Evolutionary rescue of spherical mreB deletion mutants of the rod-shape bacterium Pseudomonas fluorescens SBW25
- Institute of Natural and Mathematical Science, Massey University, New Zealand
- Laboratoire de Physique de l'ENS, Université Paris Cité, Ecole normale supérieure, UniversitéPSL, Sorbonne Université, CNRS, 75005 Paris, France
- Institut de biologie de l’Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, INSERM, PSL Research University, France
- Université Paris Cité, France
- New Zealand Institute for Advanced Study, Massey University, New Zealand
- Department of Molecular Biology, Umeå University, Sweden
- Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, SciLifeLab, Umeå Centre for Microbial Research, Umeå University, Sweden
- Department of Microbial Population Biology, Max Planck Institute for Evolutionary Biology, Germany
- Laboratoire Biophysique et Évolution, CBI, ESPCI Paris, Université PSL, France
- School of Biological Sciences, University of Canterbury, New Zealand
Figures
Figure 1 with 6 supplements
Characterisation of ancestral SBW25 and ∆mreB strains.
Figure shows (A) photomicrographs of ancestral SBW25 and ∆mreB. Scale bars, 3 μm. (B) The relationship between cell shape and estimated volume (Ve) is represented using compactness, a measure of roundness. One hundred representative cells from ancestral SBW25 and ∆mreB populations are shown as cell outlines. (C) Fitness of ancestral SBW25 GFP and the ∆mreB mutant relative to ancestral SBW25 when both are in exponential phase during pairwise competition assays. Data are means and standard deviation of 50,000 cells each of ancestral SBW25 and ∆mreB, respectively, and 100,000 cells total, after competition. (D) Elongation rate measured for SBW25 and ∆mreB SBW25. Error bars represent mean and standard deviation (NWT = 94; N∆mreB = 99). (E) Proportion of live cells in ancestral SBW25 (black bar) and ΔmreB (grey bar) based on LIVE/DEAD BacLight Bacterial Viability Kit protocol. Cells were pelleted at 2000 × g for 2 min to preserve ΔmreB cell integrity. Error bars are means and standard deviation of three biological replicates (n>100). (F) Schematic of asymmetric size production in cell lineages at generation F0 and F1. Asymmetries from differential growth rate between sister cells (i,j) are captured by Cg (see Materials and methods), while asymmetries in daughter (l,k) cell sizes at septation are measured by Cs. (G) Probability p to pass to the next generation as a function of cell size in ∆mreB cells. The shaded grey areas are indicative of regions where survival significantly drops.
Figure 1—figure supplement 1
Correlation between cell size and DNA content in SBW25 and ∆mreB cells.
DNA per cell does not vary in SBW25 cells to the degree that it varies in the ∆mreB cells as they grow larger. Cells were stained with SYTO 16, a cell-permeant green-fluorescent nucleic acid stain, and analysed using flow cytometry (n=50,000 events).
Figure 1—figure supplement 2
Cell size and DNA content by flow cytometry.
Genomic content and cell size of cells with mutations of interest. (A) Flow cytometry reveals an increase in DNA content coupled with an increase in size of SBW25 ∆mreB. In the SBW25 background pbp1A Line 1 and Line 4 mutants are slightly smaller than SBW25 cells and have comparable DNA content. The Line 7 reconstruction cells are larger by FSC than SBW25 and contain more DNA, reflective of the filamenting phenotype seen via microscopy. (B) In the ∆mreB background, all three reconstructions have smaller cells than ∆mreB, with DNA content that is similar to SBW25. A small proportion of cells with large amounts of DNA are seen in the Line 7 reconstruction; these are also observed via microscopy as clumped spherical cells forming incomplete septa.
Figure 1—figure supplement 3
Growth curves for ancestral SBW25 and ∆mreB strains as measured by OD600 in Lysogeny Broth (LB).
NB: All growth curves were produced simultaneously. Data are means and standard deviations of three biological replicates.
Figure 1—figure supplement 4
Ancestral SBW25 and ∆mreB cells, with ectopically added MreB expressed from the glmS region of the chromosome.
(A) Viability of respective strains via live-dead cell staining. (B) Phase contrast micrographs of SBW25, ∆mreB, SBW25+Tn7 mreB, and ∆mreB+Tn7 mreB. Scale bars = 3 µm.
Figure 1—figure supplement 5
Aspect ratio and orientation of elongation axes.
(a) Aspect ratio for ∆mreB cells. Aspect ratio is defined as the ratio between the short and the long axis of the cell mask fitted with an ellipse. The decrease of the aspect ratio indicates that the long axis grows faster than the short axis. (b) Orientation of the elongation axis in ∆mreB cells is constant during the division. Angles are in radians. Sudden changes correspond to phase shift (angles are expressed within the interval [-π;π] with periodic boundaries). In (a and b), time traces are plotted from cell birth until septation.
Figure 1—figure supplement 6
Rotation of elongation axis at division.
From the angle, θ, between mother and sister cells, we compute the relative orientation between mother and daughter using |sin θ | in order to get average in populations. |sin θ |=0 means that mother and daughter cells are aligned, |sin θ |=1 means that they are perpendicular. Filled or open square data points represent rod-like cells and filled or open circles represent spherical cells. WT strains are labelled in black. Open squares and open circles represent the reconstruction of the different observed mutations in the WT and ∆mreB backgrounds, respectively. Filled circles represent the evolved lines where the mutations have been identified. Colours as in Figure 3; blue is Line 1 or PBP1a D484N, green is Line 4 or PBP1a T362P, red is Line 7, grey is ectopic mreB expression. Data are means and standard deviation (NWT = 141; NL1_WT = 78; NL4_WT = 57; NL7_WT = 110; NmreB_WT = 54; N∆mreB = 154; NL1_∆mreB = 148; NL4_∆mreB = 164; NL7_∆mreB = 149; NmreB_∆mreB = 96; NL1=132; NL4=116; NL7=116).
Figure 2 with 11 supplements
Characterisation of evolved lines at 1000 generations.
(A) Relative fitness of the ∆mreB mutant, derived lines after ~1000 generations, and mutant reconstructions in ∆mreB and SBW25, relative to SBW25 (dashed line) in pairwise competition experiments. Error bars represent standard deviation. (B) Compactness versus estimated volume (Ve) of derived lines: Line 1 in blue, Line 4 in green, and Line 7 in red; derived mutations reconstructed in ∆mreB and SBW25 backgrounds. Data points are means and SD of X 100 cells. Square data points represent rod-like cells, whereas circles (both open and filled) represent spherical cells. Ancestral genotypes are labelled in black. Open squares and open circles depict the reconstruction of mutations in the SBW25 and ∆mreB backgrounds, respectively. Filled circles represent the 1000 generation evolved lines from where the mutations have been identified. Colours as follows: blue is Line 1 or Pbp1A D484N, green is Line 4 or Pbp1A T362P, red is Line 7. (C) Domain map and model of Pbp1A (PFLU0406) showing the two major domains: the glycosyltransferase (GT) domain (cyan) and the transpeptidase (TPase) domain (blue). The oligonucleotide/oligosaccharide binding (OB) domain is shown in green. The active site of the TPase domain is also shown (yellow). The locations of mutations from Lines 1 and 4 are indicated with coloured arrows in the pbp1A gene map and in the model of Pbp1A. The mutations in Lines 3 and 6 are also shown in grey but were not characterised further in this work.
Figure 2—figure supplement 1
Growth curves of evolved ∆mreB lineages after 1000 generations of evolution.
Ancestral SBW25 and ∆mreB are shown for comparison. NB: All growth curves were produced simultaneously, SBW25 (WT), and ∆mreB are shown for comparison.
Figure 2—figure supplement 2
Relative fitness of the evolved lines at distinct points during the 1000 generations of evolution.
Data are means and standard deviation of three biological replicates.
Figure 2—figure supplement 3
Phase contrast images of the 10 independent evolved lines.
Cells were harvested at log phase (OD600=0.4). Scale bars = 3 µm.
Figure 2—figure supplement 4
A Clustal Omega sequence alignment of a segment of Pbp1A amino acid sequences from seven bacterial species, including Acinetobacter baumannii, which was used for the construction of the protein model showing the location of the evolved line mutations.
NB: Streptococcus does not contain the OB/ODD. Regions of interest containing the evolved Line 1 and 4 mutations are shown. Mutated residues are in bold. The mutations are shaded in red and green respectively and labelled accordingly. Strictly conserved residues are labelled below the alignment with asterisks.
Figure 2—figure supplement 5
Growth curves of the reconstructions in (A) SBW25 and (B) ∆mreB backgrounds.
NB: All growth curves were produced simultaneously. Ancestral SBW25 (WT) and ∆mreB are shown for comparison.
Figure 2—figure supplement 6
Elongation rate measures the rate, r, at which cell volume increases V(t)=V0ert.
Filled or open square data points represent rod-like cells and filled or open circles represent spherical cells. Ancestral SBW25 (WT) strains are labelled in black. Open squares and open circles represent the reconstruction of the different observed mutations in the WT and ∆mreB backgrounds, respectively. Filled circles represent the evolved lines where the mutations have been identified. Colours as in Figure 3; blue is Line 1 or PBP1a D484N, green is Line 4 or PBP1a T362P, red is Line 7, grey is ectopic mreB expression. Error bars represent standard deviations (NWT = 94; NL1_WT = 52; NL4_WT = 38; NL7_WT = 72; NmreB_WT = 36; N∆mreB = 99; NL1_∆mreB = 92; NL4_∆mreB = 102; NL7_∆mreB = 96; NmreB_∆mreB = 64; NL1=88; NL4=74; NL7=76).
Figure 2—figure supplement 7
Fitness of SBW25 ∆mreB ∆pbp1A and ∆mreB pbp1A (T362P) relative to SBW25 ∆mreB.
Each genotype was marked separately with green and red fluorescent protein allowing reciprocal fitness measures to control for any marker effect. Data were collected by flow cytometry.
Figure 2—figure supplement 8
Cell morphologies.
Phase contrast and SEM images of reconstruction strains. Reconstructions in either the SBW25 or the ∆mreB background are depicted. SBW25 (WT) and ∆mreB are shown for comparison. Scale bars = 3 µm.
Figure 2—figure supplement 9
The cell morphologies and relationship between cell shape and estimated volume (Ve) represented using compactness as in Figure 1B of mutation reconstructions.
Cell shape for each mutation of interest in (A) Line 1 (PBP1a D484N) in ∆mreB and (B) in SBW25. (C) Line 4 (PBP1a T362P) in ∆mreB and (D) in SBW25. (E) Line 7 PFLU4921-4925 (∆5399934–5403214) in ∆mreB and (F) in SBW25. All populations are shown alongside the ∆mreB and SBW25 morphologies for reference. Colours are as Figure 2. Insets as in Figure 2B reflecting the St for mutant populations. One hundred representative cells from each population are shown.
Figure 2—figure supplement 10
Genome map of the oprD-inclusive deletion (PFLU4921–4925 ∆5399934–5403214) and surrounding region.
Genes with functional predictions are noted.
Figure 2—figure supplement 11
Reconstruction of Line 1, 4, and 7 mutations in ancestral SBW25 (WT) and SBW25 ∆mreB background stained with DAPI.
SBW25 and ∆mreB cells shown for comparison. DNA segregation defects appear to be common at septation in this population. Scale bars = 2 µm.
Figure 3 with 1 supplement
Cell size homeostasis.
(A) Average added volume for the different genotypes, which is cell volume at septation less cell volume at birth. The horizontal lines correspond to the volume of ancestral SBW25 cells and to their spherical counterparts (Rs = 2*Rc). (B) Elongation rate plotted against septation rate. The black line corresponds to size homeostasis of wild-type strains (square = P. fluorescens; star = Pseudomonas aeruginosa; diamond = E. coli). Below this line, septation is faster than elongation and cell size decreases. The dashed line corresponds to equal septation and elongation rates. (C) The probability p to pass to the next generation as a function of growth asymmetry Cg. (D) The probability p to pass to the next generation as a function of error in septum positioning measured as the relative cell size between daughters at division Cs. In (A, B, C, and D) square data points (both open and filled) represent rod-like cells, whereas circles (both open and filled) represent spherical cells. Ancestral genotypes are labelled in black. Open squares and open circles depict the reconstruction of mutations in the WT and ∆mreB backgrounds, respectively. Filled circles represent the evolved lines where the mutations have been identified. Colours as in Figure 2; blue is Line 1 or Pbp1A D484N, green is Line 4 or Pbp1a T362P, red is Line 7, grey is ectopic mreB expression. Error bars represent standard errors. In (A, B, and D): (NWT = 94; NL1_WT = 52; NL4_WT = 38; NL7_WT = 72; NmreB_WT = 36; N∆mreB = 99; NL1_∆mreB = 92; NL4_∆mreB = 102; NL7_∆mreB = 96; NmreB_∆mreB = 64; NL1=88; NL4=74; NL7=76); in (C) (NWT = 47; NL1_WT = 26; NL4_WT = 19; NL7_WT = 38; NmreB_WT = 18; N∆mreB = 55; NL1_∆mreB = 56; NL4_∆mreB = 62; NL7_∆mreB = 53; NmreB_∆mreB = 32; NL1=44; NL4=42; NL7=40).
Figure 3—figure supplement 1
The probability p to pass to the next generation as a function of division time asymmetry CT.
Division time asymmetry is measured as the relative time elapsed between cell birth and division. Error bars represent standard errors (NWT = 94; NL1_WT = 52; NL4_WT = 38; NL7_WT = 72; NmreB_WT = 36; N∆mreB = 99; NL1_∆mreB = 92; NL4_∆mreB = 102; NL7_∆mreB = 96; NmreB_∆mreB = 64; NL1=88; NL4=74; NL7=76). Filled or open square data points represent rod-like cells and filled or open circles represent spherical cells. Ancestral SBW25 genotypes are labelled in black. Open squares and open circles represent the reconstruction of the different observed mutations in the ancestral and ∆mreB backgrounds, respectively. Filled circles represent the evolved lines where the mutations have been identified. Colours as in Figure 3; blue is Line 1 or PBP1a D484N, green is Line 4 or PBP1a T362P, red is Line 7, grey is ectopic mreB expression.
Figure 4 with 3 supplements
Molecular investigation of adaptive mutations in ∆mreB evolution.
(A) Peptidoglycan cross-linking in the cell walls of mutants in SBW25 and ∆mreB genetic backgrounds. The error bars (SD) are the result of three biological replicates. ANOVA revealed a highly significant difference among means [F7,16 = 8.19, p<0.001] with Dunnett’s post hoc test adjusted for multiple comparisons showing that five genotypes (*) differ significantly (p<0.05) from SBW25. (B) Cell extracts subjected to polyacrylamide gel and SDS-PAGE labelling with Bocillin-FL gel to demonstrate penicillin binding of Pbp1A (labelled) and other Pbps in P. fluorescens SBW25. See Figure 4—figure supplement 2 for quantification of band intensities and Figure 4—source data 1 for original gel image. (C) Fluorescent images of mutations in SBW25 and ∆mreB backgrounds subjected to short-pulse labelling with fluorescent D-amino acid BADA to reveal the location and organisation of cell wall construction in the mutant backgrounds. As cells elongate in preparation to divide, septal peptidoglycan synthesis becomes more pronounced. ∆mreB cells have highly disorganised synthesis (see Figure 4—figure supplement 3 for quantitative analysis), which is alleviated in ∆mreB Pbp1A* and ∆mreB ∆PFLU4921-25 mutants. Cell outlines from phase contrast images show the cell boundaries. Scale bars = 2 µm. (D) Model of evolutionary rescue in ∆mreB mutant cells highlighting the organisation of the predicted glycan meshwork in the rod and spherical geometries. In Pbp1A* mutants (Line 1 and Line 4), a decrease in transpeptidase activity favours disorganised cell wall architecture, which is better suited to the topology of a sphere.
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Figure 4—source data 1
Original gel image shown in Figure 4B.
- https://cdn.elifesciences.org/articles/98218/elife-98218-fig4-data1-v1.zip
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Figure 4—source data 2
Labelled version of Figure 4—source data 1.
- https://cdn.elifesciences.org/articles/98218/elife-98218-fig4-data2-v1.zip
Figure 4—figure supplement 1
Peptidoglycan analysis.
(A) Peptidoglycan analysis showing the relative PG density of different P. fluorescens strains relative to SBW25 (wild-type). Error bars (SD) are the result of three biological replicates. ANOVA revealed a highly significant difference among means [F7,16 = 16.74, p<0.001] with Dunnett’s post hoc test adjusted for multiple comparisons showing that three genotypes (*) differ significantly (p<0.05) from SBW25. (B) UPLC spectra of the peptidoglycan profiles of the different P. fluorescens strains. Major muropeptide peaks were labelled as M4 (monomer disaccharide tetrapeptide), D44 (dimer disaccharide tetra-tetrapeptide), and D44N (anhydrous dimer disaccharide tetra-tetrapeptide).
Figure 4—figure supplement 2
Quantification of band intensity corresponding to PBP1A TPase activity as presented in the gel shown in Figure 4B.
Band intensity was measured from the raw image of the gel. For each condition, we measured the mean background level and subtracted the mean grey level of the band measured in ROIs of the same size.
Figure 4—figure supplement 3
Quantification of heterogeneity in cell wall synthesis as determined by BADA labelling (see Figure 4C).
Data are standard deviations divided by the square root of the mean pixel intensity (y-axis) of between 10 and 14 cells for each cell lineage. To rule out possible artefacts associated with variation of the mean intensity, we calculated BADA heterogeneity as the ratio of the standard deviation divided by the square root of the mean. ANOVA revealed a highly significant difference among means [F7,89 = 9.97, p<0.0001] with Dunnett’s post hoc test adjusted for multiple comparisons showing that SBW25 ∆mreB and SBW25 ∆PFLU4921–4925 are significantly different (*) from SBW25 (p<0.05).
Videos
Video 1
Elongation axis rotates 90° relative to the mother cell after cell division.
Video 2
Reduction in cell volume for exponentially growing SBW25 ∆mreB cells.
Video 3
Faulty septation of SBW25 ∆mreB ∆PFLU4921–4925 cells.
Additional files
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Supplementary file 1
Mutations identified in derived lines at generations 500 and 1000.
- https://cdn.elifesciences.org/articles/98218/elife-98218-supp1-v1.docx
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Supplementary file 2
Mutations in pbp1A at generation 50.
- https://cdn.elifesciences.org/articles/98218/elife-98218-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/98218/elife-98218-mdarchecklist1-v1.docx
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Evolutionary rescue of spherical mreB deletion mutants of the rod-shape bacterium Pseudomonas fluorescens SBW25
eLife 13:RP98218.
https://doi.org/10.7554/eLife.98218.4
