A Novel Cell Type Enables B. subtilis to Escape from Unsuccessful Sporulation in Minimal Medium

Abstract

Sporulation is the most enduring survival strategy developed by several bacterial species. However, spore development of the model organism Bacillus subtilis has mainly been studied by means of media or conditions optimized for the induction of sporogenesis. Here, I show that during prolonged growth during stationary phase in minimal medium, B. subtilis undergoes an asymmetric cell division that produces small and round-shaped, DNA containing cells. In contrast to wild-type cells, mutants harboring spo0A or spoIIIE/sftA double mutations neither sporulate nor produce this special cell type, providing evidence that the small round cells emerge from the abortion of endospore formation. In most cases observed, the small round cells arise in the presence of sigma H but absence of sigma F activity, different from cases of abortive sporulation described for rich media. These data suggest that in minimal media, many cells are able to initiate but fail to complete spore development, and therefore return to normal growth as rods. This work reveals that the continuation of asymmetric cell division, which results in the formation of the small round cells, is a way for cells to delay or escape from-unsuccessful-sporulation. Based on these findings, I suggest to name the here described cell type as "dwarf cells" to distinguish them from the well-known minicells observed in mutants defective in septum placement or proper chromosome partitioning.

Keywords: cell division; dwarf cell; minicell; minimal growth medium; sporulation.

Figure 1

Wild-type B. subtilis cells generate small round cells during prolonged growth. Comparative analysis of growth and spore formation (A–C) and comparative analysis of growth and small round cells formation (D–F) in different media over time. Error bars represent standard deviations of the means of two independent experiments. Small round cells counts and sporulation data are shown in Supplemental Data (Table S1–S4). (G) Representative view of cells acquired at time point 100 h grown in S7₅₀ at room temperature. White triangles indicate small round cells still attached to their larger siblings, black triangles indicate free small round cells, white asterisks indicate sporulated cells, black asterisk or empty triangles indicate a triplet or doublets of small round cells respectively from consecutive polar divisions. Empty asterisks indicate released spores. Scale bars 2 μm.

Figure 2

Wild-type B. subtilis cells generate small round cells containing DNA. (A) Wild-type cells showing that almost all small round cells contain DNA (black triangles). White triangles indicate two small round cells devoid of DNA. 98.33 ± 0.58% (mean ± SD of three independent experiments; > 250 cells counted each) of small round cells generated from wild-type cells contain DNA, while minicells generated from a B. subtilis minD/divIV double mutant (B) are all devoid of DNA. White triangles point out some minicells. The black asterisk in panel (A) indicates a cell with multiple attempts of sporulation. Cells were grown in S7₅₀ for about 100 and 28 h for wild-type and minD/divIV double mutant cells respectively. (C) Quantification of the frequency of the number of asymmetric divisions occurring per cell generating dwarf cell (s). Scale bars 2 μm.

Figure 3

Early sporulation genes (onset of sporulation) are active during formation of small round cells. (A) Cells expressing a fusion of gfp to the Spo0A-controlled spoIIG gene to visualize the activity of Spo0A. Black and white triangles indicate sporulating cells and cells generating small round cells respectively. (B) Analysis of GFP intensity in cells shown in panel (A). The difference in the GFP intensity of sporulating cells compared with cells generating small round cells was evaluated by the Mann-Whitney test and a p-value of > 0.05 (0.1765) was considered statically non-significant. 178 and 193 cells were measured for cells generating small round cells and sporulating cells respectively. Error bars represent standard deviations of the means. Fluorescence intensities are in arbitrary units (arb. units). Error bars represent standard deviations of the means. spo0A (C) or spo0H (D) mutants are unable to generate small round cells. (E) Cells expressing SpoIIE fused to GFP. Black triangles indicate polar E-rings. White asterisks indicate the helical distribution of SpoIIE-GFP. White triangles indicate the presence of E-rings at mid-cell position. Black asterisks indicate E-rings at the septum of the small round cells. (F) spoIIE mutant cells also generate small round cells (black triangles), but less frequently than the wild-type strain. Gray lines show the contour of the cells. Scale bars 2 μm.

Figure 4

Compartmentalized activation of sporulation-specific sigma factors is not established during formation of small round cells. Cells expressing a fusion of gfp to the σ^F-controlled spoIIQ (A) or to σ^E-controlled spoIID (B) genes were applied to visualize the activity of σ^F or σ^E, respectively. White triangles indicate sporulating cells with active σ^F or σ^E. Black triangles indicate cells dividing asymmetrically with no apparent activity of any sigma factor. Black asterisks indicate cells that had abortive sporulation followed by a successful attempt as judged by the attached small round cells. Gray lines show the contour of the cells. Scale bars 2 μm.

Figure 5

Chromosomal DNA is actively translocated into the small round cells. (A) B. subtilis cells expressing DNA translocase SpoIIIE fused to YFP. White triangles indicate SpoIIIE-YFP focus at the asymmetric septum. White asterisks indicate few cases where bipolar asymmetric septa persist and generate two small round cells from a single cell. Black triangles indicate small round cells without DNA lacking SpoIIIE-YFP foci at their septa. (B) spoIIIE mutant cells showing an increased number of small round cells without DNA (white triangles). Black triangles indicate small round cells apparently containing only residual amounts of DNA. (C) Cells bearing null mutations in both spoIIIE and sftA are unable to generate small round cells. Gray lines show the contour of the cells. Scale bars 2 μm.

Figure 6

Small round cells contain full chromosomes. (A) B. subtilis cells harboring a lacO cassette at 181° (terminus region of the chromosome). White triangles indicate small round cells with terminus region signal, which is a proof that the cells possess the full chromosome. The black triangle indicates a small round cell which has not completed DNA translocation (both terminus regions are still in the larger cell compartment). (B) B. subtilis cells harboring a lacO cassette at 359° (origin region of the chromosome). Nearly all small round cells contain the origin region (white triangles). Black triangle indicates a small round cell lacking origin signal. Terminus region tagged cells were grown in S7₅₀. For unknown reasons, origin region tagged cells hardly sporulated or generated small round cells in S7₅₀. However, when grown in S7₅₀ diluted 1:1 with LB, sporulation and small round cells formation could be observed (but less frequently compared with the wild-type cells grown in S7₅₀). Somehow, in this medium, cells exhibited high level of diffused fluorescence probably due to the higher expression of GFP-LacI. Gray lines show the contour of the cells. Scale bars 2 μm.

Figure 7

Time-lapse microscopy of growing small round cells. Black triangles indicate small round cells that grow back to rod shape and white triangle indicate a former mother cell resuming growth. Images were acquired every 30 min. Small round cells are of different size at the start of the time-lapse: “medium” (A), “large” (B), and “tiny” (C). In (B), white lines indicate the septum of dividing cells. Scale bars 2 μm. (D) B. subtilis cell cycle depicting the bifurcation in the spore development process. After the initiation phase of sporulation and the establishment of the asymmetric septum, the cell can escape from sporulation by generating a small round cell, which I name “dwarf” cell as it is generated by wild-type cells. However, the sporulation signals can remain stable in the former mother cell and a second round of the process is often initiated. Dwarf cells and former mother cells are able to return to rod shape and resume vegetative growth. The commitment to sporulate requires that both sigma factors are activated. The activation of sigma factors is not simultaneous and the activation of σ^F occurs first. Before σ^E is activated, cell can still abort the sporulation process, as reported by Narula et al. (2012).