Heterochronic phosphorelay gene expression as a source of heterogeneity in Bacillus subtilis spore formation

Abstract

In response to limiting nutrient sources and cell density signals, Bacillus subtilis can differentiate and form highly resistant endospores. Initiation of spore development is governed by the master regulator Spo0A, which is activated by phosphorylation via a multicomponent phosphorelay. Interestingly, only part of a clonal population will enter this developmental pathway, a phenomenon known as sporulation bistability or sporulation heterogeneity. How sporulation heterogeneity is established is largely unknown. To investigate the origins of sporulation heterogeneity, we constructed promoter-green fluorescent protein (GFP) fusions to the main phosphorelay genes and perturbed their expression levels. Using time-lapse fluorescence microscopy and flow cytometry, we showed that expression of the phosphorelay genes is distributed in a unimodal manner. However, single-cell trajectories revealed that phosphorelay gene expression is highly dynamic or "heterochronic" between individual cells and that stochasticity of phosphorelay gene transcription might be an important regulatory mechanism for sporulation heterogeneity. Furthermore, we showed that artificial induction or depletion of the phosphorelay phosphate flow results in loss of sporulation heterogeneity. Our data suggest that sporulation heterogeneity originates from highly dynamic and variable gene activity of the phosphorelay components, resulting in large cell-to-cell variability with regard to phosphate input into the system. These transcriptional and posttranslational differences in phosphorelay activity appear to be sufficient to generate a heterogeneous sporulation signal without the need of the positive-feedback loop established by the sigma factor SigH.

FIG. 1.

Main phosphotransfer routes within the Bacillus subtilis phosphorelay.

FIG. 2.

Heterogeneous expression of the main phosphorelay components and sigH. Snapshots of early, middle, and late microcolony growth phases from time-lapse experiments (see Movies S1 to S8 in the supplemental material) were selected to create histograms displaying the distribution of GFP expression during microcolony development. (A) Wild type (168 trp+, not carrying a GFP construct); (B) P_sigH-GFP (IDJ001); (C) P_kinA-GFP (IDJ002); (D) P_kinB-GFP (IDJ003); (E) P_spo0F-GFP (IDJ004); (F) P_spo0B-GFP (IDJ005); (G) P_spo0A-GFP (IDJ006); (H) P_spoIIA-GFP (IDJ007). The insets display normalized data (late time point) from cells of at least three colonies per strain (black bars). For all strains except the P_spoIIA-GFP mutant, a Gaussian-like curve could be fitted (white line). The complete data set is shown in Fig. S1 in the supplemental material.

FIG. 3.

Dynamic expression of the main phosphorelay components and sigH. GFP expression from randomly selected cells was followed through time by time-lapse microscopy (see Movies S1 to S8 in the supplemental material), resulting in single-cell trajectories of separate lineages, each leading to a sporulating (red lines) and a nonsporulating cell (black lines). After cell division, indicated by an increase in symbol size on the trace, one of the two resulting siblings was arbitrarily selected for further analysis. The traces were stopped when a spore became visible in the corresponding movie. (A) Wild type (168 trp+, not carrying a GFP construct); (B) P_sigH-GFP (IDJ001); (C) P_kinA-GFP (IDJ002); (D) P_kinB-GFP (IDJ003); (E) P_spo0F-GFP (IDJ004); (F) P_spo0B-GFP (IDJ005); (G) P_spo0A-GFP (IDJ006); (H) P_spoIIA-GFP (IDJ007).

FIG. 4.

Sporulation of cells is prevented by elevated levels of RapA. (A) GFP (green circles) and mCherry (red squares) production by IDJ039 (harboring P_rapA-GFP and P_kinA-mCherry) was followed through time during microcolony development (see Movie S9 in the supplemental material). After each cell division, indicated by gray vertical lines, one of the two resulting siblings was arbitrarily selected for further analysis. The single-cell trajectories are from randomly selected sporulating (top panel) and nonsporulating (bottom panel) cells. The dotted vertical black line indicates that the spore became visible in the corresponding movie (top panel). (B) Movie S9 in the supplemental material (IDJ039) was used to create a histogram displaying the rapA promoter activity measured by GFP levels prior to spore formation (768 min of spore development). Sporulating cells (gray bars) show significantly (P < 0.01 by Student's t test) lower P_rapA-GFP levels than their nonsporulating siblings (black bars) analyzed at the same time in microcolony development.

FIG. 5.

The activity of the phosphorelay contributes to sporulation bistability. (A) Bright-field images of the wild-type strain and strains with mutations in KinA (IDJ035), KinB (IDJ036), or both kinases (IDJ037) after 24 h of microcolony development. Arrows point to spores in microcolonies of strains able to sporulate. (B) P_spoIIA-GFP activity of cells overproducing KinA (IDJ021) or KinB (IDJ022). Cells were grown in TY medium and induced with 100 μM IPTG in early exponential phase. At 5 h after induction, samples were analyzed by flow cytometry. (C) Spore development in microcolonies of strains with induced transcription of kinA (IDJ021) or kinB (IDJ022). Cells were grown as described in Materials and Methods and spotted on agarose-based chemically defined medium containing 100 μM IPTG.

FIG. 6.

Effects of KinA overproduction on phosphorelay promoter activity. Cells were grown in TY medium, and kinA expression was induced with 100 μM IPTG in early exponential phase. at 4 h after induction, the effect of kinA overexpression on the transcription of the phosphorelay promoters was analyzed by flow cytometry. (A) P_sigH-GFP (IDJ028); (B) P_kinA-GFP (IDJ029); (C) P_kinB-GFP (IDJ030); (D) P_spo0F-GFP (IDJ031); (E) P_spo0B-GFP (IDJ032); (F) P_spo0A-GFP (IDJ033).

FIG. 7.

spo0E overexpression negatively affects sporulation even in cells overproducing KinA. (A) P_spoIIA-GFP activity of cells overexpressing Spo0E or KinA. Strain IDJ027 (P_spank-spo0E, P_xyl-kinA, P_spoIIA-GFP) was grown in TY medium and induced at early exponential phase with 1 mM IPTG for Spo0E overproduction and/or with 0.5% xylose for KinA overproduction. A noninduced culture of the same strain and the wild type (no GFP expression) were used as controls. Samples were collected for flow cytometry at 4 h after induction. (B to E) Bright-field pictures of IDJ027 taken 24 h after microcolony development. Arrows point to a selection of nonsporulating cells. (B) No induction; (C) induction of spo0E expression with 1 mM IPTG; (D) induction of kinA expression with 0.5% xylose; (E) induction of spo0E expression with 1 mM IPTG and of kinA expression with 0.5% xylose.

FIG. 8.

Positive feedback by SigH is essential for spore formation. (A) Snapshots of wild-type (168 trp+), ΔsigH (IDJ020), and P_spank-kinA ΔsigH (IDJ037) microcolonies after 24 h. The agarose-containing medium was supplemented with 100 μM IPTG to overproduce KinA. This analysis shows that SigH is required for spore development (bright-field pictures, left panels) but not for bimodal expression of P_spoIIA-GFP (GFP pictures, right panels). (B) The temporal order of activation of SigH contributes to efficient initiation of sporulation. IDJ037 harboring the P_spoIIA-GFP reporter was grown in TY medium and induced with 1 mM IPTG for SigH overproduction and/or with 0.5% xylose for KinA overproduction at early exponential phase. Samples for flow cytometry were taken 4 h after the initial induction.

FIG. 9.

Simplified transcriptional network of the Bacillus subtilis phosphorelay. Arrows and perpendiculars indicate positive and negative activities, respectively. Spo0A∼P represses transcription of abrB, which is a repressor of sigH transcription. For simplicity, this indirect positive action of Spo0A on sigH is drawn as a direct action (dotted line). The same applies for the indirect positive regulation of kinB by Spo0A∼P.