Noise in a phosphorelay drives stochastic entry into sporulation in Bacillus subtilis

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1 Article Noise in a phosphorelay drives stochastic entry into sporulation in Bacillus subtilis Jonathan R Russell 1, Matthew T Cabeen 1, Paul A Wiggins 2, Johan Paulsson 3 & Richard Losick 1,* Abstract Entry into sporulation in Bacillus subtilis is governed by a phosphorelay in which phosphoryl groups from a histidine kinase are successively transferred via relay proteins to the response regulator Spo0A. Spo0A~P, in turn, sets in motion events that lead to asymmetric division and activation of the cell-specific transcription factor r F, a hallmark for entry into sporulation. Here, we have used a microfluidics-based platform to investigate the activation of Spo0A and r F in individual cells held under constant, sporulationinducing conditions. The principal conclusions were that: (i) activation of r F occurs with an approximately constant probability after adaptation to conditions of nutrient limitation; (ii) activation of r F is tightly correlated with, and preceded by, Spo0A~P reaching a high threshold level; (iii) activation of Spo0A takes place abruptly just prior to asymmetric division; and (iv) the primary source of noise in the activation of Spo0A is the phosphorelay. We propose that cells exhibit a constant probability of attaining a high threshold level of Spo0A~P due to fluctuations in the flux of phosphoryl groups through the phosphorelay. Keywords cell fate; constant probability; phosphorelay; sporulation Subject Categories Microbiology, Virology & Host Pathogen Interaction; Signal Transduction DOI /embj Received 23 March 2017 Revised 17 July 2017 Accepted 1 August 2017 Published online 24 August 2017 The EMBO Journal (2017) 36: Introduction The process of spore formation in Bacillus subtilis is an attractive model system for investigating how genetic regulatory logic gives rise to a complex program of development. In response to nutrient limitation, cells of B. subtilis enter the pathway to form a spore, a multihour morphogenetic process in which a growing cell is converted into a dormant cell type (Stragier & Losick, 1996; Setlow, 2005). A hallmark of entry into sporulation is a switch from binary fission, in which a medially positioned septum divides the cell into two identical daughter cells, to a process of asymmetric division in which the division septum is positioned near one pole of the cell. Formation of an asymmetrically positioned septum divides the developing cell (sporangium) into a small forespore compartment, which will become the spore, and a large mother cell, which nurtures the developing spore (Stragier & Losick, 1996). Entry into the sporulation pathway is governed by the master regulator Spo0A, a member of the response regulator family of transcription factors, which is activated by phosphorylation on an aspartyl residue (Hoch, 1993). Spo0A~P controls the expression of genes and operons for multiple processes, including cannibalism and biofilm formation in addition to sporulation. However, entry into sporulation specifically requires that Spo0A~P reach a high threshold level (Fujita & Losick, 2005). High levels of Spo0A~P turn on and stimulate the expression of genes (spoiie and ftsz, respectively) that switch the position of cytokinesis to a site near a cell pole and activate genes (once again spoiie and the spoiiabc operon) that set in motion events that lead to the activation in the forespore of the sporulation-specific RNA polymerase sigma factor r F (Piggot & Losick, 2002; Molle et al, 2003; Carniol et al, 2004; Fujita et al, 2005). The r F factor, in turn, triggers a cascade of cell-specific sigma factors in the forespore and the mother cell (Carniol et al, 2004). Here, we focus on the activation of Spo0A and its behavior when cells initiate sporulation. Spo0A is phosphorylated via a multicomponent phosphorelay (Fig 1A) in which the histidine kinase KinA, responding to physiological signals from nutrient limitation, phosphorylates the relay protein Spo0F on an aspartyl group (Burbulys et al, 1991). Spo0F~P in turn transfers its phosphoryl group to a histidine residue on the next protein in the relay, Spo0B (Tzeng et al, 1998). Finally, Spo0B~P transfers its phosphoryl group to Spo0A. Much of our understanding of how cells enter the pathway to sporulate has been elucidated by analyzing bulk populations of cells, resulting in population averages. Recent studies have attempted to focus on the behavior of individual cells through the use of agarose pads (Veening et al, 2009; Eswaramoorthy et al, 2010; de Jong et al, 2010; Levine et al, 2012). The use of this technique has given rise to the idea that Spo0A is activated progressively in pulses over the course of several rounds of symmetric division before reaching a high enough level to trigger asymmetric division and sporulation. Indeed, it has been posited that this pulsatile behavior is a mechanism for cell-autonomous deferral of the commitment to sporulation (Levine et al, 2012). Subsequently, the observation of pulsed activation of Spo0A was attributed to a 1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA 2 Departments of Physics, Bioengineering and Microbiology, University of Washington, Seattle, WA, USA 3 Department of Systems Biology, Harvard Medical School, Boston, MA, USA *Corresponding author. Tel: ; losick@mcb.harvard.edu 2856 The EMBO Journal Vol 36 No ª 2017 The Authors

2 Jonathan R Russell et al Stochastic entry into sporulation The EMBO Journal A B C D stoichiometric gene dosage imbalance resulting from growth slowdown (Narula et al, 2016). However, all of these studies relied on observing cells on agarose pads, where cells may experience heterogeneity in their local environments as they deplete nutrients, grow to increasing density and secrete various factors. Thus, it is difficult to draw conclusions about the behavior of individual cells on pads independent of the influence of ever-changing microenvironments. E Figure 1. Tracking early transcriptional activation events in individual sporulating cells by fluorescence time-lapse microscopy using a microfluidic platform. A A schematic of the phosphorelay governing entry into sporulation. An autophosphorylating sensor kinase feeds single phosphoryl groups through a series of phosphotransfer reactions constituting the phosphorelay, ultimately leading to the phosphorylation of Spo0A (0A~P). Phosphate is drained from the relay by the action of the phosphatase Spo0E (0E) acting on Spo0A~P. B The cartoon depicts the construction and geometry of the microfluidic device used to image single-cell lineages over time. Small, 1-lm-wide growth channels are arrayed off a main feeding channel, and growth medium bathes cells via a 5-lm-wide shallow overlay. Growth channels are loaded with a culture of isogenic cells, which grow and divide, pushing daughter cells out into the waste stream. After a period of steady-state growth, cell behaviors are visualized after a switch to starvation medium. All of the cell lineages in a field of view were used for analysis; lineages that exited the field of view due to growth or cell lysis were truncated at the point of loss, and only lineages that were trackable past a threshold time (200 min) were used for analysis. C A representative kymograph of results for reporters of low and high levels of Spo0A~P and of r F in cells of strain JRR368 grown in the microfluidic device. A kymograph shows images of a single cell taken every 30 min after a switch to starvation conditions. The cell divides symmetrically multiple times before activating low levels of Spo0A~P (green), high levels of Spo0A~P (red), and r F (cyan), and ultimately forming a phase-bright spore (white; Phase). Time-lapse sequences of JRR368 sporulating in the microfluidic device are also shown in Movies EV1 and EV2. D Cartoon depicting the pattern, localization, and colors of the three fluorescent reporters shown in panel (C). E Single-cell lineages were constructed using a combination of SuperSegger (Stylianidou et al, 2016) and custom MATLAB scripts. An individual cell s response to a switch to sporulation conditions is plotted for each reporter channel: P low_0a~p (P sdp -mturquoise2, green curve), P high_0a~p (P spoiig - mneongreen, red curve), and P rf (P spoiiq -mneptune, cyan curve). After a delay following the switch, activation of a low-threshold Spo0A~P promoter (P sdp ) precedes the activation of a high-threshold Spo0A~P promoter (P spoiig ), which in turn precedes the activation of a r F -directed promoter (P spoiiq ). Based upon a method developed by Jun and colleagues (Wang et al, 2010), our laboratories previously used a microfluidic platform to investigate a stochastic process of cell fate decision-making in B. subtilis in which cells switch between motile and chained states (Norman et al, 2013). The ability to maintain cells under constant environmental conditions for long periods of time enabled us to show that the switch from the motile to the chaining state occurs in a memoryless manner, in which times spent in the motile state exhibit an exponential distribution. In contrast, the chaining state is timed rather than being memoryless, with cells remaining as chains for a stereotyped time period before switching back to the motile state (Norman et al, 2013). We used the microfluidic platform to investigate sporulation in a way that would enable us to visualize the behavior of individual cells for long periods of time under uniform and constant conditions after a switch to sporulation-inducing medium. Here, we report that after a period of adaptation to sporulation-inducing conditions, cells exhibit an approximately constant probability of activating Spo0A to a high threshold level, consistent with a process that is largely memoryless. We also report that Spo0A is rarely if ever activated in a progressive, pulsatile manner over multiple rounds of symmetric cell division. Rather, Spo0A is principally activated abruptly, just prior to the switch to asymmetric division. Finally, we present evidence indicating that the basis for ª 2017 The Authors The EMBO Journal Vol 36 No

3 The EMBO Journal Stochastic entry into sporulation Jonathan R Russell et al stochasticity in the activation of Spo0A is largely, if not entirely, noise in the phosphorelay. Results Visualizing r F -directed gene expression using a microfluidic platform We used r F -directed gene expression as an early, diagnostic marker for cells that had entered the pathway to form a spore, as r F activation is under the direct control of Spo0A~P, the master regulator for entry into sporulation. Synthesis of a fluorescent reporter produced under the control of r F [from the spoiiq promoter (P rf )] was visualized in individual cells using a microfluidic platform that enabled us to hold cells under constant conditions following a uniform switch to sporulation-inducing medium (Fig 1B). Cells were observed using time-lapse multi-channel fluorescence microscopy (Fig 1C and D), and gene expression for single-cell lineages was quantified using a custom MATLAB pipeline. The time-lapse results in Fig 1C and E show an example of a single cell in which r F was activated in the forespore compartment of the developing sporangium at about 4 h after the introduction of sporulation-inducing medium and about 2.5 h prior to the appearance of a phase-bright prespore, as visualized by phase-contrast microscopy. Next, we used the platform to visualize and analyze r F activation in multiple cells after the switch to sporulation medium (Fig 2C). The results showed striking heterogeneity in the time of r F activation, even though the cells were held under constant conditions after the switch. We also observed broad heterogeneity both in the number of vegetative cell divisions between the medium switch and r F activation and in the lengths of the cell cycles immediately preceding r F activation (Appendix Figs S1 and S2). These data imply that, under uniform sporulation-inducing conditions, there is no A D B C E Figure 2. The timing of r F and Spo0A~P activation is heterogeneous but shows a constant probability after a switch to constant sporulation-inducing conditions. A C The profiles of fluorescent intensities for P low_0a~p (A) P high_0a~p (B) and P rf (C) reporters are plotted for individual cell lineages of a strain (JRR368) bearing all three fluorescent reporters. Lineage behaviors are shown from 5 h before a switch into sporulation medium until approximately 15 h after the switch. For simplicity of visualization, the reporter data are smoothed by a box filter with a size corresponding to 30 min. In cases where activation occurs, lineages are plotted from the beginning of the experiment until the frame 1 h after the maximal slope of their activation. Fluorescence values are corrected for background fluorescence during the course of the experiment. D After a delay, beginning when cells experienced the switch to sporulation medium, growing cells achieved a stable probability that a division would result in activation of r F. The probability that a division at time (t) resulted in asymmetric division and r F activation was calculated by dividing the number of r F activation events by the total number of divisions, both asymmetric and symmetric. E During the period that cells exhibited a constant probability of sporulation (hours 9 16), the distribution of waiting times prior to r F activation fit well to an exponential distribution (red curve), a characteristic of a memoryless process. The single-exponential fit was calculated from a distribution of 80 events using the fit function in MATLAB. Events are plotted in bins of width corresponding to 50 min. This fit was reproduced qualitatively in an analysis of mother cell (MC) lineages where 38 events were fit to a similar exponential distribution (inset) The EMBO Journal Vol 36 No ª 2017 The Authors

4 Jonathan R Russell et al Stochastic entry into sporulation The EMBO Journal stereotyped time, number of cell cycles, or cell-cycle length associated with r F activation. We then calculated the probability over time that a cell divides asymmetrically (creating a forespore) and activates r F (Fig 2D). We found that cells held in constant conditions of sporulation-inducing medium responded with a stable probability of asymmetric division and activation of r F. Cells experienced a delay after the medium switch until the first r F activation event. We therefore focused on the period when the probability of sporulation had stabilized to calculate the distribution of waiting times prior to r F activation (Fig 2E). The bulk of observed r F activation events (80 out of 131 events) fit well to an exponential distribution in time. We conclude that once cells enter a physiological state in which they can activate r F, they do not do so synchronously. Rather, they exhibit a moreor-less constant probability of entering the pathway to sporulate. While more complex mechanisms may be at play, the observed fit to a simple exponential distribution suggests a memoryless process in the decision to sporulate. Spo0A is activated heterogeneously after the switch to sporulation-inducing medium To uncover the source of this cell-to-cell variation, we monitored the activation of Spo0A. The response regulator is known to control many genes, including genes and operons for asymmetric division (such as spoiie and ftsz) and r F activation (spoiia, spoiie and spoiig), as well as numerous genes that are not required for sporulation (such as genes for cannibalism and biofilm formation; Molle et al, 2003). Genes not involved in sporulation typically require only low levels of Spo0A~P for their activation or repression (lowthreshold genes), whereas genes for entry into sporulation require high levels of the phosphoregulator (high-threshold genes; Fujita et al, 2005). Accordingly, we used fluorescent reporter proteins produced under the control of a representative low-threshold promoter, P sdp (henceforth P low_0a~p ), and a representative highthreshold promoter, P spoiig (P high_0a~p ). Using the triple-labeled strain with reporters for P low_0a~p, P high_0a~p, and P rf described above (Fig 1), enabled us to visualize simultaneously the activation of all three promoters in single-cell lineages over time (Fig 2A C). As in the case of transcription from P rf, we saw striking heterogeneity in the activation of the low- and high-threshold Spo0A~P reporters over time after the switch to sporulation-inducing medium (Fig 2A and B). Activation of both the low- and high-threshold reporters was completely dependent on Spo0A, as judged by the use of a spo0a deletion mutant (Movie EV3). High levels of Spo0A~P predict r F activation with stereotyped timing We used MATLAB scripts to classify the types of behaviors we observed among cells responding to the switch to starvation medium. Consistent with previous observations, not all cells activated r F within the duration of observation. We sorted cell lineages into two classes: those that activated r F (Fig 3A C) and those that did not (Fig 3D F). Cells activating the low-threshold reporter (P low_0a~p ) were broadly distributed across both classes (Fig 3A and D), whereas cells that expressed the high-threshold reporter (P high_0a~p ) were restricted to the class that subsequently activated r F (Fig 3B and E). Indeed, activation of the high-threshold reporter invariably preceded and correlated with activation of r F. We aligned these cell lineages in time by their activation of r F (defined as 5r above background variation; Fig 3G). The activation of P high_0a~p preceded the activation of r F with a mean time of 1.12 h and a CV of 0.56 (Fig 3H). Activation of the low-threshold P low_0a~p showed little correlation with r F activation, exhibiting a mean time delay of 3.03 h and a CV of 0.75 (Fig 3I). We conclude that activation of r F is a deterministic consequence of Spo0A~P reaching a high threshold level and that the basis for heterogeneity in the activation of the forespore sigma factor lies in the activation of Spo0A or in events upstream of it. Activation of Spo0A does not increase progressively with cycles of cell division A widely held model for the activation of Spo0A posits that the levels of the phosphoprotein rise in progressively higher pulses in conjunction with, and coupled to, successive rounds of symmetric cell division that precede asymmetric division and entry into sporulation (Veening et al, 2009; Levine et al, 2012; Narula et al, 2016). To test this model in the microfluidic device, we analyzed strains bearing reporters for promoters with various sensitivities to levels of Spo0A~P (P sdp,p spo0f,p spo0a,p spo0ado1 O3,P sini,p spoiiaa,p spoiie, and P spoiig ; Fujita et al, 2005; Veening et al, 2009; Chastanet & Losick, 2011; Levine et al, 2012; Narula et al, 2016). By analyzing the relative timing of promoter activation and the proportion of activating cells in a population, we found that P sdp was the most sensitive to Spo0A~P levels, whereas P spo0f and P spo0a responded in time similarly to the high-threshold promoters P spoiiaa and P spoiig (Movie EV4). The behavior of cells plotted until their first activation of P sdp is shown in Fig 2A. The results show that the majority of cell lineages activate rapidly in response to a switch to starvation medium. We constructed cell lineages in an effort to detect cell cycledependent pulsing in Spo0A~P activation in a P sdp reporter strain that also harbored a reporter for r F activation. We restricted our analysis to lineages that culminated in entry into sporulation and aligned the lineages relative to the timing of r F activation. Amidst broad heterogeneity in the response of cells, we found that cells in the microfluidic device did not exhibit a consistent, detectable behavior resembling pulsed activation of Spo0A~P, a result contrary to what had been previously reported. Example lineages of cells exhibiting Spo0A~P-directed gene expression and their respective cell divisions are shown in Fig 4A. Whereas very few cells did accumulate Spo0A~P over multiple cell cycles (Fig 4A, bottom panel), most others did not. In most cases, the predominant increase in Spo0A~P-directed reporter expression was seen during or just prior to the final, asymmetric division. To control for the possibility that our results were due to an idiosyncratic feature of the P sdp promoter (even though we found it to be the most sensitive reporter for detecting Spo0A~P activity), we repeated the experiment using P spo0a, which had been used in prior work that reported pulsing of Spo0A~P levels (Levine et al, 2012). Again, we found no consistent evidence for pulses tied to cell cycles across multiple cell divisions (Movies EV1 and EV4). To quantify these behaviors across many cells, we aligned and time-normalized lineages of r F -activating cells in the generation before r F activation (G -1 ) and two generations before (G -2 ). If cells ª 2017 The Authors The EMBO Journal Vol 36 No

5 The EMBO Journal Stochastic entry into sporulation Jonathan R Russell et al G A D B E H C F I Figure 3. Cell lineage sorting by r F activation reveals two classes of behavior, with stereotyped timing between high-threshold Spo0A activation and r F activation. A F Lineage data from a triple-labeled strain (JRR368) was sorted into two classes: those that activated r F (A C) and those that did not (D F). While low-threshold activation of Spo0A was broadly distributed between these two classes (A and D), high-threshold activation occurred only in cells that went on to activate r F (B and E). Activation of r F was defined as the signal crossing a threshold 5r above the background variation prior to the switch. Lineages were plotted from 5 h before a switch to starvation medium until 15 h after the switch. For simplicity of visualization, reporter data are smoothed by a box filter with a size corresponding to 30 min. In cases where activation occurred, lineages were plotted from the beginning of the experiment until the frame 1 h after the maximal slope of their activation. G I Cell lineages (JRR368) were aligned in time to the point at which they crossed the threshold for r F activation. The data corresponding to the fluorescent channels for each reporter were then plotted on the same time axis. (G) r F activation events were well-aligned in their profiles of activation. (H) Activation of a highthreshold reporter for Spo0A correlated with and preceded activation of r F by a mean time (l) of1.12 h (CV 0.53). (I) Activation of a low-threshold reporter was poorly correlated with but tended to precede activation of r F (l = 3.03 h, CV 0.75). Activation timing was calculated as when a reporter signal crossed a threshold 5r above the background variation prior to the switch. Mean activation timing (l) was calculated as an average of the time differences between activation events with a standard deviation of r. The coefficient of variation (CV) was calculated as CV = r/l. experienced pulses of higher levels of Spo0A~P prior to r F activation, we would have expected to see a trend toward a significant increase in Spo0A~P activity in cells from generation G -2 to G -1.As stated above, while we did occasionally see some behaviors that resembled pulses of Spo0A~P (3 cells), such examples were far outnumbered by those that did not pulse prior to asymmetric division (41 cells; Fig 4B). Neither the Spo0A~P level nor Spo0A~P activity (calculated as d(spo0a~p level)/dt) showed a significant pulsatile pattern in the generations preceding r F activation. We next constructed a strain harboring a Dspo0A mutation (JRR399) to investigate the relationship between Spo0A activation and a slowdown in cell growth rate, as reported by Narula et al (2016). Whereas wild-type cells displayed a heterogeneous growth-rate slowdown in response to a switch to sporulationinducing medium (corresponding to the onset of sporulation in a subset of cells), Dspo0A mutant cells continued to grow and divide in the sporulation medium without exhibiting significant slowdowns (heterogeneous or otherwise; Appendix Fig S3 and 2860 The EMBO Journal Vol 36 No ª 2017 The Authors

6 Jonathan R Russell et al Stochastic entry into sporulation The EMBO Journal A B Figure 4. Spo0A activity rarely exhibits cell cycle-dependent pulsing prior to activation of r F. A Profiles of low-threshold P low_0a~p (green) and P rf (cyan) fluorescence intensity are plotted for single-cell lineages (JRR424) after a medium switch. Vegetative cell divisions during growth are marked in time with dotted lines. There was no consistent evidence of pulsatile Spo0A activation preceding activation of r F. Some cells did exhibit behavior in which Spo0A activity increased between cell cycles (bottom plot), but this pattern was exceptional. Reporter data were smoothed with a box filter with a size corresponding to 30 min. B Cell cycles were extracted from lineages of cells that ultimately activated r F. The profile of low-threshold Spo0A reporter activity for the two cell cycles (G -2 and G -1 ) preceding the cycle in which r F becomes activated are plotted (top row). The lifetimes of each cell cycle (as defined by its preceding and subsequent divisions) are normalized to a unit vector length. The majority of cells did not exhibit progressively higher levels of Spo0A activity, even as measured by the most sensitive reporter we tested (P sdp ). The derivative of average reporter intensity with respect to time is a rough proxy for the concentration of Spo0A~P in a given cell. The derivative is plotted (bottom row) over the same normalized time for the cell cycles preceding r F activation. Potential pulses of Spo0A~P were detected in a few (3) cells in generation G -1, but the remaining cells observed (41) did not exhibit pulsing in the two cell cycles preceding r F activation. Data are presented as raw traces corresponding to individual cell lineages. Movie EV3). Further, when we examined the cell-cycle lengths immediately preceding r F activation and maximal Spo0A~P activity using both low- and high-threshold reporters, we found no substantial correlation between longer cell cycles (i.e., slowed growth) and greater Spo0A~P activity (Appendix Fig S4). We conclude that growth slowdown is not responsible for activating Spo0A; rather, and just the opposite, activation of the response regulator is itself responsible for causing the slowdown in the growth rate of wild-type cells. KinA is the principal kinase responsible for r F activation in cells held under constant conditions As a next step in investigating the basis for the heterogeneous activation of r F, we tested mutants of the histidine kinases KinA, KinB, and KinC to determine which one or ones contributed most importantly to entry into sporulation under the conditions of our microfluidic device. Cell lineages from these mutants were observed following a switch to starvation medium in the microfluidic device. ª 2017 The Authors The EMBO Journal Vol 36 No

7 The EMBO Journal Stochastic entry into sporulation Jonathan R Russell et al The most striking effect was seen for the kina mutation, for which high-threshold levels of Spo0A~P were rarely achieved, whereas the kinb and kinc mutations had relatively little effect (Appendix Fig S5). The efficiency of the mutants to support r F activation relative to wild-type cells was also determined using resuspension in batch culture. After 8 h, kina mutant cells showed little or no activation of r F, whereas kinb and kinc mutant cells exhibited activation efficiencies on the same order of magnitude as wild-type cells (Appendix Fig S5B). Thus, cells in the microfluidic device mimic the behavior of cells in batch culture in being principally, if not exclusively, dependent on KinA for entry into sporulation. The rate of KinA synthesis is low and narrowly distributed in response to starvation Given that KinA is the predominant kinase that feeds phosphoryl groups into the phosphorelay, we next asked whether heterogeneity could be attributed to transcriptional or translational noise in the process of KinA synthesis. We measured KinA levels in sporulating cells using a translational fusion of KinA to GFP that is known to be functional under both wild type and inducible control (Fujita & Losick, 2005). Under the control of the native kina promoter, KinA levels were low and relatively unchanging across the population of cells over time (Fig 5A). The maximal level of KinA as produced from its native promoter in a given cell lineage was also uncorrelated with the activation of r F (Fig 5B). We artificially induced KinA-GFP expression concomitant with the starvation switch to address the hypothesis that a given input (i.e., level of KinA-GFP) determines a specific output (i.e., level of Spo0A~P activation). Maximal KinA-GFP expression varied 10-fold over a narrow range of IPTG concentrations (Fig 5C). As in the case of the native kina promoter, synthesis of KinA-GFP from the IPTG-inducible P hyperspank promoter did not determine activation of r F, even at high levels of KinA-GFP induction (Fig 5D). We conclude that cell-to-cell variation A B C D Figure 5. KinA-GFP levels do not predict r F activation. A KinA-GFP levels produced under the control of the native P kina promoter were low and stable in time after a medium switch to starvation conditions. Highlighted (cyan) are cell lineages that ended in r F activation. Cell lineages were plotted for a strain (JRR425) harboring a construct for inducible synthesis of KinA-GFP (amye::p hyperspank -kina-gfp) in addition to a reporter for P rf (P spoiiq -mneptune). B The maximum value of KinA-GFP achieved in each intact cell lineage in (A) is plotted against the corresponding maximal value of the r F reporter reached in that cell lineage. Activation of r F (cyan) is defined as signal 5r above the background variation prior to the medium switch (dotted line). Data shown are from a single experiment with 121 intact lineages from the same strain as in (A). The result was reproduced qualitatively in at least three other experiments. C Under the control of an inducible P hyperspank promoter, KinA-GFP levels were tuned across a range of concentrations by the addition of various concentrations of IPTG (0 10 lm) concomitant with the switch to sporulation medium. KinA-GFP levels reached a maximum after induction before decreasing, presumably as some cells began to enter sporulation. D Despite variation in induction, KinA-GFP levels were uncorrelated to the activation of r F, although high levels of induction did result in predominantly r F -activating cell lineages (10 lm, dark green). Activation of r F is defined as signal 5r above the background variation prior to the medium switch (dotted line). Data shown are from single experiments for at least 100 intact lineages from each induction condition. The result was reproduced qualitatively in two separate experiments The EMBO Journal Vol 36 No ª 2017 The Authors

8 Jonathan R Russell et al Stochastic entry into sporulation The EMBO Journal in KinA protein levels does not explain the variation in the timing of entry into sporulation. The growth medium influences the level of KinA needed to activate Spo0A To investigate whether heterogeneous activation of r F could be attributed to cell-to-cell variation in the activation (rather than the level) of KinA, we repeated the experiment of Fig 5 but this time compared the profiles of cells that had been switched to sporulation medium with those of cells that had been maintained in rich growth medium. We reasoned that if heterogeneity could be attributed to cell-to-cell variations in cell physiology, then cells might exhibit a more uniform response in rich growth medium than in sporulation medium. In rich medium, higher levels of inducer (and hence of KinA-GFP protein) were necessary to trigger the activation of r F than in sporulation medium (Fig 6A and B). Nonetheless, activation of r F remained heterogeneous in rich medium over a wide range of inducer concentrations. The activity of KinA is subject to inhibition by Sda, a known inhibitor of KinA autophosphorylation (Rowland et al, 2004; Ruvolo et al, 2006). To investigate whether Sda has a role in generating heterogeneity, we tested the effects of an sda deletion. We found no measurable difference in the pattern of heterogeneity in the mutant relative to that of wild-type cells; that is, the mutant cells activated Spo0A~P and r F heterogeneously in time, in a manner indistinguishable from the wild type (Movie EV5). In sum, the simplest interpretation of these results and those of Figs 5 and 6 is that heterogeneity arises at a step downstream of the synthesis and activation of KinA. A B Bypassing the phosphorelay largely eliminates heterogeneity If, as the above results seem to suggest, heterogeneity arises upstream of Spo0A and downstream of KinA, then an appealing hypothesis is that the basis for heterogeneity is noise in the flux of phosphoryl groups through the phosphorelay. To investigate this hypothesis, we took advantage of a previously described mutant of Spo0A that can act as a direct substrate for KinA~P, bypassing the otherwise strict requirement for the transfer of phosphoryl groups to Spo0A via the relay components Spo0F and Spo0B. The Spo0A E14A mutant was originally identified as a suppressor of a spo0f mutation (sof-3, herein called spo0a E14A ) that was able to support spore formation in the absence of a functioning relay (Spiegelman et al, 1990; Quisel et al, 2001). Spo0A E14A was also shown to accept phosphate from overexpressed KinA (Quisel et al, 2001). Taking advantage of these earlier findings, we sought to determine the extent of heterogeneity in r F activation in a Spo0A E14A mutant containing or lacking an intact spo0f gene. In keeping with earlier work, we included in our constructs a deletion (Dspo0E) of the Spo0A~P phosphatase Spo0E, which enhanced the levels of Spo0A~P-directed gene expression when the phosphorelay was bypassed. Importantly, we verified that deletion of spo0e did not significantly change the timing of heterogeneous r F activation in the microfluidic device (Movie EV6). In a spo0a E14A (and Dspo0E)-bearing strain with an intact phosphorelay (spo0f + ), r F activation was heterogeneous over a wide range of inducer concentrations (Fig 7, top row). In striking contrast, however, in an otherwise identical strain that was mutant for the phosphorelay (spo0f), activation of r F was much more switch-like, resembling an all-or-nothing response (Fig 7, bottom row), although it required somewhat higher levels of KinA than when the relay was intact. Representative kymographs of these behaviors are shown in Fig 8. A more switch-like response by relay-bypassed cells was further supported by logistic regression analyses, which showed a much better fit (i.e., a sharper transition) for relay-bypassed cells than for relay-intact cells (Appendix Fig S6). This sharper transition persisted even at high levels of KinA-GFP induction (Appendix Fig S7). In toto, the results of Figs 7 and 8, and of Appendix Fig S6 together with those of Figs 5 and 6 indicate that heterogeneity in the activation of Spo0A and r F can be attributed in whole or in part to noise in the flux of phosphoryl groups through the phosphorelay. The sporulation septum is placed with similar probability at either pole Figure 6. Growth medium affects the level of KinA required to activate r F but not heterogeneity. A, B Expression of KinA-GFP was induced by the addition of IPTG to JRR425 cells bearing a construct for inducible synthesis of KinA-GFP (kina::p hyperspank -kina-gfp). Cells were either switched to sporulation medium (A, green) or maintained in growth medium (B, blue). Higher levels of KinA were needed to activate r F in cells that did not experience a concomitant medium switch. The maximal value of KinA-GFP achieved in each intact cell lineage is plotted against the corresponding maximal value of the r F reporter reached in that cell lineage. Data shown are from single experiments for at least 100 intact lineages from each induction condition. The result was reproduced qualitatively in two separate experiments. Finally, we sought to take advantage of the microfluidic platform to investigate the role of noise in the choice of the cell pole at which septation takes place after Spo0A is activated. This question has been addressed previously with the conclusion that the choice of pole (new versus old) is largely stochastic (Veening et al, 2008), but the primary images leading to that conclusion were not published, and the earlier work was not conducted with cells held under constant conditions. Specifically, we asked whether in wild-type cells the sporulation septum is preferentially placed at the new or old pole, as defined by the previous round of division in the parental cell. Kymographs constructed from time-lapse experiments revealed the placement of the forespore (as a proxy for the polar septum) relative to the old and new poles of the parental cells (Appendix Fig S8). Indeed, as had been previously reported, among 237 ª 2017 The Authors The EMBO Journal Vol 36 No

9 The EMBO Journal Stochastic entry into sporulation Jonathan R Russell et al A B C Figure 7. Bypass of the phosphorelay reduces heterogeneity in r F activation over a range of KinA levels. A C A strain with a point mutation in the spo0a gene (spo0a E14A, also known as sof-3) was assessed for its response to a range of induced KinA-GFP levels in the presence (JRR497) or absence of spo0f (JRR500). The maximal value of KinA-GFP achieved in each intact cell lineage is plotted against the corresponding maximal value of the r F reporter in that cell lineage. Over a range of KinA-GFP levels, activation of r F was more switch-like in the relay mutant (B) than in cells with an intact phosphorelay (A). Representative time-lapse sequences of KinA-GFP cells at different induction levels are shown in Movies EV7 EV9 (JRR497, relay-intact cells) and in Movies EV10 EV12 (JRR500, relay-bypassed cells). Activation of r F is defined as a signal 5r above the background variation prior to the medium switch (dotted line). The data shown are from a single experiment with at least 100 intact lineages per induction condition. The result was qualitatively reproduced in at least two experiments. The proportion of cells activating r F is plotted for cells expressing a range of KinA-GFP levels (C). Data are shown for cells with an intact relay (JRR497, black) and in the absence of an intact relay (JRR500, red). The transition between KinA-GFP levels yielding no activation and levels yielding uniform activation is sharper in the absence of an intact phosphorelay (red points). The proportion was calculated as a ratio of the number of cells activating r F to the total number of cells observed during the observation window. observed events, 108 sporangia exhibited a forespore at the old pole and 129 at the new pole, suggesting that pole choice is largely if not entirely stochastic (mean proportion of new-pole events, within a 95% confidence interval). Appendix Fig S8 also shows representative examples in which a forespore arose at a new pole, an old pole, or at both poles. We also investigated pole bias in cells that had been artificially induced to sporulate using the P hyperspank - kina construct. This analysis showed a small preference for the new pole: Among 306 observed events, 184 were at the new pole and 122 were at the old pole ( within a 95% confidence interval). We conclude that pole choice during asymmetric division and forespore formation is largely, if not entirely, stochastic The EMBO Journal Vol 36 No ª 2017 The Authors

10 Jonathan R Russell et al Stochastic entry into sporulation The EMBO Journal Figure 8. Kymographs showing the effect of phosphorelay bypass on the activation of r F. A strain with a point mutation in the spo0a gene (spo0a E14A, also known as sof-3) was visualized in the microfluidic device as it responded to a range of KinA-GFP levels in the presence (JRR497) or absence (JRR500) of spo0f. In the top row, heterogeneous behavior can be seen in spo0f + cells across a range of inducer (IPTG) concentrations. In the bottom row, Dspo0F cells failed to activate r F at low levels of inducer, but activated r F in almost all cells at high levels of inducer. Kymographs were constructed from images captured at 30-min intervals for 8 h after a medium switch. Images are a merge of CFP fluorescence (r F reporter) images and phase-contrast images. The figure is a representative example of lineages on which the results shown in Fig 7 are based. Discussion The use of a microfluidics-based platform has enabled us to investigate the activation of Spo0A and r F in individual cells after a switch to constant, tightly controlled, sporulation-inducing conditions. One of our principal findings is that entry into sporulation, as judged by the activation of r F, is heterogeneous in time and occurs with an approximately constant probability after a period of adaptation to the sporulation-inducing conditions. We also found that this heterogeneous behavior could be largely if not entirely attributed to the activation of Spo0A, which reached a high threshold level in a stochastic manner that preceded the activation of r F by just over an hour. A widely held view in the sporulation field is that Spo0A is activated in a pulsatile manner over the course of multiple rounds of symmetric division preceding the switch to asymmetric division. ª 2017 The Authors The EMBO Journal Vol 36 No

11 The EMBO Journal Stochastic entry into sporulation Jonathan R Russell et al Pulses of Spo0A~P were initially reported as a mechanism by which cells coordinate activation of the response regulator with cycles of DNA replication prior to entry into sporulation via the production of Sda, an inhibitor of KinA (Veening et al, 2009). Progressive pulsatile activation of Spo0A with increasing magnitude over multiple rounds of symmetric division was later reported by Levine et al (2012), who interpreted this behavior as a mechanism for deferring the commitment of cells to sporulation. Most recently, Narula et al (2016) have also reported pulsing; they proposed that it is caused by a slowdown in the growth rate of individual cells and a resulting transient gene dosage imbalance. However, all of these investigations were conducted using agarose pads, on which cells were likely experiencing ever-changing variations in their microenvironments due to varying proximities to other cells and changing physiology as cells grew into microcolonies and depleted local nutrients. We speculate that growth of the local cell population may also have resulted in the relatively stereotyped number of cell divisions before sporulation observed by Levine et al (2012). Because the population doubles at each generation, even a gradual effect of the population size could create relatively sharp apparent timing effects, illustrating the difficulty in interpreting results on a changing agarose pad. As a further example of the importance of holding cells under constant conditions, in other work we used our microfluidic platform to visualize the response of B. subtilis cells to energy stress (Cabeen et al, 2017). Unlike a previous investigation based on the use of agarose pads (Locke et al, 2011) that reported that cells respond by means of frequency modulation, we found that when cells are observed under the tightly controlled conditions of the microfluidic device, the response to energy stress is largely if not entirely amplitude modulated. Furthermore, with regard to the report of Veening et al (2009), we saw little or no effect of an sda mutation on the pattern of Spo0A activation in our microfluidic device. With regard to Narula et al (2016), cells that activate Spo0A to high levels do exhibit a slowdown in growth rate and do so heterogeneously, but the slowdown is dependent upon the presence of spo0a itself. That is, the slowdown is a consequence of entry into sporulation, not its cause. Finally, and most importantly, we observed few events that could be interpreted as progressive increases in Spo0A~P levels over successive rounds of symmetric division in cells held under constant conditions (Fig 4). Instead, Spo0A~P was principally seen to be activated over the course of a single round of cell division, often the same cycle in which cells divided asymmetrically and activated r F. We cannot exclude the possibility that under certain conditions and with certain strains, Spo0A appears to be activated in a pulsatile manner. If so, our microfluidic experiments enable us to conclude that this is not an intrinsic feature of the mechanism of Spo0A activation under sporulationinducing conditions. Our use of a microfluidic platform enables us to conclude that the activation of Spo0A is a noisy process once cells have acclimated to sporulation-inducing conditions. In particular, the ability of Spo0A~P to reach a high threshold level in cells held under constant conditions is stochastic, occurring with an approximately constant probability in time. What is the source of this stochasticity? Efforts to assign the origin of noise to the synthesis, accumulation or activity of KinA were unsuccessful. Instead, and in keeping with previous speculation (Chastanet et al, 2010; Kothamachu et al, 2013), our evidence points to the phosphorelay as the source of stochasticity, based on experiments in which we short-circuited the relay by using a Spo0A mutant (Spo0A E14A ) that is subject to direct phosphorylation by KinA. When KinA was restricted to phosphorylating Spo0A E14A directly (in a mutant lacking the relay protein Spo0F), activation was more switch-like over a range of KinA levels than when the kinase was also able to channel phosphoryl groups through the relay. As previously suggested, the four-layered His-Asp phosphorelay of B. subtilis could in principle generate heterogeneity in the flux of phosphoryl groups via the sum of forward and reverse reactions during phosphate transfer as well as by the hydrolysis of labile, aspartyl-phosphate linkages (as in Spo0F~P and Spo0A~P) both spontaneously and by the action of known phosphatases (Rap phosphatases and Spo0E; Kothamachu et al, 2013). It is also conceivable that noise arises from cell-to cell variation in the levels of the Spo0F and Spo0B relay proteins (de Jong et al, 2010), although previous work showed that heterogeneous activation of Spo0A is independent of Spo0F levels (Chastanet et al, 2010; Chastanet & Losick, 2011). We speculate that the structure of the phosphorelay is a conserved motif that cells use to inject noise into cellular decisionmaking. It has been noted that noise and phenotypic plasticity can be advantageous for a population of isogenic cells (Wolf et al, 2005). Such bet-hedging strategies can allow a population to adapt quickly to fluctuations in the environment. Whereas some measure of noise generation is advantageous, the level of noise must be tunable so that cells can compete in both favorable and unfavorable environments. The phosphorelay is appealing in this regard as its architecture suggests multiple steps that could be modified by evolution to adjust noise levels. The decision to metamorphose into an endospore is a major commitment of time and physiological resources. Committing heterogeneously to this cell fate can therefore be thought of as a bet-hedging strategy that prevents the entire population of cells from entering the pathway to produce an endospore should an adverse environment prove to be transient. We propose that, rather than serving as a delay mechanism or a signal-integration system, the KinA-Spo0F-Spo0B-Spo0A phosphorelay is best understood as a noise generator that prevents all cells in the population from committing to sporulation over a narrow window of time. As phosphorelays are found in many microbial systems, it is appealing to imagine that they serve a previously unappreciated, widespread role in noise generation. Potential examples include the aerobic respiration sensor ArcAB in Escherichia coli (Kato et al, 1997); the master regulator of virulence BvgS in Bordetella pertussis (Uhi & Miller, 1994); the osmoregulators Sln1, Ypd1, and Ssk1 in Saccharomyces cerevisiae (Posas et al, 1996); and the stalk biogenesis regulators ShkA, ShpA, and TacA in Caulobacter crescentus (Biondi et al, 2005). Notably, each of these relays is composed of multiple proteins or protein domains that shuttle a single phosphoryl group alternately between histidine and aspartate residues before a final transfer to a response regulator. While no experimental evidence has yet been published to implicate these phosphorelay structures in generating noise, we speculate that the phosphorelay could be a convergently evolved mechanism by which cells generate noise as a bet-hedging strategy The EMBO Journal Vol 36 No ª 2017 The Authors

12 Jonathan R Russell et al Stochastic entry into sporulation The EMBO Journal Materials and Methods Bacterial strains All strains were constructed in the wild-type B. subtilis NCIB3610 background using standard molecular biology techniques. The strains used in this study are listed in Table 1 and Appendix Table S1. Detailed methods for cloning and strain construction, including strain names and primers used (Appendix Table S2), are listed in the Appendix. All strains used in the device included a straight flagellar mutation (hag A233V ) to keep cells from swimming out of the channels (Norman et al, 2013). Microfluidic device construction The construction of the microfluidic device was conducted largely as described previously (Norman et al, 2013). PDMS casts of a silicon master were prepared prior to each experiment. PDMS devices with side-channel features of 1.0 lm width were plasma bonded to a mm glass coverslip that had been cleaned with isopropanol. Clean surfaces of featured PDMS and glass were treated with an oxygen plasma exposure of 15 s, 30 W, at 170 mtorr O 2. The devices were baked at 60 C for at least 1 h to ensure proper bonding. Devices were passivated with growth medium and used within 12 h of bonding to prevent channels from becoming overly hydrophobic. Medium and cell culture preparation Cells were grown in Spizizen s Minimal Medium (SMM; 0.5% glucose, 17 mm K 2 HPO 4, 8 mm KH 2 PO 4, 3 mm (NH 4 ) 2 SO 4, 1.2 mm tri-sodium citrate2h 2 O, 0.16 mm MgSO 4 7H 2 O, ph 7.0 adjusted with 10 N NaOH) until late stationary phase (OD ) before loading into the microfluidic device. Cell culture was first passed Table 1. Strains used in this study. Strain name Genotype Reference JRR hag A233V phleor amye::p spoiiq -mneptune Cm, ywrk::pspoiig-mneongreen Spc, ylnf::psdp-mturquoise2 Tet This study JRR399 JRR401 JRR424 JRR425 JRR497 JRR hag A233V phleor amye::p spoiiq -mneptune Cm, ywrk::pspoiig-mneongreen Spc spo0a::kan 3610 hag A233V phleor amye::p spoiiq -mneptune Cm, ywrk::pspoiig-mneongreen Spc ylnf::amye::psdp-mturquoise2 Tet, spo0a::kan 3610 hag A233V phleor amye::p spoiiq -mneptune Cm ylnf::psdp-mturquoise Tet 3610 hag A233V phleor kina::p Hyperspank -kina-gfp Kan Spc amye::p spoiiq -mneptune Cm ylnf::p sdp -mturquoise Tet 3610 hag A233V phleor spo0e::kan spo0a::spo0a(e14a) amye::p spoiiq -mneptune Cm kina::p Hyperspank -kina-gfp Kan Spc 3610 hag A233V phleor spo0e::kan spo0a::spo0a(e14a) amye::p spoiiq -mneptune Cm kina::p Hyperspank -kina-gfp Kan Spc spo0f::tet This study This study This study This study This study This study through a 5-lm filter (Pall Acrodisc ) to remove large chains so as to facilitate loading into side channels. Filtered cell culture was centrifuged at 4,000 g for 10 min and resuspended in 1 ml of remaining SMM. Devices were passivated with 1 SMM prior to loading. Resuspended cell culture was injected into the feeding channels in each lane of the device, and cells were spun into the side channels at 5,000 g for 10 min using a custom-machined platform (Norman et al, 2013). Syringes containing SMM (for the initial growth phase) or A+B sporulation medium (0.01% solution A [0.089% FeCl 3 6H 2 O, 0.83% MgCl26H 2 O, 1.979% MnCl 2 6H 2 O], 1% solution B [5.35% NH 4 Cl, 1.06% Na 2 SO 4, 0.68% KH 2 PO 4, 0.97% NH 4 NO 3 ], 40 mm MgSO 4, 0.2% L-glutamate, 1 mm CaCl 2 ) were loaded onto syringe pumps and attached to the microfluidic device using Tygon tubing. Binder clips were used on sections of flexible silicon tubing attached to polyethylene Y-junctions to achieve the medium switch. In cases where inducer was added to medium, 1 M IPTG (isopropyl b-d-1-thiogalactopyranoside) was diluted into the appropriate medium at the specified concentrations and similarly loaded into syringes. Cells were allowed to equilibrate and grow in the device for up to 3 h before the commencement of image acquisition. The medium flow rate was set to 1.5 ll/min for both growth and sporulation media. Time-lapse fluorescence imaging Devices were imaged using a Nikon Eclipse Ti inverted microscope equipped with an Orca R2 (Hamamatsu) camera, a 60 Plan Apo oil objective (NA 1.4, Nikon) an automated stage and a Lumencor SOLA fluorescent illumination system. The stage was housed within an environmental chamber maintained at 37 C. Positions within each lane of the device were selected and imaged using lmanager via a custom MATLAB image acquisition script (Edelstein et al, 2014). Images were acquired every 10 min for 2 3 h prior to a medium switch and for at least 12 h following the medium switch. Cells are particularly sensitive to phototoxicity during sporulation, so the LED intensity and exposure times were determined empirically such that sporulation efficiency was comparable between positions that were imaged and positions that were not (LED intensity set to 30% and exposure times of 100 ms for the CFP channel, 150 ms for the GFP channel, 150 ms for the RFP channel, and 150 ms for phase contrast). The following filter sets were used for acquisition: GFP (Semrock GFP-1828A), RFP (Semrock mcherry-b), and CFP (Semrock CFP-2432C). To achieve phase imaging in lmanager during time-lapse acquisition, we built a custom TLL switch using an Arduino board and basic electronic components. Focal drift was corrected for using a previously developed autofocus routine (Norman et al, 2013) that relies on the Nikon PerfectFocus system and phase images collected at a sacrificial position. Image analysis Images acquired from time-lapse experiments were analyzed by adapting a previously described set of scripts (Stylianidou et al, 2016). The software uses a watershed-based algorithm to segment and outline individual cells in each frame. A vertical mean correction is applied to all phase images to remove features associated with the microfluidic channels. Fluorescence data are extracted as ª 2017 The Authors The EMBO Journal Vol 36 No