-GFP, anti-Pbp2B, anti-MreB and anti-DivIVA). Finally, the membrane were developed using the Pierce ECL 2 Western Blotting substrate reagent. Chemiluminescence was detected using an ImageQuant LAS4000mini GE Healthcare system.      Table S1. E-values and identity homologies identified using the basic local alignment search tool (BLAST) (Altschul et al., 1990).