Table 1.
List of strains used in the present study.
Fig 1.
FI and scatter signals of B. subtilis vegetative cells and purified spores measured separately.
To measure cells, the non-sporulating Bs02005 strain was employed. Spores were isolated from a culture of Bs02003 according to Nicholson and Setlow, 1990 [35] resulting in a yield of approx 90%.
Fig 2.
Comparison of different dyes and channels used for optimal separation of cells and spores (time = 30 minutes, concentration = 2 fold).
(A) Raw data and cutoff values based on GMM. X-axis shows translated signal, so that the threshold is always at 0. (B) Distance between normal distributions as predicted by GMM in the respective channels with error bars showing the pooled standard deviation. The same metric was used to evaluate the effect of staining time, stain concentration (S1 Fig).
Fig 3.
Scatter plots with color indicating the classification.
Hyperbolic-sine transformed side scatter (SSC) and fluorescence in FL1-A channel are shown in (A), whereas (B) shows front- and side scatter (FSC, SSC). Color of the event indicates the respective cluster as predicted by GMM. Cluster centers are shown in blue and red lines show 2d kernel densities. (C) Corresponding microscopy images of the respective subpopulations are shown. Subpopulations were subsequently isolated by FACS.
Fig 4.
Analysis of sporulation dynamics of B. subtilis strains harboring different genome modifications.
(A) Shift of culture subpopulations containing cells, endospores and spores over time. Strains Bs02003, Bs02018 and Bs02020 are double mutants for cwlD [24] and sleB [25] whereas strain Bs02018 additionally expresses sfGFP as a fusion protein utilized for spore surface display. Strain Bs02020 additionally harbors deletion of cotA [29]. (B) Comparison of culture subpopulations of two B. subtilis mutants after 24 hours of cultivation. Strain Bs02025 contains deletions of cwlD [24] and sleB [25] and additionally harbors deletions of spo0E [31] and skfA [30] whereas Bs02003 serves as control.