Figures and data
Mature M. xanthus spores induced by either glycerol or starvation do not contain significant muropeptides.
UPLC muropeptides 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 labelled. 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.
LtgA and LtgB are required for glycerol-induced sporulation, whereas MurA overexpression inhibit sporulation.
A) Bright field 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. Other putative LTGs do not affect either sporulation pathway significantly, which is shown in Figure 2 – figure supplement 1.
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. 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).
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 bright field microscopy. White arrows point to the sacculi in irregular shapes. BF, bright field. B) The overexpression of LtgA, but not LtgB, collapses rod-shape in vegetative cells. C) Purified 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 ⁰C. 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.
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 and C) the absence of LtgB reduces the diffusion of LtgA, which is reflected in the increase of 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 derivation of 1,000 bootstrap samples and * indicates a difference of > 0.005.
