Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects
- Institut Pasteur, France
- Université Paris Descartes, Sorbonne-Paris-Cité, France
- Umeå University, Sweden
- Université Paris Diderot, Sorbonne-Paris-Cité, France
- Proteomics Platform, Institut Pasteur, France
Figures
Figure 1 with 6 supplements
aPBPs have no role in maintaining rod-like cell shape.
(A) Sketch of the strain AV44 (LC69 mrcB::gfp-mrcB, mrcA::rfp-mrcA) with tunable levels of RFP-PBP1a and GFP-PBP1b. CRISPR guides are expressed either as crRNA (top) or as sgRNA (bottom), see also Fi…
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Figure 1—source data 1
Data used to generate Figure 1 and its supplements.
- https://cdn.elifesciences.org/articles/51998/elife-51998-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Passage probability of the different CRISPR guides used in this study.
This is measured by fluorescence microscopy in strain AV47 (LC69 HK022::P127-msfgfp, λ::P127-mcherry)/pCRRNAcos for crRNA or pAV20 for sgRNA. When used to repress the RFP-PBP1a and GFP-PBP1b fusions …
Figure 1—figure supplement 2
The RFP-PBP1a and GFP-PBP1b fusions are the only forms of aPBPs present in AV44.
(A) Fluorescent bocillin binds specifically to Penicillin Binding Proteins (PBP). The change in band intensity after repression by CRISPR does not reflect the change in fluorescence measured by …
Figure 1—figure supplement 3
Quantification of GFP-PBP1b by semi-quantitative SDS-PAGE.
(A) Purified msfGFP-6xHis. Left: Elution fraction loaded in a 4–20% acrylamide gel stained with Coomassie blue. The predicted msfGFP-6xHis molecular weight is ≈28.42 kDa. Right: visualization of the …
Figure 1—figure supplement 4
Residual PBP1a and PBP1b levels in response to CRISPR-based repression, measured by fluorescence microscopy.
Left: The same CRISPR guides produce different repression strength on GFP, depending on whether it is expressed constitutively in AV47 (186::Ptet-dCas9, HK022::P127-msfgfp, λ::P127-mcherry) or fused …
Figure 1—figure supplement 5
Dimensions of individual cells before lysis due to PBP1ab repression, in LB Strain: AV44/pAV20 G20-R20.
Imaging starts 4h45 after the induction of the CRISPR system. Cell length and cell diameter are normalized with respect to the dimensions of the cell in the first frame of the movie. Solid lines are …
Figure 1—video 1
Video of a cell of AV44/pAV20 with sgRNA G20-R20, bulging then lysing.
Timestamps start at the beginning of the movie, 5 hr after CRISPR induction. Corresponds to Figure 1E.
Figure 2 with 4 supplements
Repression of PBP1ab reduces mechanical stiffness while maintaining a high rate of peptidoglycan insertion.
(A) Top: Steady-state amount of peptidoglycan per cell, measured in AV84 (AV44 ΔLysA)/pAV20 and AV105 (AV44 ΔPBP1b ΔLysA) as annotated. Bottom: Fraction of the peptidoglycan that is cross-linked in …
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Figure 2—source data 1
Data used to generate Figure 2 and its supplements.
- https://cdn.elifesciences.org/articles/51998/elife-51998-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Amount of incorporated 3H-mDAP per optical density as a function of time.
Strains are AV84 (AV44 ΔlysA)/pAV20 GØ-RØ, AV84/pAV20 G14-R20, AV84/pAV20 G20-RØ and AV105 (AV84 ΔPBP1b)/pAV20 GØ-RØ, from left to right. The incorporated 3H-mDAP per cell is fit with formula , …
Figure 2—figure supplement 2
UPLC-UV chromatograms of the peptidoglycan after digestion by muramidase.
From top to bottom, strains are AV84/pAV20 GØ-RØ, AV84/pAV20 G14-R20, AV84/pAV20 G20-RØ and AV105/pAV20 GØ-RØ (ΔPBP1b). Abs: absorbance.
Figure 2—figure supplement 3
Growth curves before and after osmotic shock in minimal medium.
Strains are AV44 or AV93 (AV44 ΔmscSL) with either pAV20 GØ-RØ or G14-R20. Vertical lines mark the time of centrifugation, medium removal and resuspension in a medium of lower osmolarity. Normalized …
Figure 2—video 1
Video of sample cells in phase-contrast microscopy during an osmotic downshock.
Cells are expanding when the lower-osmolarity medium is flushed in the tunnel. The 1 osm/L osmotic downshock happens at 6 s. Corresponds to Figure 2B.
Figure 3 with 9 supplements
PBP1b facilitates quick recovery from transient inhibition of peptidoglycan synthesis.
(A) Cell elongation before lysis from D-cycloserine treatment (1 mM) under the microscope, including sample snapshots. Strain is MG1655. Length is normalized to the length at the beginning of the …
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Figure 3—source data 1
Data used to generate Figure 3 and its supplements.
- https://cdn.elifesciences.org/articles/51998/elife-51998-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Effect of depletion of peptidoglycan precursors measured by MreB motion.
Top: Probability distributions of the instantaneous velocity of MreB-msfGFP measured upon D-cycloserine treatment using strain B172 (MG1655 mreB::mreB-msfgfp) (A–C), or during depletion of mDAP in …
Figure 3—figure supplement 2
UPLC-UV analysis of the peptidoglycan, either on cells grown without treatment, or after 1 hr of 1 mM D-cycloserine treatment in MG1655 in LB.
Figure 3—figure supplement 3
Damage sensitivity and recovery in LB.
(A) Sensitivity to 1 mM D-cycloserine, for MG1655 (WT) and B150 (ΔPBP1b)/pBC03 (pBAD33-ParaPBP1b) with arabinose (overexp.) or without arabinose (non-induced). (B) Recovery after washout from 22 min …
Figure 3—figure supplement 4
Detection of lysis events during D-cycloserine recovery.
Sample images from movies taken during recovery from 32 min of 1 mM D-cycloserine treatment, with PBP1b never induced (top) or induced 10 min before drug washout (bottom). Two frames are …
Figure 3—figure supplement 5
Alleviating effect of PBP1b expression after transient D-cycloserine treatment.
(A,C) Instantaneous effective growth rate (sum of rates of growth and lysis) of B150/pBC03 as a function of time after drug washout following 32 min (A) or 22 min (C) of D-cycloserine treatment (1 …
Figure 3—figure supplement 6
The E313D mutant of PBP1b (PBP1b*) enables growth without LpoB, but does not rescue D-cycloserine recovery.
(A) Verification of PBP1b*’s functionality in ΔLpoB, in AV130 (GFP-PBP1b*, RFP-PBP1a, ΔLpoB) with single-guide RNAs carried by pAV20. With pAV20 GØ-RØ, both PBP1a and PBP1b* are expressed to high …
Figure 3—figure supplement 7
Sensitivity to 100 μM D-cycloserine at different levels of PBP1b.
Growth curves of AV29 (ΔPBP1b), AV31 (GFP-PBP1b), MG1655 and AV128 (GFP-PBP1b*, ΔLpoB) in LB, measured with a plate reader. The fusions to GFP are expected to be over-expressed to about 370% of WT …
Figure 3—video 1
Movie of MG1655 cells lysing following D-cycloserine treatment in LB.
Timestamps start at the moment when the cells are transfered on the agarose pad with D-cycloserine. Corresponds to Figure 3A.
Figure 3—video 2
Sample of movies used for single-molecule tracking of MreB-GFPSW, with overlaid trajectories.
Top: Untreated cells, Middle: D-cyc treatment (20 min time point), Bottom: mDAP depletion (30 min time point). Corresponds to Figure 3—figure supplement 1.
Figure 4 with 5 supplements
PBP1b localizes depending on the need for peptidoglycan synthesis.
(A-C) Calculated bound fraction of PBP1b at different levels of PBP1b, PBP1a and LpoB, using strains AV44, AV51 (ΔPBP1a) or AV110 (ΔLpoB). For GFP-PBP1b, sgRNA G14 (in pAV20), crRNA G10 (in …
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Figure 4—source data 1
Data used to generate Figure 4 and its supplements.
- https://cdn.elifesciences.org/articles/51998/elife-51998-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Tracking of single molecules and quantification of the fraction of bound molecules.
Left: Sample tracks corresponding to bound and diffusive GFP-PBP1b molecules, overlaid on a brightfield image using strain AV44/pCRRNAcos G10-R18 (280% PBP1a, 130% PBP1b). Right: Observed and fit …
Figure 4—figure supplement 2
Localization of bound molecules.
(A-B) Mean positions of bound molecules with respect to a normalized cell-coordinate system, using 195 tracks in 67 cells of AV51 (AV44 ΔPBP1a)/pCRRNAcos with crRNA G10 (130% PBP1b with respect to …
Figure 4—figure supplement 3
Calculated bound fraction of the PBP1b* mutant, compared to PBP1b with and without LpoB.
The strains are AV44 (GFP-PBP1b, RFP-PBP1a), AV110 (GFP-PBP1b, RFP-PBP1a, ΔLpoB) and AV130 (GFP-PBP1b*, RFP-PBP1a, ΔLpoB). The PBP1a and PBP1b variants are repressed to 280% and 130% of WT level …
Figure 4—figure supplement 4
Diffusion coefficient of non-bound molecules during D-cycloserine treatment or recovery.
free of GFP-PBP1b at different times during 1 mM D-cycloserine treatment (A) and during recovery from 30 min of 1 mM D-cycloserine treatment (B) in the strain AV51/pCRRNAcos G10-RØ. Corresponding …
Figure 4—video 1
Sample of TIRF-microscopy movies used for single-molecule tracking of GFP-PBP1b, with overlaid trajectories.
Left: AV51/pAV20 G14-RØ (PBP1b at 30% of WT, ΔPBP1a). Right: AV44/pAV20 G14-RØ (PBP1b at 30% of WT, PBP1a at 1300% of WT). These samples correspond to the conditions in Figure 4B.
Tables
Table 1
Levels of PBP1ab expressed from different cassettes, and repressed using different sgRNA or crRNA.
The levels are determined using either fluorescence microscopy, SDS-PAGE with fluorescence detection, or mass spectrometry (DIA: Data-Independent Acquisition or PRM: Parallel Reaction Monitoring.), …
| Relative and absolute quantification of PBP1b | ||||||||
|---|---|---|---|---|---|---|---|---|
| Strain | Promoter | System | Guide | Fluorescence (% of AV44) | Fluorescence (% of LC69) | Dia (%) | SDS-PAGE (copy/cell) | PRM (copy/cell) |
| LC69 | Wild-type | n.d. | n.d. | 100 | n.d. | 166 ± 28 (100%) | ||
| AV44 | Native fusion | sgRNA | G20 | 1.0 ± 0.04 | 3.8 ± 0.4 | n.d. | n.d. | n.d. |
| AV44 | Native fusion | sgRNA | G14 | 6.6 ± 0.79 | 24 ± 2.9 | 27 ± 2 | 40 ± 5 | 46 (28%) |
| AV51 | Native fusion | sgRNA | G14 | n.d. | n.d. | 33 ± 3 | 67 ± 14 | 56 ± 7 (33%) |
| AV51 | Native fusion | crRNA | G20 | 4.1 ± 2.0 | 15 ± 7.6 | n.d. | n.d. | n.d. |
| AV51 | Native fusion | crRNA | G14 | 12 ± 3.1 | 44 ± 12 | n.d. | n.d. | n.d. |
| AV51 | Native fusion | crRNA | G10 | 36 ± 2.8 | 131 ± 15 | n.d. | n.d. | n.d. |
| AV51 | Native fusion | crRNA | GØ | 97 ± 13 | 356 ± 57 | n.d. | n.d. | n.d. |
| AV44 | Native fusion | crRNA | GØ | 100 ± 5.4 | 367 ± 38 | 367 ± 32 | 688 ± 115 | 547 ± 52 (330%) |
| AV58 | Para | crRNA | GØ | 509 ± 57 | 1870 ± 265 | n.d. | n.d. | n.d. |
| Relative quantification of PBP1a | ||||||||
| Strain | Promoter | System | Guide | Fluorescence (% of AV44) | Fluorescence (% of LC69) | Dia (%) | ||
| LC69 | Wild-type | n.d. | n.d. | 100 | ||||
| AV44 | Native fusion | sgRNA | R20 | n.d. | n.d. | 20 ± 2 | ||
| AV50 | Native fusion | crRNA | R20 | 3 ± 4 | 43 ± 56 | n.d. | ||
| AV50 | Native fusion | crRNA | R18 | 21 ± 4 | 278 ± 139 | n.d. | ||
| AV50 | Native fusion | crRNA | R11 | 46 ± 6 | 620 ± 298 | n.d. | ||
| AV50 | Native fusion | crRNA | RØ | 100 ± 1 | 1337 ± 615 | n.d. | ||
| AV44 | Native fusion | crRNA | RØ | 100 ± 7 | 1337 ± 622 | 1337 ± 615 | ||
| AV63 | Para | crRNA | R18 | 166 ± 14 | 1549 ± 786 | n.d. | ||
| AV63 | Para | crRNA | R11 | 355 ± 22 | 4750 ± 2204 | n.d. | ||
| AV63 | Para | crRNA | RØ | 691 ± 50 | 9243 ± 4304 | n.d. | ||
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Table 1—source data 1
Data used to generate Table 1.
- https://cdn.elifesciences.org/articles/51998/elife-51998-table1-data1-v2.xlsx
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (E. coli) | LC69 | Cui et al., 2018 | 186::Ptet-dcas9 | |
| Strain, strain background (E. coli) | AV03 | Vigouroux et al., 2018 | 186::Ptet-dcas9, HK022::P, λ::P | |
| Strain, strain background (E. coli) | AV04 | Vigouroux et al., 2018 | 186::Ptet-dcas9, λ::P-mcherry | |
| Strain, strain background (E. coli) | AV08 | Vigouroux et al., 2018 | 186::Ptet-dcas9, mrdA::mcherry-mrdA | |
| Strain, strain background (E. coli) | AV29 | This work | 186::Ptet-dcas9, ΔmrcB | Supplementary file 1 |
| Strain, strain background (E. coli) | AV31 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB | Supplementary file 1 |
| Strain, strain background (E. coli) | AV44 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, mrcA::mcherry-mrcA | Supplementary file 1 |
| Strain, strain background (E. coli) | AV47 | This work | 186::Ptet-dcas9, HK022::P-msfgfp, λ::P-mcherry | Supplementary file 1 |
| Strain, strain background (E. coli) | AV50 | This work | 186::Ptet-dcas9, mrcA::mcherry-mrcA, ΔmrcB | Supplementary file 1 |
| Strain, strain background (E. coli) | AV51 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, ΔmrcA | Supplementary file 1 |
| Strain, strain background (E. coli) | AV58 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, ΔmrcA, HK022::Para-msfgfp-mrcB | Supplementary file 1 |
| Strain, strain background (E. coli) | AV63 | This work | 186::Ptet-dcas9, mrcA::mcherry-mrcA, ΔmrcB, HK022::Para-mCherry-mrcA | Supplementary file 1 |
| Strain, strain background (E. coli) | AV67 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, mrcA::mcherry-mrcA, ΔpbpC | Supplementary file 1 |
| Strain, strain background (E. coli) | AV80 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, mrcA::mcherry-mrcA, ΔpbpC, ΔmtgA | Supplementary file 1 |
| Strain, strain background (E. coli) | AV84 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, mrcA::mcherry-mrcA, ΔpbpC, ΔmtgA, ΔlysA | Supplementary file 1 |
| Strain, strain background (E. coli) | AV88 | Dion et al., 2019 | 186::Ptet-dcas9, mreB::mreB-msfGFP | |
| Strain, strain background (E. coli) | AV92 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, mrcA::mcherry-mrcA, ΔpbpC, ΔmtgA, ΔmscS | Supplementary file 1 |
| Strain, strain background (E. coli) | AV93 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, mrcA::mcherry-mrcA, ΔpbpC, ΔmtgA, ΔmscS, ΔmscL | Supplementary file 1 |
| Strain, strain background (E. coli) | AV100 | This work | 186::Ptet-dcas9, ΔmrcA, ΔmrcB, HK022::Para-msfgfp-mrcB | Supplementary file 1 |
| Strain, strain background (E. coli) | AV101 | This work | 186::Ptet-dcas9, ΔmrcA, ΔmrcB, HK022::Para-mcherry-mrcA | Supplementary file 1 |
| Strain, strain background (E. coli) | AV105 | This work | 186::Ptet-dcas9, ΔmrcB, mrcA::mcherry-mrcA, ΔpbpC, ΔmtgA, ΔlysA | Supplementary file 1 |
| Strain, strain background (E. coli) | AV109 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, mrcA::mcherry-mrcA, ΔlpoA | Supplementary file 1 |
| Strain, strain background (E. coli) | AV110 | This work | 186::Ptet-dcas9, mrcB::msfgfp-mrcB, mrcA::mcherry-mrcA, ΔlpoB | Supplementary file 1 |
| Strain, strain background (E. coli) | AV124 | This work | 186::Ptet-dCas9, mrcB::msfgfp-mrcB(E313D) | Supplementary file 1 |
| Strain, strain background (E. coli) | AV128 | This work | 186::Ptet-dCas9, mrcB::msfgfp-mrcB(E313D), ΔlpoB | Supplementary file 1 |
| strain, strain background (E. coli) | AV130 | This work | 186::Ptet-dCas9, mrcB::msfgfp-mrcB(E313D), mrcA::mcherry-mrcA, ΔlpoB | Supplementary file 1 |
| Strain, strain background (E. coli) | NO34 | Ouzounov et al., 2016 | mreB::mreB-msfgfpsw-kanR | |
| Strain, strain background (E. coli) | B150 | This work | ΔmrcB | Supplementary file 1 |
| Strain, strain background (E. coli) | B151 | van Teeffelen et al., 2011 | FB83, asd-1 | |
| Strain, strain background (E. coli) | B157 | This work | FB83, asd-1, ΔmrcB | Supplementary file 1 |
| Strain, strain background (E. coli) | B172 | This work | mreB::mreB-msfgfpsw-kanR | Supplementary file 1 |
| Strain, strain background (E. coli) | B174 | This work | ΔmrcB, mreB::mreB-msfgfpsw-kanR | Supplementary file 1 |
| Strain, strain background (E. coli) | B176 | This work | FB83, asd-1, mreB::mreB-msfgfpsw-kanR | Supplementary file 1 |
| Strain, strain background (E. coli) | B178 | This work | FB83, asd-1, ΔmrcB, mreB::mreB-msfgfpsw-kanR | Supplementary file 1 |
| Software, algorithm | Trackmate | Tinevez et al., 2017 | ||
| Software, algorithm | ThunderStorm | Ovesný et al., 2014 | ||
| Software, algorithm | SpotOn | Hansen et al., 2018 | ||
| Software, algorithm | Morphometrics | Ursell et al., 2017 | ||
| software, algorithm | TrackPy | Allan et al., 2016 | ||
| Software, algorithm | MicroManager | Edelstein et al., 2010 |
Additional files
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Supplementary file 1
Strains, plasmids, DNA fragments and oligonucleotides used in this study.
All gene deletions were done by P1 transduction from the Keio collection (Baba et al., 2006). Ptet-dcas9 refers to the cassette described in Cui et al. (2018) that minimizes the ‘bad seed’ effect. MG1655 is a gift from Didier Mazel.
- https://cdn.elifesciences.org/articles/51998/elife-51998-supp1-v2.xlsx
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Supplementary file 2
Relative abundance of the different peaks of muramidase-digested peptidoglycan, measured by UPLC.
- https://cdn.elifesciences.org/articles/51998/elife-51998-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/51998/elife-51998-transrepform-v2.docx
