Strains and plasmids

E. coli strains Frag1, MJF429ΔyggB ΔkefA::kan and MJF465 ΔyggB ΔkefA::kan ΔmscL::cm were kindly provided by I. R. Booth (University of Aberdeen, Aberdeen, UK). Frag1 is a wt strain, a derivative of E. coli K-12. E. coli strains BW25113 F, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ^-, rph-1, Δ(rhaD-rhaB)568 and isogenic strain JW2891-2 carrying deletion of mscS gen (ΔmscS775::kan) were obtained in E. coli Genetic Stock Center (http://cgsc.biology.yale.edu/). MscS alleles were expressed in MJF429 throughout the work unless stated otherwise.

Construction of expression vectors

pTRC-mscS construct is mscS ORF coding for wild-type MscS in pTRC99a vector. It was constructed by making STOP mutation in position 287 in the previously described construct pYggB-His₆. The primers used were:

5’-GGTGAAAGAAGACAAAGCTGCGTAACACCATCACCATCACTAAGG-3’ and

5’-CCTTAGTGATGGTGATGGTGTTACGCAGCTTTGTCTTCTTTCACC-3’. Basing on this construct the following constructs were made by site-directed mutagenesis. pTRC-mscSΔ266–286 has STOP mutation in position 266 (primers used:

5’-GAATTTGATGCCGCCGGTTAAAGCTTCCCGTACCCGC-3’ and

5’-GCGGGTACGGGAAGCTTTAACCGGCGGCATCAAATTC-3’).

pTRC-mscS51N,68N has A51N mutation (primers used:

5’-CGCGCGGATGATTTCCAACAACGTGAATCGCCTGATGATCTC-3’ and

5’-GAGATCATCAGGCGATTCACGTTGTTGGAAATCATCCGCGCG-3’)

and F68N mutation (primers used:

5’-ATCGATGCCACTGTTGCTGATAATCTTTCTGCATTAGTCCG-3’ and

5’-CGGACTAATGCAGAAAGATTATCAGCAACAGTGGCATCGAT-3’).

pTRC-mscS51N,68NΔ266–286 has all the three mutations: F266STOP, A51N and F68N. All mutation reactions were performed using QuikChange Site-Directed Mutagenesis Kit (Stratagene).

pTRC-abdom construct was created by amplifying DNA fragment coding for the ABDOM domain of MscS (aa form I175 to S261, with MAF fragment added N-terminally) with pTRC-mscS as the template (primers used:

5’-TTACCATGGAATTCATTATTAACTTCTCCCGCGA-3’ and

5’-GCCTAGGTTAGCTGATACCGGCGGCA-3’)

and cloning the product in pTRC99a vector in EcoRI-BamHI. pGEX-abdom construct for GST-ABDOM expression was made by amplifying the fragment coding for ABDOM domain with pTRC-mscS as the template (primers used:

5’-TTACCATGGAATTCATTATTAACTTCTCCCGCGA-3’ and

5’-AGTCGCGGCCGCTTAGCTGATACCGGCGGCATCAAATTC-3’)

and cloning the product in pGEX4T-1 in EcoRI-NotI. pMAL-abdom construct for MBP-ABDOM expression was made by amplifying the fragment coding for ABDOM domain with pTRC-mscS as the template with primers

5’-.ATTGAATTCTCCCGCGAGCCAGTTCGCCGTAAC-3’ and

5’-CGGGGATCCTTAGCTGATACCGGCGGCATCAAATTCAC-3’

and cloning the product in pMAL-C2E vector in EcoRI-BamHI.

Both pET11-ftsZ and pJSB2-ftsZYF plasmids were kind gifts from Prof H. P. Erickson (Duke University Medical Center, Durham, NC).

pBAD-ftsZ construct was made by amplifying ftsZ gene with pET11-ftsZ as the template (primers used:

5’-TAGAGCTCATGTTTGAACCAATGGAACTTACC-3' and

5’-ATAAGCTTAATCAGCTTGCTTACGCAGG-3')

and cloning the PCR product in pBAD33 in SacI-HindIII.

GST pull-down assay

The GST-ABDOM fusion protein was expressed in MJF429 strain at a very low level (uninduced promoter). The GST-ABDOM fusion protein was purified on Glutathione Sepharose 4B (GE Healthcare) according to the manufacture instructions with modifications The cells where harvested by centrifugation, suspended in the binding buffer containing: PBS pH 7.5, 1mM MgCl₂, 10% glycerol, 3mM PMSF, 10l/ml Protease Inhibitor Cocktail (Sigma), lysozyme 1mg/ml, DNase 1mg/ml and sonicated. Triton X-100 was added to concentration 0.5% and the lysate was cleared by centrifugation at 21,000 x g at 4^°C. The supernatant was incubated with Glutathione Sepharose 4B beads (Amersham Biosciences) for 25 minutes at 23^°C. A column was formed and the beads were washed with 30 bead volumes of binding buffer with 0.5% Triton X-100 and inhibitors followed by 15 bead volumes of binding buffer with 0.5% Triton X-100. The fusion protein was eluted with glutathione elution buffer (100mM Tris-HCl pH 8.0, 125mM NaCl, 3 mg/ml glutathione). The fusion protein was concentrated with Microcon Centrifugal Filter Device with NMWL 3,000 Da (Millipore). Identical procedure was applied to cells expressing GST as a control experiment. Eluates were analyzed by mass spectrometry.

Protein expression and purification

E. coli FtsZ was purified as described previously [23], using GTP-precipitation method. BL21(DE3) cells carrying pET11-FtsZ vector were disrupted using a French press (Thermo Scientific) at 120,000 psi. Lysate was centrifuged at 100,000 x g for 1h at 4°C. GTP induced precipitation was performed on supernatant and repeated four times. The Q-sepharose column step was omitted. Final FtsZ pellet was suspended in 50 mM PIPES, pH 6.5, 1 mM EGTA, 5 mM MgCl₂ at concentration of 10 mg/ml.

ABDOM domain we expressed and purified it with N-terminally fused maltose binding protein (MBP) using amylose resin (Pharmacia) column chromatography. Maltose eluate was dialyzed against 20 mM Tris pH 7.4, 1 mM EGTA. MBP protein for control experiments was also purified as described above. The protein concentration was determined using a Biorad Protein Assay (BioRad). SDS-PAGE analysis of purified proteins is shown in S2 Fig.

Light scattering

FtsZ polymerization was monitored by 90° angle light scattering in a Fluorog 3 fluorometer (Jobin Yvon Spex) with both the excitation and emission wavelengths set at 350 nm. FtsZ (12 μM) alone or in the presence of MBP-ABDOM (12 μM) in buffer containing (in mM) 50 PIPES pH 6.0, 1 EDTA, 5 MgCl₂ was added to fluorometer cuvette with a 5-mm path length placed in a thermostated at 30°C chamber equipped with a magnetic stirrer. FtsZ polymerization was induced by addition of 1 mM GTP (Sigma) under constant stirring. Data points were collected every second and plotted with the Microcal Origin 4.1 software.

Co-sedimentation assay

FtsZ (0–40 μM) was incubated with 8 μM MBP-ABDOM in 50 mM PIPES, pH 6.5, 1 mM EDTA, 5 mM MgCl₂, 5mM CaCl₂ for 5 min. Then, 2 mM GTP was added to reaction mixture and incubated at 30°C for 1 min. Samples were centrifuged at 250,000 x g (Sorvall Discovery M120). Equal volumes of suspended pellets were separated on SDS-PAGE and Western blotted with monoclonal mouse anti-MBP antibody (Abcam).

Microscopy

Aliquots of 10μl of cell culture were dropped on 1% agarose pads made on glass slides, left to dry and covered with coverslips. The images where obtained using Olympus IX70 inverted microscope with Olympus UPlanSApo 60x/1.20 lens (water immersion) and CCD camera Sensicam 12BIT (PCO CCD Imaging). All images were analyzed with the Adobe Photoshop 7.0 software.

For cell filamentation assay depicted in Fig 1, MJF429 cells carrying different constructs (all being pTRC99a derivatives) were induced with 1 mM IPTG at OD₆₀₀ of 0,1 and grown at 37^°C and 200rpm shaking. The images were taken 3 h after the induction. At this point OD₆₀₀ of the three cultures of filamentous cells was about 0.9 compared to 1.2 in case of the rest of cultures.

For fluorescence microscopy cells of MJF429 strain carrying pJSB2-ftsZYFP plasmid [24] and either pTRC-mscSΔ266–286 or pTRC99A were diluted from overnight culture and grown in LB media with chloramphenicol and ampicillin to OD₆₀₀ of 0.1. Subsequently arabinose and IPTG were added to 0.0002% and 1mM, respectively. Cells were grown at 37^°C and 200 rpm shaking and images where taken 1 h after the induction. Cells were fixed with 2.5% paraformaldehyde in PBS for 15 min at room temperature and 45 min on ice.

Growth in ampicillin

MJF429 strain carrying pWG1 vector (kanR derivative of pTRC99A) alone, or expressing MscS, or MscS-K258A/R259A, or MscS-YFP was grown in LB170 (control) or in LB170 with 1.6 μg/ml or 4.1μg/ml ampicillin.

D-cysteine labeling and immunolabelling of peptidoglycan sacculi

MJF429 cell cultures carring pTRC-MscSΔ(268–286) or pTRC-ABDOM vectors grown overnight were diluted in LB broth containing 100 μg/ml ampicillin. After 10 min 200μg/ml D-cysteine (USBiological) was added and incubated for 30 min at 37°C. Then, 1 mM IPTG was added. After 1h incubation cells were collected by centrifugation (4 min, 5,000 x g) and resuspended in D-cysteine-free medium prewarmed to 37° and incubated for additional 30 min. Then, cells were centrifuged for 4 min at 5,000 x g and suspended in 0.9% NaCl. Sacculi were isolated and D-cysteine residues were biotinylated using (N-(6-(Biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide (Pierce) as described previously (de Pedro et al., 1997). Biotin was detected using monoclonal mouse anti-biotin antibody conjugated with Alexa Fluor 488 (Invitrogen) diluted 1:300 into 0.2% gelatin and 0.5% BSA in PBS, pH 7.3.

Mass spectrometry

There were two independent GST pull-down experiments performed. In the first one the concentrated eluates were directly subjected to standard trypsin digestion, while in the second, the eluted proteins were first separated on 12% SDS-PAGE gel. The gel was silver-stained, the bands were excised and in-gel trypsin digestion was performed. The peptide mixtures obtained in each experiments was applied to a RP-18 precolumn (LC Packings) using water containing 0.1% trifluoroacetic acid as a mobile phase and then transferred to a nano-HPLC RP-18 column (LC Packings, 75 μM i.d.) using an acetonitrile gradient (0%- 50% acetonitrile in 30 minutes) in the presence of 0.05% formic acid at a flow rate of 150 nl/min. The column outlet was directly coupled to an ion source of the LTQ FTICR spectrometer (Thermo) working in the regime of data dependent MS to MS/MS switch.

The mass spectra obtained in both experiments were used to search the non-redundant protein database of National Center of Biotechnology Information (NCBI) using the MASCOT search engine (Matrixscience.com). Search parameters were set as follows: taxonomy restriction—all entries, enzyme—trypsin; fixed modification—carbamidomethylation (C); variable modifications—carbamidomethylation (K), oxidation (M); protein mass—unrestricted; peptide mass tolerance +/- 40 ppm; fragment mass tolerance—+/-0.8 Da; max missed cleavages—1. Only protein hits characterized by the presence of at least one peptide with a score > 75 were accepted. All peptide hits with score > 75 were also validated by manual inspection of the MS/MS data. Hits were accepted if all strong peaks in the MS/MS spectrum were assigned either to y- or b- series ions and the spectrum contained y- or b- series signals corresponding to at least one stretch of three or more consecutive amino acids.

Flow cytometry

Cells were grown in LB supplemented with kanamycin and with or without ampicillin. For flow cytometry measurements (FASCalibur, BD Biosciences) cell cultures were diluted in the same media if needed. For cell death determination cells were incubated for 10 min before measurement in 10μg/ml propidium iodide (PI). PI fluorescence was measured with Ex533nm/>Em670nm.

Data were presented as means ± SD of three experiments.

Patch clamp

All patch-clamp experiments were done as previously described [19]. Pressure to the recording pipette was applied manually or by the high speed pressure clamp (HSPC-1, ALA Scientific Intruments). Purified proteins, FtsZ or MBP were added to 16 μM.

Cell filamentation is induced by expression of either the isolated ABDOM or of MscSΔ266–286 mutant

In our previous work we demonstrated that the MscS cytoplasmic chamber changes its shape upon channel kinetic transitions [17]. We wanted to know if the movement of its cytoplasmic domains is eventually involved in an interaction with other proteins. We focused on the ABDOM (residues 175–265) and the region that follows immediately after (Fig 1, panel A).

Overexpression of the 90-residues-long ABDOM in E. coli induced a striking cell filamentation (Fig 1, panel B). We asked whether a filamentation would also be induced by overexpression of the full-length MscS channel and we found that it was not the case (Fig 1, panel B). We wondered whether this lack of effect was due to the membrane-associated localization of the ABDOM in the intact channel, or to a limitation of its exposure to possible interaction partners. We addressed this question using a truncated mutant MscSΔ266–286, from which the last 20 amino acids of the C-terminus immediately following the ABDOM were missing. These amino acids form a separate ß-barrel structure and its deletion shouldn’t affect the structure of ABDOM. MscSΔ266–286 mutant opens at slightly higher threshold and inactivates normally, but has defective recovery from inactivation [15], indicating that it stabilizes a non-conducting, possibly inactivated-like state. Nevertheless, overexpressed MscSΔ266–286 protects cells against hypo-osmotic shock, indicating that the channel is physiologically functional but the configuration of its cytoplasmic domain is different from the full-length MscS [15]. We found that overexpression of the MscSΔ266–286 mutant channel resulted in cell filamentation that was similar or stronger to what we saw in cells overexpressing the ABDOM alone (Fig 1, panel B).

To be sure that the conduction of solutes through the channel was not responsible for the cell filamentation we introduced a loss-off-function mutation A51N/F68N [25] into the transmembrane domain of the MscSΔ266–286 mutant. In both the wild-type background and the MscSΔ266–286 background introduction of the A51N/F68N mutations eliminated the channel’s ability to protect cells from osmotic down-shocks, consistently with their disability to conduct solutes (not shown). Nevertheless, the impaired version of MscSΔ266–286 was fully proficient in inducing cell filamentation (Fig 1, panel B). This result demonstrates that current flow through the channel was not responsible for cell filamentation. Instead, the simplest explanation appears to be that the MscSΔ266–286 mutation stabilized a conformation of the channel that exposed the ABDOM and thus mimicked the effect of expression of ABDOM alone. However, the effect on cell length suggests that ABDOM may interact with regulators of bacterial cell shape and/or cell division.

ABDOM interacts with FtsZ

In an attempt to identify possible interaction partners of ABDOM, we carried out a pull-down assay from bacterial lysates followed by mass spectrometry, using the ABDOM fused to glutathione-S-transferase (GST) as the bait. To avoid non-specific protein interactions within the cell, the GST-ABDOM fusion was expressed at low level, at which no cell filamentation was observed. In two independent experiments we identified several proteins that bound to the GST-ABDOM fusion, but not to GST alone. These were FtsZ, ClpX, OmpC, RecA, HCP, and MreB (S1 Table). Several of these proteins are found together in complexes that probably regulate cell division and cell shape [26]. Among the ABDOM-interacting proteins we selected FtsZ for further investigation for the following reasons: i. it was identified with the highest confidence score; ii. we expected that excessive ABDOM binding may interfere with a primary function of the partner and that, therefore, its expression effect on cell filamentation could be accounted for by a block of FtsZ function [21]; iii) ClpX interacts with FtsZ [27,28] suggesting that its binding to ABDOM may be indirect (i.e. mediated through FtsZ), and, moreover, we observed the ABDOM-induced filamentation in clpX^—strain (data not shown); iv. we observed the ABDOM-induced filamentation in recA^—strain (data not shown); v. MreB was identified with a low score and interference with its function results in round cells and not in the cell filamentation [29]. Moreover, MreB possibly associates with FtsZ [26], so that its interaction with ABDOM could be indirect.

To determine if FtsZ interacts with ABDOM directly, we assayed co-sedimentation of FtsZ with a fusion of ABDOM to maltose binding protein (MBP). In contrast to dimeric GST, MBP is monomeric, therefore advantageous in co-sedimentation experiments. MBP-ABDOM, MBP and FtsZ were each purified (S2 Fig), combined in a pair-wise fashion and tested in vitro. We found that MBP-ABDOM, but not MBP alone, co-sedimented with GTP-polymerized FtsZ (Fig 2, panel A), indicating that ABDOM interacts directly with a polymerized form of FtsZ. No co-sedimention of MBP-ABDOM was observed in the absence of either GTP (S3 Fig, panel B) or FtsZ (S3 Fig, panels A and C).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. ABDOM binds FtsZ but does not interfere with its polymerization neither in vitro nor in vivo.

A. Purified MBP-ABDOM bound assembled FtsZ in vitro. The MBP-ABDOM fusion protein, but not MBP alone, co-sedimented with polymerized FtsZ. Fixed concentration of FtsZ (16 μM) was co-sedimented by addition of 1mM GTP in the presence of increasing concentration of MBP or MBP-ABDOM. For reference, equal amounts of MBP-ABDOM and MBP supernatant without FtsZ are shown in rightmost lane. Proteins were detected with anti-MBP antibody. B. MBP-ABDOM had no effect on FtsZ polymerization/depolymerization in vitro, as detected by 90° angle light scattering. FtsZ was polymerized after addition of 1 mM GTP (arrow) in the presence of either MBP-ABDOM (black line) or MBP (red line) C. Filaments produced by MscSΔ266–286 expression contain multiple nonfunctional Z-rings (upper row), which resemble Z-rings arrested by the cephalexin inhibition of PBP3 (middle row), control cells (lower row). FtsZ-YFP was expressed to visualize Z-rings. Scale bar represents 10 μm. D. Peptidoglycan synthesis detected by immunofluorescent D-cysteine labeling. Overexpression of MscSΔ266–286 resulted in cell filamentation, with clear dark rings of newly synthesized murein in septal areas (left). A similar pattern of murein segregation was observed in cells treated with 1μg/ml aztreonam, a selective inhibitor of PBP3 whose septal localization depends on functional Z-rings (right). Experiments were carried out over one cell cycle in D-cysteine, followed by 30 min. chase. The labeled, older murein is seen as bright spots, while newly made murein is seen as dark areas. Scale bar represents 1 μm.

https://doi.org/10.1371/journal.pone.0127029.g002

Proteins that inhibit FtsZ polymerization induce cell filamentation when over-expressed [30]. Knowing that ABDOM expression results in cell filamentation, we wondered if ABDOM binding influences FtsZ polymerization. FtsZ polymerizes into long protofilaments in a GTP-dependent manner. This polymerization can be monitored by 90° angle light scattering [31]. Proteins that inhibit FtsZ polymerization decrease light scattering [32]. We found that light scattering was not affected when MBP-ABDOM was added to media containing FtsZ and GTP, indicating that neither polymerization nor depolymerization of FtsZ is favored in vitro (Fig 2B). This indicates that ABDOM is not an inhibitor of FtsZ polymerization.

Z-ring formation and murein synthesis in cells overexpressing MscSΔ266–286 mutant

Overexpression of some proteins that interact with FtsZ impair the formation of Z-rings or cause Z-ring dysfunction, thereby leading to cell filamentation [33]. We observed that Z-rings were formed in cell filaments that were induced by MscSΔ266–286 overexpression (Fig 2, panel C) and we found that their number and the distances between them appeared to be similar to those found in cell filaments induced by cephalexin treatment. Cephalexin blocks activity of penicillin binding protein 3 (PBP3) which acts downstream of FtsZ and thereby does not interfere with Z-ring formation. Our observation suggests that ABDOM does not interfere with FtsZ polymerization in vivo. Thus, although the ABDOM interacts with polymerized FtsZ it does not appear to influence the degree of polymerization either in vitro or in vivo.

We then investigated the effect of the ABDOM-FtsZ interaction on the distribution of newly synthesized peptidoglycan (PG), also known as murein, in cells overexpressing MscSΔ266–286. This was done over one cell cycle by pulse labeling with a D-amino acid, followed by a chase without this amino acid and immunomicroscopy [34]. The labeled, older PG was seen as bright spots, while the more recently synthesized murein was seen as dark background areas (Fig 2, panel D). Overexpression of MscSΔ266–286 resulted in cell filamentation, with clear dark rings of newly synthesized murein in septal areas (Fig 2, panel D). A similar pattern of murein segregation was observed in cells treated with aztreonam, a selective inhibitor of PBP3 whose septal localization depends on functional Z-rings (Fig 2, panel D). These results indicate that, although, Z-ring function is disrupted and cell division is inhibited by the overexpression of MscSΔ266–286, the formation of the Z-rings and murein pre-septal rings is not affected.

The cell filamentation induced by overexpression of ABDOM or by MscSΔ266–286 is a non-physiological phenomenon; however, it represents a useful indication of the MscS-FtsZ interaction.

Impaired MscS-FtsZ binding is associated with compromised growth on β-lactam antibiotics

In order to better understand the interaction between MscS and FtsZ we searched for mutations in ABDOM that would abolish the binding. We constructed several double and triple mutants of MBP-ABDOM with alanines substituting amino acids exposed to its external surface (accordingly to the location in the crystal structure representing the nonconductive state of the channel [35]). We purified these proteins and tested their binding to FtsZ in vitro (S3 Fig, panel A). Among the mutations tested the double mutation K258A/R259A (S3 Fig, panel A) had the most significant effect, reducing binding affinity by approximately 3-fold (from Kd 5.3 μM in MscS to 14.6 μM in the mutant) (Fig 3, panel B and S3 Fig, panel B). Introduction of K258A/R259A to MscS did not change channel activation threshold (not shown) nor protein expression level (S1 Fig) and its ability to protect cells from osmotic downshocks (S4 Fig) indicating that the mutation did not change MscS channel properties significantly. To check whether K258A/R259A mutation impairs FtsZ-binding in vivo we wanted to investigate cell filamentation induced by MscSΔ266–286 overexpression, which we consider a non-physiological hallmark of the MscS-FtsZ interaction. Unfortunately, the K258A/R259A mutation introduced into MscSΔ266–286 reduced the level of protein expression dramatically (not shown) and we were unable to determine its effect on the cell filamentation in vivo. Since mutations K258 and R259 are situated at the very bottom of the ABDOM (Fig 3, panel A) and thus indicate a possible FtsZ binding site, we decided to introduce there a bulky tag that might sterically interfere with FtsZ binding. We fused YFP to the C-terminus of MscS and found out that MscS-YFP did not change the MscS channel properties (S4 and S5 Figs). However, we noticed that the wild-type and the YFP-tagged MscS responded in a different way to the addition of FtsZ to the cytoplasmic side of the membrane. FtsZ slowed down adaptation as well as the recovery from inactivation by 12% in the wild-type MscS and did not in the tagged MscS (S6 Fig).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Alanine substitutions of K258 and R259 in ABDOM impair its binding to FtsZ in vitro.

A. A crystal structure of nonconductive MscS (PDB ID: 2OAR), amino acids K258 and R259 are shown in red spacefill. B. Double alanine substitution K258A/R259A impaired ABDOM binding to FtsZ. Co-sedimentation experiments were carried out similarly to those shown in Fig 2, panel A. The data were fitted with Hill model (y = xⁿ/(Kd+xⁿ) with Kd = 5.3 μM, n = 2.6 and Kd = 14.6 μM, n = 4.3 for MBP-ABDOM and MBP-ABDOM-K258A/R259A respectively.

https://doi.org/10.1371/journal.pone.0127029.g003

Next we fused YFP to MscSΔ266–286 to investigate whether cells expressing MscSΔ(266–286)-YFP show a non-physiological filamentation. As we expected overexpression of MscSΔ(266–286)-YFP did not induce cell filamentation, unlike overexpression of the untagged MscSΔ266–286 (S7 Fig). Both lines of evidence: the one using the double K258A/R259A mutation and the other one introducing the bulky tag to the end of MscSΔ266–286 confirmed that putative ABDOM—FtsZ interaction is a prerequisite for the observed cell filamentation and show that the binding site of FtsZ is situated at the bottom of the ABDOM.

FtsZ is known to be responsible for cell division but also directs cell wall synthesis [36]. We wondered, whether MscS could be responsible for protection against cell wall damage and whether this function requires interactions with FtsZ. Ampicilin is a broad spectrum ß-lactam penicillin that binds to all penicillin binding proteins [37] and by inhibiting PG cross-linking induces general lesions of peptidoglycan [38]. Penicillin treatment often results in membrane protrusions emanating from midcell before they rupture [39, 40]. An accepted hypothesis to explain this is that penicillin causes a defect in the splitting of septal PG during division. Consistent with this idea, blocking of cell division has been shown to have protective effects when E. coli cells are treated with ß–lactams [41]. FtsZ is a central division protein that directs the septal as well as the lateral wall PG synthesis [34, 36]. We expected that if indeed MscS functionally interacts with FtsZ, it could also have protective effect via this interaction against ampicillin treatment. To induce cell wall lesions without cell lysis we used of ampicillin in concentrations below minimal inhibitory concentration (subMIC) in which we did not observe any apparent cell morphology changes. We transformed mscS^- strain MJF429 with pWG1 plasmids (kanR derivatives of pTRC99A) carrying mscS alleles. We found that overexpression of MscS, but not MscS-K258A/R259A nor MscS-YFP, increases the growth rate of cells cultivated in the presence of subMIC of ampicillin (lower—1.6 μg/ml or higher—4.2 μg/ml) (Fig 4, panels A and B). These results suggest that MscS protects cells against ampicillin and the lack of this effect in MscS-K258A/R259A or in MscS-YFP suggests that the mechanism of protection is associated with the MscS binding of FtsZ. To get more insight into this mechanism we investigated the ampicillin treated cells by flow cytometry (FC). We measured forward scatter (FSC) which is proportional to the cell length [42]. We noticed that ampicillin treatment resulted in a new population of cells with increased length in all strains tested (Fig 4, panels C and D, see insets above the plots). Fractions of elongated cells in the presence or in the absence of ampicillin are shown as bars on X axes in Fig 4, panels C and D. Larger number of the elongated cells was observed in higher concentration of ampicillin (Fig 4, panel D). It was demonstrated previously that subMIC of ampicillin arrested E. coli cell division by inducing SOS system [41] what in turn resulted in cell elongation [43]. Curiously, in our experiments expression of wt-MscS partially relieved this effect (Fig 4, panels C andD; S8 Fig, panels A-C). In cells expressing MscS, both the percent of elongated cells and the median cell scatter (S2 Table) were lower compared to those in control, while the protective effect of expression of MscS-K258A/R259A was weaker (Fig 4, panels C and D; S8 Fig, panels A-C). In the presence of 1.6 μg/ml ampicillin expression of MscS-YFP resulted in the largest number of elongated cells (Fig 4, panel C) as a consequence of almost complete cell division arrest (Fig 4, panel A). Similar phenomenon was seen for all constructs in 4.2 μg/ml ampicillin (cell elongation is shown in Fig 4, panel D and cell division arrest is shown in Fig 4, panel B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. MscS protects cells from β-lactam antibiotics.

Experiments were conducted on plasmid transformed MJF429 strain in two different concentrations of ampicillin: 1.6 μg/ml (A, C, E) and 4.1μg/ml ampicillin (B, D, F). A, B. Overexpression of MscS but not MscS-K258A/R259A or MscS-YFP supports the cell growth in the presence of subMIC of ampicillin. Inset in B: growth of ampicillin treated cells shown using expanded Y axis scale. C, D. The number of elongated cells estimated from forward scatter (FSC) was reduced for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample FSC histograms are presented in insets, for complete FSC analysis see S8 Fig, panels A-C. E, F. The number of cells with compromised membrane integrity estimated using PI staining was decreased for bacteria expressing MscS (green) as compared to MscS-K258A/R259A (pink) or MscS-YFP (blue). Sample scatter plots are presented for complete FC analysis see S8 Fig, panels D-F. Cells grown in A, B were then used in experiments presented in C, D, E, F. For statistically different results p-values < 0.05 of paired Student`s t-test are shown above the graphs (n = 3).

https://doi.org/10.1371/journal.pone.0127029.g004

Growth in ampicillin results in PG damage. Therefore we wondered whether we could see decreased cell envelope integrity under our experimental conditions. To this end we stained cells with propidium iodide (PI) [44] which is excluded from live cells but stains the damaged and dead cells (Fig 4, panels E andF see insets above the plots, and S8 Fig, panels D-F). In the higher concentration of ampicillin (Fig 4, panel F) the fractions of stained cells (displayed as bars on X axes) were larger than those in the lower ampicillin concentration (Fig 4, panel E). In the higher ampicillin concentration expression of MscS resulted in fewer dead or damaged cells (18.42% ± 3.24% vs. 32.58% ± 2.80% in control), while expression of MscS-K258A/R259A mutant did not have any effect (33.23% ± 3.06%) (Fig 4, panel F). It is worth to note that expression of MscS-YFP sensitized cells against ampicillin treatment (40.89% ± 6.01% of PI stained cells). This effect was even more pronounced in the lower ampicillin concentration: expression of MscS-YFP resulted in 21.76% ± 4.27% of PI stained cells as opposed to 3.52% ± 0.42% and 1.35% ± 0.33% of PI stained cells carrying vector and expressing MscS, respectively.