Broadly heterogeneous activation of the master regulator for sporulation in Bacillus subtilis

Abstract

A model system for investigating how developmental regulatory networks determine cell fate is spore formation in Bacillus subtilis. The master regulator for sporulation is Spo0A, which is activated by phosphorylation via a phosphorelay that is subject to three positive feedback loops. The ultimate decision to sporulate is, however, stochastic in that only a portion of the population sporulates even under optimal conditions. It was previously assumed that activation of Spo0A and hence entry into sporulation is subject to a bistable switch mediated by one or more feedback loops. Here we reinvestigate the basis for bimodality in sporulation. We show that none of the feedback loops is rate limiting for the synthesis and phosphorylation of Spo0A. Instead, the loops ensure a just-in-time supply of relay components for rising levels of phosphorylated Spo0A, with phosphate flux through the relay being limiting for Spo0A activation and sporulation. In addition, genes under Spo0A control did not exhibit a bimodal pattern of expression as expected for a bistable switch. In contrast, we observed a highly heterogeneous pattern of Spo0A activation that increased in a nonlinear manner with time. We present a computational model for the nonlinear increase and propose that the phosphorelay is a noise generator and that only cells that attain a threshold level of phosphorylated Spo0A sporulate.A challenge in developmental biology is to understand how cells in an apparently homogeneous population adopt different fates. An attractive organism in which to address this challenge is Bacillus subtilis, which can adopt a variety of alternative fates depending on growth conditions (1-3). In some cases, cell population heterogeneity is generated stochastically. That is, fluctuations in gene expression due to noise can be amplified by feedback loops to lock cells in alternative stable states, resulting in a bimodal distribution of cell types. This is exemplified by genetic competence in which a positive feedback loop acting as a bistable switch creates such a distribution (2, 4). We use bimodal to mean systems that exhibit two discrete states and bistable to specify a class of bimodal systems in which nonlinear reinforcement stabilizes the alternative states. Here we are concerned with bimodality in the capacity of B. subtilis to sporulate.The master regulator for entry into sporulation, Spo0A (0A), accumulates gradually over the first 90 min of sporulation (5) (Fig. S1.) and is only active in its phosphorylated form (0A∼P) (6). Some genes under its control, such as those involved in biofilm formation and cannibalism, have strong binding sites for 0A∼P and are switched ON at low levels of 0A∼P. Other genes, such as those for spore formation, have weak binding sites and are only activated when 0A∼P accumulates to high levels (5, 7). The accumulation of 0A∼P is governed by a regulatory network built around a four-component cascade in which the relay protein Spo0F (0F) is phosphorylated by KinA and other kinases (6,8). 0F∼P, in turn, transfer...

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).…”