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.When nutrient sources are dwindling, the Gram-positive bacterium Bacillus subtilis can utilize a number of adaptive phenotypes such as the secretion of proteases and the development of genetic competence (for a recent review, see reference 49). The most sophisticated survival strategy that B. subtilis employs is the formation of a highly resistant endospore (34). Initiation of spore formation is governed by a complex multicomponent phosphorelay (Fig. 1) including the primary kinases KinA and KinB and two intermediate phosphotransferases, Spo0B and Spo0F (33). The phosphorelay activates Spo0A, the master sporulation regulator of sporulation, by phosphorylation (4), upon which phosphorylated Spo0A (Spo0AϳP) can directly regulate more than 100 genes (29). This induces a chain of events that takes several hours to complete and involves the activation of a set of alternative sigma factors that give unidirectionality to the cascade, culminating in the formation of the endospore (10).Interestingly, within isogenic populations of B. subtilis grown under identical conditions, not all cells enter this timely and costly developmental pathway. This type of phenotypic bifurcation is known as sporulation bistability or sporulation heterogeneity (9, 38). How populations of genetically identical cells bifurcate into phenoty...

Ultrasensitivity of the Bacillus subtilis sporulation decision

“…Thus, our results agree with several recent reports that have shown cell fate heterogeneity during sporulation is not associated with bimodality of Spo0A∼P activity (16,17). Altogether, these results lead us to conclude that the phosphorelay response or Spo0A∼P concentration does not increase steeply around the KinA threshold and that the ultrasensitivity in the sporulation response must be associated with network components that reside downstream of the phosphorelay.…”

“…Furthermore, our results (Fig. 2D) regarding the unimodality of the expression of Spo0A∼P target genes are in agreement with those of previous reports that studied the phosphorelay response of WT cells in starvation conditions (16,17,21). Taking all these results into consideration, we expect that the ASI system can effectively mimic the natural sporulation network despite differences in KinA regulation and a lack of other environmental inputs.…”

“…We note that we have no direct control of the Spo0A activity in this setup, and therefore cannot probe the input-output response of the network directly at the population level. Nevertheless, because Spo0A activity is broadly heterogeneous during starvation (16,17,21,22), correlations between activities of Spo0A and sigma factor can be examined in single cells over a wide range of values. Therefore, to determine if activation of σ F and σ E is ultrasensitive to increases in Spo0A activity, we simultaneously measured activities of Spo0A and sigma factors during starvation.…”

“…This hypothesis is motivated by the observation that an increase in Spo0A∼P is sufficient to shift the site of cell division to an asymmetrical polar site, and thereby to initiate sporulation (19,20). However, the hypothesis of a single Spo0A∼P threshold has been undermined by recent studies showing that (i) starving populations of B. subtilis exhibit broad and unimodal distributions of Spo0A activity (16,17,21) and (ii) there is significant overlap in distributions of Spo0A activity in sporulating and nonsporulating cells (22). Moreover, cells that form an asymmetrical septum but do not activate σ F and/or σ E are able to resume vegetative growth (23)(24)(25).…”

“…Several previous studies have hypothesized that sporulation is triggered in cells reaching a threshold level of Spo0A∼P (14)(15)(16)(17)(18). This hypothesis is motivated by the observation that an increase in Spo0A∼P is sufficient to shift the site of cell division to an asymmetrical polar site, and thereby to initiate sporulation (19,20).…”