A novel mechanism for morphological change during bacterial sporulation based on programmed peptidoglycan degradation
- Department of Biology, Texas A&M University, United States
- The Genetics and Genomics Interdisciplinary Program, Texas A&M University, United States
- Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, SciLifeLab, Umeå University, Sweden
Figures
Figure 1
Mature M. xanthus spores induced by either glycerol or starvation do not contain significant muropeptides.
UPLC muropeptide profiles of vegetative cells, glycerol- and starvation-induced spores indicate that the major muropeptides species, M4 and D44, in vegetative cells are diminished in both spore types, while spores are enriched in anhydro-muropeptides (Anh). Only muropeptides that could be confidently identified by MS/MS analysis are labeled. The characteristic peaks are labeled as follows: M, monomeric muropeptide (uncrosslinked); D, dimeric muropeptide (crosslink connecting two muropeptides), T, trimeric muropeptide (crosslink connecting three muropeptides). Numbers refer to the status of the peptide side chain (3, tripeptide; 4, tetrapeptide). Red characters mark the muropeptides only detected in spores.
Figure 2 with 2 supplements
LtgA and LtgB are required for glycerol-induced sporulation, whereas MurA overexpression inhibits sporulation.
(A) Brightfield images of cells at different time points after glycerol induction. Black arrows point to lysing cells. (B) Quantitative analysis of glycerol-induced sporulation using the length/width ratio (L/W) of cells. Whiskers indicate the 25th–75th percentiles and red dots the median. The total number of cells analyzed is shown on top of each plot. (C) LtgA and LtgB are homologous to E. coli MltE. The asterisk marks the conserved active site.
-
Figure 2—source data 1
Cell morphology measurements for Figure 2B.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Besides LtgA and LtgB, other putative LTGs do not affect either sporulation pathway significantly.
Brightfield images of cells were obtained after 4 h of glycerol induction.
Figure 2—figure supplement 2
In the absence of vanillate, Pvan-driven MurA expression does not affect glycerol-induced sporulation.
(A) Brightfield images of cells at different time points after glycerol induction. (B) Quantitative analysis of glycerol-induced sporulation using the length/width ratio (L/W) of cells. Whiskers indicate the 25th–75th percentiles and red dots the median. The total number of cells analyzed is shown on top of each plot.
-
Figure 2—figure supplement 2—source data 1
Original cell morphology measurements for Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig2-figsupp2-data1-v1.xlsx
Figure 3
LtgA, LtgB, and MurA regulate starvation-induced sporulation.
(A) The absence of LtgA and LtgB, and the overexpression of MurA do not affect the formation of fruiting body-like aggregates on starvation agar. The inset shows that the Pvan-driven MurA expression does not affect starvation-induced fruiting body formation in the absence of vanillate,. The image was taken after 96 h of incubation on a CF agar. (B) Both LtgA and LtgB are essential for forming starvation-induced spores, while MurA overexpression negatively regulates this process. Sonication-resistant spores were counted as colony formation units (cfu) after 96 h of incubation on CYE agar. Data are shown as mean ± standard deviation (n=3). Scale bars, 1 mm.
-
Figure 3—source data 1
Spore counts for Figure 3B.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig3-data1-v1.xlsx
Figure 4 with 1 supplement
LtgA and LtgB play distinct roles in the two sporulation pathways.
(A) While LtgA is required for PG degradation during glycerol-induced sporulation, LtgB is the major LTG for forming starvation-induced spores. PG was detected using an anti-PG serum and a fluorescence-conjugated secondary antibody. PG sacculi were purified from cells after 6 h and 120 h of glycerol-induced and starvation-induced sporulation, respectively. Flattened sacculi are not visible under brightfield microscopy. White arrows point to the sacculi in irregular shapes. BF, brightfield. (B) The overexpression of LtgA, but not LtgB, collapses rod shape in vegetative cells. (C) Purified periplasmic domain of LtgA and LtgB solubilize dye-labeled PG sacculi at different rates. Lysozyme and buffer serve as the positive and negative controls, respectively. Absorption at 595 nm was measured after 18 h incubation at 25 °C. Data are presented as mean values ± SD from three technical replicates. The inset shows purified LtgA and LtgB in a Coomassie-stained gel. Scale bars, 5 μm.
-
Figure 4—source data 1
Unlabeled SDS-PAGE image of purified periplasmic domain of LtgA and LtgB for Figure 4C.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig4-data1-v1.zip
-
Figure 4—source data 2
Absorption at 595 nm in the Remazol brilliant (RBB) assay for Figure 4C.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Vanillate-induced expression of LtgA-PAmCherry and LtgB-PAmCherry.
Protein expression was visualized through immunoblotting using mCherry antibodies (α mCherry). The MreB protein was used as the loading control, detected by the MreB antibodies (α MreB). ‘—’, no vanillate; ‘+’, 200 μM sodium vanillate.
-
Figure 4—figure supplement 1—source data 1
Unlabeled western blot images.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig4-figsupp1-data1-v1.zip
-
Figure 4—figure supplement 1—source data 2
Labeled western blot images.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig4-figsupp1-data2-v1.zip
-
Figure 4—figure supplement 1—source data 3
Original western images for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig4-figsupp1-data3-v1.pdf
Figure 5 with 1 supplement
LtgB regulates the PG-binding of LtgA during glycerol-induced sporulation.
(A) LtgB exhibits a response to glycerol induction earlier than LtgA. Representative trajectories of LtgA and LtgB before (uninduced) and after (1 min and 30 min) glycerol induction. The overall distribution of both LTGs is displayed using the composite of 100 consecutive frames taken at 100 ms intervals. Single-particle trajectories of PAmCherry were generated from the same frames. Individual trajectories are distinguished by colors. Scale bars, 5 μm. (B, C) The absence of LtgB reduces the diffusion of LtgA, which is reflected in the increase in immobile population (B) and the decrease in diffusion coefficient (D) (C), and these effects are especially prominent in the cells before (uninduced) and immediately after (1 min) glycerol induction. For each protein and condition, particles were identified from at least 100 cells and three independent experiments. The total number of particles analyzed is shown on top of each plot. Error bars were the standard deviation of 1000 bootstrap samples, and * indicates a difference of >0.005.
-
Figure 5—source data 1
Diffusion coefficient data for Figure 5C.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
PAmCherry-labeled LtgA and LtgB express as full-length and fully functional proteins.
(A) PAmCherry-labeled LtgA and LtgB are expressed as full-length proteins in vegetative cells (53 kDa and 103 kDa, respectively), which were detected by an anti-mCherry antibody. (B) The PAmCherry labels do not affect glycerol-induced sporulation. Quantitative analysis of glycerol-induced sporulation using the length/width ratio (L/W) of cells. Whiskers indicate the 25th–75th percentiles and red dots the median. The total number of cells analyzed is shown on top of each plot. p values were calculated against the wild-type data at the same time points using a single-tailed ANOVA test on unweighted samples. (C) The PAmCherry labels do not inhibit starvation-induced fruiting body formation on agar surfaces. The images were taken after 96 h of incubation on CF agar. (D) The PAmCherry labels do not inhibit the formation of starvation-induced spores. Sonication-resistant spores were counted as colony formation units (cfu) after 96 h of incubation on CYE agar. Data are shown as mean ± standard deviation (n=3).
-
Figure 5—figure supplement 1—source data 1
Unlabeled image of Figure 5—figure supplement 1A.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig5-figsupp1-data1-v1.tif
-
Figure 5—figure supplement 1—source data 2
Cell morphology measurements for Figure 5—figure supplement 1B.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig5-figsupp1-data2-v1.xlsx
-
Figure 5—figure supplement 1—source data 3
Spore count for Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/108250/elife-108250-fig5-figsupp1-data3-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Myxococcus xanthus) | ltgA (K1515_20820) | Aramayo and Nan, 2022 | MXAN_RS16290 MXAN_3363 | Slt/MltE LTG family 1 |
| Gene (M. xanthus) | ltgB (K1515_17460) | Aramayo and Nan, 2022 | MXAN_RS19615 MXAN_4034 | Slt/MltE LTG family 1 |
| Gene (M. xanthus) | murA (K1515_13170) | Aramayo and Nan, 2022 | MXAN_RS28325 MXAN_4909 | |
| Gene (M. xanthus) | agmT | Aramayo and Nan, 2022 Ramirez Carbo et al., 2024 | K1515_04910 MXAN_6607 | MltG/YceG LTG family 5 |
| Gene (M. xanthus) | K1515_37860 | Aramayo and Nan, 2022 | MXAN_RS00570 MXAN_0114 | Slt/MltE LTG family 1 |
| Gene (M. xanthus) | K1515_37385 | Aramayo and Nan, 2022 | MXAN_RS01035 MXAN_0210 | Slt/MltE LTG family 1 |
| Gene (M. xanthus) | K1515_34725 | Aramayo and Nan, 2022 | MXAN_RS03640 MXAN_0754 | LTG family unknown |
| Gene (M. xanthus) | K1515_24775 | Aramayo and Nan, 2022 | MXAN_RS12365 MXAN_2558 | RlpA LTG family 6 |
| Gene (M. xanthus) | K1515_22185 | Aramayo and Nan, 2022 | MXAN_RS14935 MXAN_3081 | Slt/MltE LTG family 1 |
| Gene (M. xanthus) | K1515_20905 | Aramayo and Nan, 2022 | MXAN_RS16205 MXAN_3344 | Slt/MltE LTG family 1 |
| Gene (M. xanthus) | K1515_14545 | Aramayo and Nan, 2022 | MXAN_RS22465 MXAN_4628 | Slt/MltE LTG family 1 |
| Gene (M. xanthus) | K1515_09355 | Aramayo and Nan, 2022 | MXAN_RS27580 MXAN_5690 | Slt/MltD/MltE, LTG family 1 |
| Gene (M. xanthus) | K1515_06035 | Aramayo and Nan, 2022 | MXAN_RS30855 MXAN_6370 | Slt/MltE LTG family 1 |
| Gene (M. xanthus) | K1515_01490 | Aramayo and Nan, 2022 | MXAN_RS35365 MXAN_7308 | MltA LTG family 2 |
| Gene (M. xanthus) | K1515_01440 | Aramayo and Nan, 2022 | MXAN_RS35415 MXAN_7318 | Slt/MltD/MltE, LTG family 1 |
| Strain, strain background (M. xanthus) | DZ2 | Campos and Zusman, 1975 | DZ2 | Wild-type strain |
| Strain, strain background (M. xanthus) | ΔltgA | This paper | BN361 | Request from Nan lab |
| Strain, strain background (M. xanthus) | ΔltgB | This paper | BN362 | Request from Nan lab |
| Strain, strain background (M. xanthus) | ΔltgA ΔltgB | This paper | BN363 | Request from Nan lab |
| Strain, strain background (M. xanthus) | ΔltgA attB::Pvan-ltgB | This paper | BN364 | Request from Nan lab |
| Strain, strain background (M. xanthus) | attB::Pvan-ltgA | This paper | BN365 | Request from Nan lab |
| Strain, strain background (M. xanthus) | attB::Pvan-ltgB | This paper | BN366 | Request from Nan lab |
| Strain, strain background (M. xanthus) | attB::Pvan-ltgA-PAmCherry | This paper | BN367 | Request from Nan lab |
| Strain, strain background (M. xanthus) | attB::Pvan-ltgB-PAmCherry | This paper | BN368 | Request from Nan lab |
| Strain, strain background (M. xanthus) | attB::Pvan-murA | This paper | BN369 | Request from Nan lab |
| Strain, strain background (M. xanthus) | ltgA-PAmCherry | This paper | BN370 | Request from Nan lab |
| Strain, strain background (M. xanthus) | ltgB-PAmCherry | This paper | BN371 | Request from Nan lab |
| Strain, strain background (M. xanthus) | ΔltgB ltgA-PAmCherry | This paper | BN372 | Request from Nan lab |
| Strain, strain background (M. xanthus) | ΔltgA ltgB-PAmCherry | This paper | BN373 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_37860::kan | This paper | BN374 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_37385::kan | This paper | BN375 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_34725::kan | This paper | BN376 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_24775::kan | This paper | BN377 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_22185::kan | This paper | BN378 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_20905::kan | This paper | BN379 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_14545::kan | This paper | BN380 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_09355::kan | This paper | BN381 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_06035::kan | This paper | BN382 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_01490::kan | This paper | BN383 | Request from Nan lab |
| Strain, strain background (M. xanthus) | K1515_01440::kan | This paper | BN384 | Request from Nan lab |
| Strain, strain background (Escherichia coli) | DH5α | Lab collection | ||
| Strain, strain background (E. coli) | BL31 (DE3) | Lab collection | ||
| Antibody | Rabbit polyclonal anti-mCherry | Rockland Immunochemicals | 600-401-P16 | (1:2000) |
| Antibody | Rabbit polyclonal anti-MreB | Mauriello et al., 2010 | (1:20,000) | |
| Antibody | Goat polyclonal anti-rabbit IgG H+L (HRP) | Fisher Scientific | 31460 | (1:20,000) |
| Software, algorithm | MATLAB | MathWorks | ||
| Software, algorithm | Single-particle analysis | Fu et al., 2018; Zhang et al., 2023; Ramirez Carbo and Nan, 2026 | https://doi.org/10.5281/zenodo.8234126 | |
| Software, algorithm | ImageJ | https://imagej.net |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/108250/elife-108250-mdarchecklist1-v1.pdf
-
Supplementary file 1
Plasmids and primers used in this study.
- https://cdn.elifesciences.org/articles/108250/elife-108250-supp1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A novel mechanism for morphological change during bacterial sporulation based on programmed peptidoglycan degradation
eLife 14:RP108250.
https://doi.org/10.7554/eLife.108250.4
