1 Dormancy as a spectrum measuring spore ' s proximity to death and to 1 replicative life 2 3 4

ABSTRACT How organisms with their lives ceased stay alive and what sets their lifespans are fundamental questions relevant for microbial spores. Starved microbes can form spores whose metabolism and gene-expressions are active for hours-to-days but nearly cease upon entering dormancy. Although dormant spores can wake-up (germinate) when nutrients reappear, they die - cannot germinate - after prolonged nutrient-absences. Previous studies identified several factors that affect spore revival. But how these and as-yet-unknown intracellular factors collectively encode a dormant spore’s lifespan remains poorly understood. Here we reveal an easy-to-measure, systems-level metric - a quantity that combines many intracellular factors - that accurately predicts dormant yeast-spores’ lifespans by establishing dormancy as a quantity that, without nutrients, decreases at a predictable rate, thereby revealing how dormant spores approach death. We discovered different glucose-concentrations germinating distinct percentages of yeast-spores, with low glucose-concentrations priming un-germinated spores to accelerate their germinations when more glucose appears hours-to-days later. Using a synthetic circuit, we quantified dormant spores’ gene-expressing ability without nutrients - a systems-level metric - whose value determines a minimum glucose-concentration required for guaranteeing germination and, for glucose concentrations below it, probability of germinating. Dormant spores’ gene-expressing ability predictably decreases over days-to-months, causing glucose-concentration required for germination to increase with predictable rates until going beyond a saturating value - spore’s moment of death. By introducing “dormancy spectrum” - a ruler that measures spore’s proximity to death (lifespan) and to replicative life (germination capacity) - and finding dormancy’s systems-level indicators, we unveiled hidden dynamics of dormant spores approaching death.


INTRODUCTION
6 dormancy as a continuous "ruler", rather than a single state, that simultaneously 7 one spore that replicates after glucose reappears. With a saturating glucose concentration (2%), 158 nearly every spore bag in the population germinated (Fig. 1c). But with lower glucose 159 concentrations (i.e., less than ~0.01%), a noticeable percentage of spore bags in the population 160 (i.e., ~10% or more) did not germinate regardless of how many hours we waited after adding the 161 glucose (Fig. 1c). The percentage of spore bags that germinated followed a sharp, step-like 162 (sigmoidal) function of the glucose concentration ( Fig. 1d -red points) with the step-up located at 163 a glucose-concentration of ~0.003% (i.e., at this concentration, ~50% of the spore bags 164 germinate). In contrast, the average time taken to germinate weakly depended on the glucose 165 concentration, increasing by at most 2-folds despite a 10,000-fold decrease in the glucose 166 concentration from 2% to 0.0002% (Fig. 1d -blue points), indicating that glucose weakly affects 167 the speed of germination. Importantly, the germinations did not stop because the spores ran out 168 of glucose for any of the glucose concentrations that we used because when we measured the 169 glucose concentrations in the media after no more germinations occurred (i.e., ~10 hours (~600 170 minutes) after adding glucose for all glucose concentrations), we found that there was always a 171 large fraction of the original glucose left in the media and, importantly, that the glucose 172 concentration hardly decreased for the very low glucose concentrations that we used (e.g., 173 0.002% and 0.001%) ( Supplementary Fig. 2). Moreover, we observed that the wild-type diploid, 174 vegetative cells -the same cells that formed the spores -could replicate multiple times even at 175 the lowest glucose concentration (i.e., 0.0002%) ( Supplementary Fig. 3), meaning that even the 176 lowest glucose concentration was ample enough for a cell to divide multiple times. These results 177 establish that yeast spores do not necessarily germinate in an environment with ample glucose.

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Spores that do not germinate after encountering ample glucose are not necessarily dead

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As an answer to why some spores do not germinate with ample glucose, we considered two 182 possibilities. One was that the spore bags that did not germinate (i.e., "un-germinated spore 183 bags") died while trying to germinate and thus will not germinate even after encountering more 184 glucose. The other possibility was that the un-germinated spore bags were still able to germinate 185 and thus not dead. To distinguish these two possibilities, we repeated the above experiments but 186 now by adding glucose in two steps (Fig. 2a). First, we gave a relatively low concentration of 187 glucose to the spores. We then waited, typically ~16 hours (~1000 minutes), by which point no 188 more germinations occurred. Then, we added more glucose to increase the total glucose 189 concentration and then observed if any more germinations subsequently occurred. We found that 190 some of the spore bags that did not germinate after receiving the first glucose germinated after 191 8 192 Figure 2 | Un-germinated spores, primed for days by a low glucose concentration, germinate faster upon encountering more glucose. a, Wild-type spores are first incubated in a low glucose-concentration before we add more glucose at a later time to increase the glucose concentration. b, Time taken by each spore bag to germinate for the experiment in (a). First glucose concentration is 0.0005% (from 0 to 16 hours) (green bars) and the final concentration is 0.002% (from 16 to 32 hours) (orange bars). n = 143 spore bags (representative data). c, For experiment in (a), percentage of spore bags that germinated as function of time after glucose addition in two steps (concentrations as indicated). Second glucose added at 1000 minutes (purple vertical line). n = 3; error bars are s.e.m. d, Average time taken for a spore bag to germinate in the experiment shown in (a), due to the second added glucose (denoted Δ in bottom panel of (c)). The final glucose concentration was varied and the final concentration was 2%. n = 3; error bars are s.e.m. 9 receiving more glucose . Yet, some of the spore bags still To better understand why only some spore bags germinated for a given glucose-concentration, 208 we examined whether the un-germinated spore bags had any measurable response to the 209 glucose that they encountered. When we added glucose in two steps so that the final 210 concentration was 2% (Fig. 2a), we found that the spore bags took less times to germinate to the 211 second batch of glucose than they would have if they had received the entire 2%-glucose all at 212 once without encountering a lesser amount of glucose first (Fig. 2d). Specifically, if a spore bag 213 was in a minimal medium without any glucose for 16 hours and then encountered a 2%-glucose,

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it needed an average of ~200 minutes to germinate. But this time decreased by about half (i.e., 215 to ~120 minutes) if a spore bag was first in a minimal medium with a low glucose concentration -216 ranging from 0.0002% to 0.002% -for 16 hours and then received more glucose so that the final 217 e, Spore bags that do not germinate after encountering the first glucose-concentration are "primed" to germinate faster upon encountering more glucose. f, Average time taken for a spore bag to germinate (Δ ) in the experiment shown in (a) after adding the second glucose (final glucose concentration is 2%) as a function of the first, low glucose concentration. Different colors represent different times at which the second glucose was added: 16 hours (yellow), 48 hours (blue) and 96 hours (red). "Relative Δ " is the average time Δ divided by the Δ for spore bags that were incubated in minimal media without any glucose (0%) for the same duration of time, before they received 2%-glucose. n = 3; error bars are s.e.m. g, Heat map showing transcriptome-wide changes in un-germinated, primed spores at 0, 16, 48, and 96 hours after being primed by a 0.002%-glucose (obtained with RNA-Seq). See Supplementary Table 1 for a list of genes for each transcriptional module (rows of heat map) and also see Supplementary Fig. 8. For each transcriptional module, we first divided the expression level of each gene in that module by its expression level at 0 hours -this yields "normalized expression level" for that gene for primed and unprimed spores (the latter were incubated in minimal media without glucose for 0, 16, 48, and 96 hours). We then averaged these values over all genes in a given transcriptional module, yielding one value of "normalized expression (primed)" and one value of "normalized expression (unprimed)" for each transcriptional module. Colors represent ratio of these two values, averaged over three biological replicates (n = 3). concentration was 2% (Fig. 2d). Thus, encountering a very low amount of glucose "primes" some 218 spores so that, upon encountering a saturating level of glucose later, they would germinate faster 219 -up to two times faster on average -compared to spores that did not previously encounter any 220 glucose (Fig. 2e). Furthermore, when we primed the spores with a very low glucose concentration 221 and then waited between 16 hours to 4 days before increasing the glucose concentration to 2%, 222 we still observed the sign of primed dormancy -a faster germination compared to the spores that 223 were kept in minimal media without any glucose for the same amount of time -up to two days but 224 not four days after the first glucose-addition ( Fig. 2f and Supplementary Fig. 6). Thus, primed 225 dormancy lasts for and decays over days. Before turning to the question of what causes only some spore bags to germinate for a given 230 glucose concentration, we sought gene-expression signatures of primed dormancy. To do so, we 231 first primed the spores by incubating them with a low glucose concentration (0.002%) for either 232 16 hours, 1 day, 2 days, or 4 days. We then used zymolyase, as is the standard (33), to isolate 233 the un-germinated spores from the surrounding vegetative cells (Supplementary Fig. 7) and 234 analyzed their transcriptomes with RNA-seq. As a control, we also analyzed the transcriptome of 235 un-primed spores, which were incubated in minimal media without glucose for the same amounts 236 of time as the primed spores. Following an insightful previous study (32) that analyzed the yeast 237 spores' transcriptome as they germinated after receiving a 2%-glucose over several hours, we 238 grouped multiple genes together into a set, called a "transcriptional module" (32,34), if those 239 genes are involved in the same process (e.g., protein synthesis) (Supplementary Table 1). We 240 averaged the expression levels of all genes in a given module to obtain one expression-level for 241 that module, for both the primed and un-primed spores. For six of nine transcriptional modules, 242 we found that the primed spores had higher expression levels than the un-primed spores after 16 243 hours and 48 hours of incubations whereas both types of spores had nearly the same expression 244 level after four days of incubation ( Fig. 2g -last six rows and Supplementary Fig. 8). This trend 245 mirrors the trend that we observed in the average time taken by primed spores to germinate (i.e., 246 accelerated germinations up to 48 hours after being primed but no accelerated germinations after 247 four days) (Fig. 2f). Two transcriptional modules showed this trend in a particularly pronounced 248 manner. One of them is the module for mating (35), which the resulting haploid cells carry out 249 after the germination (Fig. 2g -seventh row). The other is the module for transitioning from cell 250 cycle's G2-phase to mitosis (Fig. 2g -last row and Supplementary Fig. 8A), which is a crucial final 251 11 step of germination. These results make sense for accelerating germinations. Together, these 252 results establish that very low glucose concentrations can trigger transcriptome-wide changes in 253 un-germinated spores to accelerate their potential, future germinations up to days later.

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Hypothesis on why only some yeast spores germinate for low glucose-concentrations

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Although we now understand how un-germinated spores respond to glucose, we have not 258 addressed the question of what determines, in the first place, which spore bags germinate and 259 which do not. As an answer, we hypothesized that each diploid cell forms a spore bag with a 260 distinct "internal state". An internal state may be defined by a broad set of factors, including the 261 amounts of ATPs or amino acids or ribosomes that are stored inside the spore bag or 262 combinations of these or other stored molecules that serve as "starting materials" for entering 263 replicative life. We then hypothesized that, for each glucose concentration, only some of the spore 264 bags have the "right" internal states that allow for germination. Our experiments thus far involved 265 giving glucose to spores and then observing their subsequent actions, including for primed 266 dormancy. But in such experiments, due to all measurements occurring after the spores receive 267 glucose, we cannot infer the spores' internal states that existed before they encountered glucose 268 and thus we sought to manipulate the internal states without glucose. In particular, we reasoned 269 that depleting any internal resources (e.g., ATPs or amino acids) that are stored inside spores 270 before adding glucose would either decrease -or alter in more complex ways -the percentage of 271 spore bags that germinate for each glucose concentration.

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To test our hypothesis, we first built a synthetic gene-circuit in vegetative diploid yeast cells so 276 that doxycyline -an inducer molecule -would cause the cells to produce the Green Fluorescent 277 Protein (GFP). This synthetic circuit functioned in such a way that increasing the doxycyline 278 concentration increased the cell's GFP production. We formed spores out of these engineered 279 diploid cells (Fig. 3a). We reasoned that if doxycyline can induce GFP expression in these spores 280 without any nutrients (e.g., in plain water without amino acids and glucose), then we might deplete 281 their stored resources and thereby alter the percentage of spore bags that germinate for a given 282 glucose concentration. But it was unclear whether it was possible to induce the expression of GFP 283 or any arbitrary gene in dormant yeast-spores without nutrients in the first place. For one, if that 284 were possible, then it is unclear why, apparently, the expression of almost all the genes in dormant 12 Figure 3 | Synthetically inducing gene-expression in dormant yeast-spores without nutrients leads to germination landscape, which shows that ability to express a gene (e.g., GFP) without nutrients quantifies how likely a spore bag will germinate for each glucose concentration. a, A synthetic gene-circuit that constitutively expresses a transcription factor, rtTA (with ADH1-promoter) and an inducible promoter (TET-promoter) controlling GFP expression. Increasing doxycycline increases GFP production. b, Engineered spore bags (shown in (a)) transcribe and translate GFP in plain water with 25 µg/ml of doxycycline (top row) and in a saline solution (PBS) with 50 µg/ml of doxycycline (bottom row) without nutrients. Snapshots of GFP expression shown at 22 hours after adding doxycyline. c, GFP levels of individual spore bags (grey curves) over time (measured every 10 minutes with a wide-field epifluorescence microscope) after incubation in PBS with 10 µg/ml of doxycycline (top panel: n = 104 spore bags) and 100 µg/ml of doxycycline (bottom panel: n = 150 spore bags). d, Engineered spore bags (shown in (a)) were first incubated for 22 hours in either PBS without any doxycyline or with 100 µg/ml of doxycycline before they were transferred to minimal media with various glucose concentrations. Plot shows the total percentage of the engineered spore bags that germinated (measured 20 hours after incubating with glucose) for those pre-incubated in PBS without doxycycline (black points) and in PBS with 100 µg/ml of doxycycline (orange points). n = 3; error bars are s.e.m.

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A previous, bulk-level (population-level) study (12) has shown that there are transient, constitutive 295 expressions of two genes -PGK1 (involved in gluconeogenesis) and SPS100 (involved in forming 296 spore walls during spore formation) -that turn off a few days after spore formation (i.e., during 297 entrance into dormancy). But it remains unclear whether their expressions completely turn off or 298 just decrease to vanishingly low, but non-zero levels a few days after spore formation. This is 299 because these bulk-level measurements were based on finding ribosomes bound to mRNAs in 300 lysates of populations of yeast-spores, which does not have the necessary sensitivity. Moreover, 301 the ribosomes bound to mRNAs may be from the macroscopic aggregates that formed before the 302 spores entered dormancy (14,15), which may disable translation of those ribosome-bound 303 mRNAs (16-21). Adding to the ambiguity is the fact that a bulk-level study revealed a depletion of 304 a minute fraction of radioactive uracil and methionine from an extracellular medium by a dense 305 population of yeast spores (12). This finding indicates that transcription (proxied by the depleted 306 uracil) and translation (proxied by the depleted methionine) may be possible in dormant yeast-307 spores, though these are indirect measurements since they did not directly visualize gene-308 expression dynamics inside spores. Imaging gene-induction dynamics in individual yeast-spores 309 would provide a definitive answer that resolves these ambiguities. To that end and to test if GFP-310 induction was possible for testing our hypothesis for why only some spores germinate for a given 311 glucose concentration, we incubated the engineered spores in either water or a saline solution 312 (PBS) with only doxycyline. Surprisingly, we discovered that doxycycline fully induced 313 e, Top: Percentage of spore bags with the same GFP-level (in experiment in (d)) that germinated after receiving a 0.001%-glucose. Percentages are averaged over all spore bags with the same binned GFP-level (corresponding histogram shown below). Data from a representative population of spore bags (n = 145 spore bags in a population). f, Germination landscape: Colors represent the probability that a spore bag with a particular steady-state GFP-level germinates for each glucose concentration (i.e., data in top panel of (e) represents a single row of this heat map). To measure each pixel, as in the experiment described in (d), we incubated spore bags in PBS with 100 µg/ml of doxycycline for 22 hours before giving glucose concentrations indicated along the rows. Columns indicate steady-state GFP-level of a spore bag at 22 hours after adding the doxycyline. Each pixel is an average over 3 replicate populations (n = 3). g, Given a spore bag, its steady-state GFP-level is a read-out of both its intrinsic ability to express a gene without any nutrients and the minimal glucose concentration that it needs for germination. Spore bags with lesser geneexpressing abilities without nutrients require more glucose to germinate.
14 transcription and translation of GFP in these spores .

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Crucially, varying the doxycycline concentration in water and PBS tuned the spores' GFP levels 315 over a similarly wide-range of values as in the vegetative cells with the same synthetic circuit 316 . We found that both the rate of GFP-production and the final (steady-317 state) level of GFP widely varied among spore bags in the same population ( Fig. 3c and 318 Supplementary Fig. 12). The most striking feature, however, was that all spore bags expressed 319 GFP very slowly -GFP levels plateaued at steady-state values only after ~20 hours of doxycyline 320 induction whereas they plateaued after ~8 hours in vegetative cells with the same circuit 321 ( Supplementary Fig. 11). But a more puzzling discovery was that the spores' GFP-levels stabilized 322 at steady-state values in the first place. After all, the spores were not dividing and hence their 323 GFP -a highly stable protein -could not be diluted away by cell divisions. In replicating yeasts,

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highly stable proteins such as GFP reach steady-state levels because their production rate

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To test the hypothesis that depleting the spores' internal resources by expressing GFP without 338 nutrients would hinder spore bags' ability to germinate, we incubated the spores in PBS with a 339 high doxycycline concentration (100 µg/ml) for 24 hours so that their GFP levels would reach 340 steady-state values ( Fig. 3c and Supplementary Fig. 13). We then removed the PBS with 341 doxycycline and transferred the spores to a minimal medium with a fixed glucose concentration.

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We then measured the percentage of these spore bags that germinated and, as a control, 343 compared it with the germination percentage of spore bags that received the same glucose 344 concentration after being incubated for 24 hours in PBS without doxycyline ( Fig. 3d and

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Supplementary Fig. 14). For all glucose concentrations, we found that inducing GFP expression 346 did not appreciably alter the percentage of spore bags that germinated and that it also did not 347 15 appreciably alter the average times taken for germinations ( Supplementary Fig. 15). Thus, our 348 hypothesis is incorrect -expressing GFP does not alter the spores' ability to germinate. While it 349 may now appear that we are "back to square one" -since we still have not yet uncovered what 350 causes only some of the spore bags to germinate for a given glucose concentration -we 351 discovered the answer, as we will next show, by measuring the GFP levels of individual spore In the above experiment, by measuring the steady-state GFP levels of individual spore bags just 358 before they encounter glucose, we discovered that spore bags that produced more GFP were 359 more likely to germinate . As an example, after 360 encountering a 0.001%-glucose, nearly 100% of the spore bags that had the highest, steady-state 361 GFP levels germinated whereas, in the same population, only ~10% of the spore bags with half 362 of this GFP-level germinated. In fact, for each glucose concentration, we could precisely 363 determine the probability of germinating for a spore bag once we knew its GFP level 364 ( Supplementary Fig. 17). We thereby established a quantitative relationship between the GFP 365 level and the ability to germinate, rather than a qualitative relationship such as "spores that can 366 express GFP can germinate whereas those that cannot express GFP do not germinate".

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Importantly, this quantitative link between the steady-state GFP-level and the probability of 368 germinating establishes that the stochastic variability (36,37) in the induced GFP-expression 369 among dormant spores is meaningful and predictive, despite GFP not having any obvious 370 connection to the complex, multi-step process that leads to germinations. Crucially, since GFP is 371 a generic gene without a functional role in germinations, it is reasonable to view the GFP-level as 372 quantifying the spore bag's intrinsic ability to express an arbitrary, generic gene that is induced 373 without nutrients, in accordance with how one defines the "extrinsic noise" by using cell-to-cell

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To visually represent our results, we plotted a "germination landscape" -a heat map whose color 380 represents a probability that a spore bag with a given steady-state GFP-level germinates for each 381 16 glucose concentration ( Fig. 3f and Supplementary Fig. 18). For brevity, from now on, we will refer 382 to "steady-state GFP level" simply as "GFP level". In the germination landscape, yellow 383 represents a near-certain germination (i.e., germination probability of nearly 1), green represents 384 a germination probability of ~0.5, and dark blue represents a germination probability of nearly 385 zero. The germination landscape (Fig. 3f) shows a "coastline" of nearly-yellow pixels moving up 386 towards higher rows (i.e., towards higher glucose concentrations) as one moves from right to left 387 (i.e., as GFP level decreases), meaning that more glucose is required to guarantee a germination 388 for a spore bag with a lesser GFP. The blue-green pixels are almost immediately below the 389 coastline of yellow pixels, indicating that the probability of germinating, for a fixed GFP-level, is a 390 sharp step-like function of the glucose concentration. We confirmed this by quantitatively 391 extracting (by log-regression), from the germination landscape, the minimum glucose-392 concentration required for a spore with a given GFP level to have a 99%-chance of germinating 393 ( Supplementary Fig. 19). We call this concentration, given the sharpness of the nearly step-like 394 probability function, the "minimum glucose-concentration required for germination". We 395 determined that as a spore bag's ability to express GFP decreases, the minimum glucose-396 concentration required for germination increases (Fig 3g and Supplementary Fig. 19). Importantly, 397 since inducing GFP-production does not alter the total percentage of spore bags that germinate 398 for any glucose concentration (Fig. 3d), inducing GFP-production in a spore bag does not change 399 (increase or decrease) its probability of germinating. The GFP level is thus merely an indicator for 400 which spore bags are more likely to germinate and, as previously mentioned, a quantifier of an 401 ability to express a generic gene without nutrients. Thus, we can state that spore bags with a 402 higher ability to express a generic gene without nutrients, compared to those with a lesser ability, 403 are more likely to germinate and require lesser glucose to germinate (

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Having identified a quantifiable intrinsic capacity of a spore bag to germinate -the spore bag's 411 intrinsic ability to express a generic gene -we now turn to how this capacity changes over time 412 and whether it can reveal how and when dormant spores die. Being a dead spore means that it 413 17 414 Figure 4 | Dormancy as a quantifiable spectrum ("ruler") that measures and forecasts dormant yeast-spore's proximity to death (lifespan) and proximity to entering replicative life (germination capacity). a, Percentage of wild-type and GFP-inducible spore-bags ( Fig. 3a) that germinate due to a saturating glucose-concentration (2%) that they encounter after 0 -85 days of incubation in water (grey and black data) or minimal medium with essential amino acids (red data) at 30 O C. n = 3; error bars are s.e.m. b, Hypothesis on how dormancy-to-death transition gradually occurs: Dormant spore bag loses its gene-expressing ability over time (Top left), thus it needs a higher glucose concentration to germinate (Top right), which in turn causes it to require an ever-more increasing glucose concentration for germination. Eventually the glucose concentration goes above the saturating level (2%), which is impossible to obtain and thus the spore bag will never germinate (i.e., dead) (Bottom). no longer has any capacity to germinate for any glucose concentration, including for the saturating 415 2%-glucose. While a 2%-glucose caused nearly every spore bag to germinate in the experiments 416 described thus far ( Fig. 1c), noticeable fractions of spore bags did not germinate even after 417 receiving a 2%-glucose if we incubated them for days or weeks in either water or minimal medium 418 without glucose at 30 O C (Fig. 4a). Concretely, regardless of whether we kept the spores in water 419 or minimal medium (which has all the essential amino acids), we found that the number of dormant 420 (alive) spore bags -the ones that germinated after receiving a 2%-glucose -decreased by similar 421 rates over several weeks. Specifically, about half of the spore bags in a population died after ~20 422 days without glucose and almost everyone died after ~60 days without glucose (Fig. 4a).

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Furthermore, we observed that spore bags needed more time to germinate as the number of days 424 without nutrients increased ( Supplementary Fig. 20), suggesting that a spore bag's germination 425 ability gradually deteriorates rather than suddenly, in an all-or-none type manner, going to zero.

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Motivated by these observations, we hypothesized that if the germination landscape ( Fig. 3f) still 427 applies to spores that are incubated for days without nutrients, then the dormant spore bags with 428 nearly zero GFP levels -these would have a close-to-zero ability to express genes -may not 429 germinate even after receiving a 2%-glucose. This is because the minimum glucose- 433 Dormant spores would gradually lose their ability to express genes without nutrients due to, for 434 example, their intracellular components naturally (thermally) degrading over time (  c, Top row: Steady-state GFP-levels of individual spore bags measured as described in Supplementary Fig. 21, after 0, 5,10 or 20 days of incubation (left to right) at 30 °C in plain water (see Supplementary Fig. 22 for data up to 80 days). Dead spore bags' GFP levels are not shown. Bottom row: germination landscape measured for these spores for 0, 5, 10 or 20 days (left to right). Each pixel's color is from averaging over 3 replicate populations (n = 3). d, Mean GFP-level of dormant (purple) and dead (grey) spore bags that were incubated in water without nutrients at 30 O C over ~80 days (also see Supplementary Fig. 21). The purple data points are the average GFP-levels from the histograms shown in (c) and Supplementary Fig. 22. Grey data points are averages of the dead spore bags' histograms shown in Supplementary Fig. 22. n=3, error bars represent the average standard deviation from three biological replicates. e, Average, minimum glucose-concentration required for germination, 'h' (in equation (1)) extracted from the four germination landscapes shown in (c) as a function of the gene-expressing ability (GFP-level) of dormant spore bags (see Supplementary Fig. 19 for the extraction method) incubated in water for 0 days (black), 5 days (brown), 10 days (yellow), and 20 days (orange). n=3, error bars are s.e.m. Dead spore bags represented by a grey point (at above saturating glucose levels). f, Testing the hypothesis posed in (b): $%& ⁄ in equation (1) after combining results in (d-e) (see Supplementary Fig. 23 for details). n = 3, error bars are s.e.m. g, Dormant spore bag's ability to express a gene without nutrients defines its position on the "dormancy spectrum" (bottom cartoon). The spore bag's position on the spectrum determines the minimum glucose-concentration that it needs for germination (red curve is the day-10 data in (e)) and its lifespan (blue curve is from the same mathematical analysis as in (c-f) (see Supplementary Fig. 24-25 for details)). be considered dead since it can never germinate regardless of how much glucose it encounters. The main challenge in testing the above hypothesis is that we cannot directly measure, for the 444 same spore bag, the minimum glucose-concentration that it requires for germination (denoted

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Measuring the three terms that appear on the right side of equation (1)

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purple points) and approaching the GFP-levels of the dead spore bags -the ones that did not 468 germinate to a 2%-glucose (Fig. 4a). Importantly, we found the dead spore bags' GFP levels,

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which were low but non-zero for many of them ( Combining all three measured terms that appear in equation (1)

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Notably, by looking at how "green" the yeast spores are now, one can determine how much longer 499 they can live (remain dormant) before dying. Our study began with two broad, related questions: how an organism whose life has ceased can 506 still remain alive and how we can monitor its approach to death given that it either completely 507 lacks any discernable intracellular dynamics (i.e., all intracellular processes have halted) or may 508 have faint intracellular dynamics that exist but are difficult to clearly measure. The difficulty in 509 addressing these questions lies in the fact that we usually associate being alive with being 510 dynamic on multiple fronts, such as being metabolically active, having active gene-expressions,

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and possibly being motile. This is often not the case for dormant cells. A microbial spore, while it 512 can have active intracellular dynamics for hours-to-days after forming (11)(12)(13), eventually enters 513 dormancy during which it either completely lacks any discernible intracellular dynamics (1) or has 514 greatly reduced levels of dynamics that are challenging to unambiguously measure in individual, 515 dormant spores (11,12). In a practical sense, being alive now as a dormant spore means that -516 despite appearing static -it has the potential to wake-up and re-enter replicative life if nutrients 517 were to suddenly reappear at this very moment. Thus, a brute-force approach to addressing 518 whether a given spore is dormant or dead would be to infer -if one can measure the abundances 519 of all of its key intracellular factors -whether its static contents can, in the future, achieve complex

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In our study, we circumvented this challenge for yeast spores by taking a different approach: we 525 identified a single, systems-level metric -the dormant yeast-spore's ability to express a gene 526 without nutrients. This is a systems-level metric because the ability to express a gene depends 527 on many factors (e.g., abundances of stored ribosomes). The metric measures the dormant 528 spore's capacity for achieving a dynamic behavior -expressing a gene -when induced to do so 529 in a non-invasive manner (i.e., the spore's ability to germinate remains unchanged after we 530 measure its gene-expressing ability with a synthetic gene-induction). This systems-level quantity 531 then decays over time in a way that we could monitor, enabling us to non-invasively reveal how 532 the loss of this capacity occurs in a predictable manner, thereby allowing us to predict dormant 533 spores' lifespans based on their current gene-expressing ability and reveal a previously hidden 534 dynamics by which the dormant yeast-spores approach their deaths.

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Identifying the dormant spore's ability to express a gene without nutrients as a quantity -537 which we can think of as an "amount of dormancy" -allows us to introduce the concept of a 538 "dormancy spectrum" (Fig. 4g). We can think of the dormancy spectrum as a ruler that 539 22 simultaneously measures in how many hours-to-months dormant yeast-spores will die (i.e., their 540 lifespans) and how readily they can re-enter replicative life (i.e., their "proximity" to replicative life).

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By inducing gene-expression in dormant yeast-spores and then using GFP to quantify their 542 inherent ability to express genes without nutrients, we can place dormant yeast-spores on this 543 spectrum based on their varied gene-expressing abilities and then determine how they 544 continuously slide over time along the spectrum towards their death with a predictable speed.
545 Surprisingly, despite GFP having no functional links to cell death and spore revivability, the 546 variability in GFP-levels among genetically identical yeast-spores allowed us to precisely predict 547 their lifespans and probabilities of germinating for every glucose concentration. Taken    Euroscarf. These haploid strains were "20000A" (isogenic to another standard laboratory strain 613 called "W303" with mating-type "a") and "20000B" (isogenic to W303 with mating-type "alpha").

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Spore formation (sporulation). We used a standard protocol for sporulating yeasts. In short,

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we first grew diploid yeasts (homozygous diploid wild-type or GFP-inducible strains) to saturation 648 overnight. We then transferred these cells to a "pre-sporulation media" (i.e., YPAc: consists of

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Yeast Peptone media with a 2% potassium acetate) which we then incubated for 8 hours at 30°C.

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We subsequently transferred the diploid yeasts to a "sporulation medium" (i.e., 2% potassium 651 acetate) and left them to sporulate for 5 days at 20 °C, while rotating as a liquid culture in a tube.

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Afterwards, we transferred the resulting spores to water and stored them at 4°C. Through 653 measurements, we found that we could store these spores for several months without loss of 654 viability (i.e., 2%-glucose still germinates ~100% of the spore bags).    Data availability:

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The authors declare that all data supporting the findings of this study are available within the 738 paper and its supplementary information files. The data that support the findings of this study are 739 available from the corresponding author upon reasonable request.            975 g, Given a spore bag, its steady-state GFP-level is a read-out of both its intrinsic ability to express 976 a gene without any nutrients and the minimal glucose concentration that it needs for germination.

977
Spore bags with lesser gene-expressing abilities without nutrients require more glucose to  for germination e Hypothesis is correct: Mean GFP 6.6x 13.2x 0x 3.3x 9.9x GFP before glucose added (folds)  Fig. 1b-c). We incubated the spores in minimal media (which contains all essential amino acids and nitrogenous bases) that we supplemented with a desired concentration of glucose (denoted above each histogram). We then used a wide-field microscope to observe individual spore bags over time and Before incubating the wild-type spores in a minimal medium with glucose (experiment shown in Fig. 1c), we measured the initial glucose-concentration in the medium (blue bars -"0 hours of incubation") with a hexokinase-based assay (see Materials and Methods). In all the glucose concentrations that we studied (from 0.0002% to 2%), all germinations have either stopped or were about to stop after ~10 hours (~600 minutes) of incubation. This is evidenced by the plateauing of all the curves -representing the % of spore bags germinated as a function of time for various initial glucose-concentrations -beginning at around 600 to 700 minutes in Fig. 1c. We sought to determine whether the germinations were stopping due to vegetative cells, which result from germinated spores, having consumed appreciable amounts of glucose during their continuous cell divisions (vegetative yeasts divided once every 2-3 hours at these glucose concentrations). We measured the remaining glucose-concentration after ~10 hours of incubation for various initial glucose-concentrations (orange bars -"10 hours of incubation"). For the relatively high initial glucose-concentrations (e.g., 0.01% and 0.005% shown here), germinated 5 spores and the resulting vegetative cells depleted nearly half of the initial glucose-concentration after 10 hours. But for the relatively low initial glucose-concentrations (e.g., 0.002% and 0.001% shown here), the germinated spores and the resulting vegetative cells have not consumed appreciable amounts of glucose -the glucose-concentration remained nearly unchanged after 10 hours. These results show that the germinations do not stop at any of the glucose-concentrations that we studied (Fig. 1c) because the spores ran out of glucose. This is also true for the relatively high glucose-concentrations (e.g., 0.01% and 0.005%) since these conditions still had high amounts of glucose remaining after 10 hours -these remaining glucose-concentrations were still higher than some the low initial glucose-concentrations (e.g., 0.002% and 0.001%) which were enough to germinate spores. For all bars shown: n = 3, error bars are s.e.m.

Supplementary Figure 3 | A vegetative yeast cell can divide multiple times even with the
lowest glucose-concentration (0.0002%) that we used, which could not germinate any spores (complements Fig. 1c-d). We sought to test if the lowest concentration of glucose that we used in Fig. 1c-d, which was 0.0002% and could not germinate any spores, was enough to allow divisions of vegetative, diploid wild-type cells that formed the spore bags. We incubated the vegetative, diploid wild-type cells in a minimal medium with 0.0002% glucose and used a widefield microscope to observe them over 8 days. We found that these cells could divide multiple times. Specifically, we took pictures of 10 micro-colonies on different days and measured the area of each micro-colony over time. We then combined all their areas into a single number, on each day, and thus determined the fold-change in the total (combined) area of the colonies over time (i.e., the combined area of all micro-colonies on each day divided by the combined area of all the micro-colonies on day 0). On the fourth day, the colonies stopped growing, at which point the total area had increased by ~19-fold compared to the initial total area (corresponds to ~4.2 divisions).
As a control, we incubated the vegetative cells in a minimal medium without any glucose, which led to a lesser, transient growth that stopped after two days (data not shown). These results establish that 0.0002% of glucose is enough to sustain multiple divisions of a single, isolated, vegetative wild-type cell despite it not germinating any spores.
(complements Fig. 2b). In the experiment described in Fig. 2a, we first incubated the wild-type spores in a minimal medium with a relatively low concentration of glucose. We used a wide-field microscope to observe the spores and count the number of spore bags that germinated as a function of time. A set number of hours after incubating the spores in the low glucoseconcentration (16 hours in (A), 48 hours in (B), and 96 hours in (C)), we added more glucose to the medium and then counted how many more spore bags germinated as a result -these spore bags did not germinate when we gave them the first, low concentration of glucose. Shown here are typical histograms from these experiments.  . 2g). In a mixture of spore-bags and vegetative yeasts, one typically isolates the spore bags by using zymolyase, which lyses vegetative yeasts but not spore bags due to the spore bags' thick, protective outer walls. For this reason, typical (but not all) sporulation procedures (i.e., procedures for forming spores from diploid yeasts) involve adding zymolyase at the end to isolate spore bags and kill off any diploid yeasts that failed to form spore bags. We did not use zymolyase at the end of our sporulation procedure because we typically had high yields of spore bags and, more importantly, zymolyase hurts the spore bags by causing them to lose their protective walls (seen in (B)). We did not want to hurt the spore bags in our experiments. Since our experiments involved using a microscope to track individual spore bags, we could always distinguish vegetative cells from spores. Thus, we did not need to add zymolyase at the end of our sporulation procedure (zymolyase is necessary for population-level experiments in which one does not track individual spore bags). But we used zymolyase to isolate primed, un-germinated spores (Fig. 2e) from the vegetative cells that resulted from the spore bags that did germinate. To be sure, we checked by microscopy that zymolyase indeed lysed vegetative cells and left behind only un-germinated spore bags. (A) A representative microscope-image that shows un-germinated spore bags in the absence of zymolyase (scale bar, 5 µm). (B) A representative microscope-image that shows intact, un-germinated spore bags after the zymolyase treatment (not the same field of view as (A)). As seen here, spore bags appear smaller after encountering zymolyase than they did before they encountered zymolyase because zymolyase partially degrades their protective walls (note the lack of white-outline in (B) that exists around the spore bag in (A). But the spores are still intact and kept together as one unit inside a bag, as seen here (scale bar, 5 µm). After adding gene-expression levels for individual genes within each transcriptional module whereas Fig. 2g shows a single, normalized gene-expression for an entire transcriptional module at each time point that we obtained by averaging the expression levels of all genes in that module.
"Normalization" for (A-C) means that we divided the expression level of a given gene for spores that received a low concentration of glucose -which did not germinate them -by the expression level of the same gene for spores that were kept in minimal media without glucose for the same amount of time as the spores that received the glucose (for Fig. 2g, we normalized in the same way except that we used the average expression level of a module instead of individual genes).
Here we show representative transcriptional modules that reveal how studying individual genes can give a different perspective from the one provided by averaging over all genes in a module Normalized gene-expression profiles of the "gluconeogenesis" module (list of genes in Supplementary Table 1). When we average the expression levels of all genes in this module for each time point (Fig. 2g), we observe no clear trend. However, when we study the expression levels of individual genes in this module, as shown here, we observe diverging trends (i.e., some genes have a red pixel while others have a green pixel at the same time point), explaining the absence of any observable trends when we average over all genes to get a single expressionlevel for this module (Fig. 2g). (C) Normalized gene-expression profiles of the "stress" module (list of genes in Supplementary Table 1). When we average the expression levels of all genes in this module for each time point (Fig. 2g), we observe a clear trend (i.e., elevated expression-level over time) that is qualitatively different from the temporal trend in the average time taken by the primed spores to germinate (Fig. 2f). When we study the expression levels of individual genes in this module, as shown here, we see a homogeneous expression profile (i.e., all genes have red pixels at and after 16 hours -no temporal undulations in the expression levels over time).  Fig. 11C). Interestingly, while spore bags in PBS reach half of their saturating GFP levels after 10 hours of induction (see Fig. 3c or Supplementary Fig. 12), we see here that the spore bags in water take ~30 to 40 hours to reach steady-state GFP levels. In particular, as seen in (A), spore bags in water have nearly undetectable levels of GFP even ~10 hours after encountering doxycycline. On the other hand, for the same doxycycline concentration, the average steady-state GFP-levels are similar between spore bags in water and spore bags in PBS.

Supplementary Figure 11 | Steady-state GFP levels of vegetative (replicating) cells that
have the same synthetic gene-circuit as the GFP-inducible spores (Fig. 3a) (for comparison with the steady-state GFP levels of spore bags) (complements Fig. 3c). We sought to compare how the GFP levels of spores compare with the GFP levels of replicating cells that have the same gene circuit. Shown here are the steady-state GFP-levels of individual, replicating, diploid cells that have the same GFP-inducing gene-circuit as the GFP-inducible spores (Fig. 3a).
In fact, we sporulated these cells to form the GFP-inducible spores. We measured these GFP levels after 8 hours of incubation in minimal media with a 2%-glucose and (A) 100 µg/ml of doxycycline (n = 112 cells), or (B) 10 µg/ml of doxycycline (n = 113 cells), or (C) 1 µg/ml of doxycycline (n = 80 cells), or (D) 0.01 µg/ml of doxycycline (n = 108 cells). Data obtained by using wide-field epifluorescence microscopy as in Supplementary Fig. 9. By comparing these GFP levels of diploid, replicating cells with the GFP levels of spores ( Supplementary Fig. 9), we see that even without nutrients, spores can produce GFP at levels that are similar to those of replicating cells. The two main differences between the spores and vegetative cells is that (1) the spores without nutrients requires more time (~24 hours) to reach steady-state GFP levels where as the vegetative cells require only ~ 8 hours to reach steady-state GFP levels and that (2) spores need more doxycycline (100 µg/ml) than the vegetative cells (1 µg/ml) to reach similar GFP levels. population-level average (solid colored curves) over time while incubated in PBS (i.e., without any nutrients such as glucose and amino acids). We incubated the GFP-inducible spores in PBS with a set doxycycline-concentration (indicated above each graph) and then used a wide-field, epifluorescence microscope to measure the GFP levels of each spore bag for the next 22 hours.

Supplementary
As seen in the grey curves plateauing over time, the GFP level of each spore bag reached a doxycycline (n = 101 spore bags). The red curve shows the average GFP-level for these spore bags over time. We first induced GFP expression in these spores for 24 hours with 100 µg/ml of doxycycline. At the end of the 24-hour incubation, the GFP levels reached their steady-state values ( Fig. 3c and Supplementary Fig. 12). Then, we removed the doxycycline and washed away any residual doxycycline with PBS several times. We then incubated these spores in PBS at 30 O C (start of this incubation marks "0 hours"). By doing so, we sought to understand why the GFPlevels reach steady-state values given that spores are not dividing to dilute away their accumulated copies of GFP -a vegetative cell's GFP level would reach a steady-state value because the production rate of GFP matching the dilution rate of GFP (dilution by cell divisions).
If a spore bag's GFP level reached a steady-state value because of its GFP-production rate matching the GFP-degradation rate, then stopping the production of GFP by removing the doxycycline should cause decreases in its GFP-level. This is because GFP can then only degrade stochastically (thermally) and it cannot be replenished. As seen in the grey curves and the red curve, we did not observe any significant decreases in the GFP levels during the 42-hours that followed the removal of doxycycline. Thus, the reason that the GFP levels reached steady-state values is not because of the GFP-production rate matching the GFP-degradation rate during the 24 hours of induction with doxycyline. In fact, we see here that the GFP-degradation rate is nearly zero inside the spores. Thus, we can conclude that it is the eventual stopping of GFP-production, while doxycycline is still present, that causes the GFP-levels to reach steady-state values during the 24-hours of induction.

Supplementary Figure 14 | Comparing efficiency of germination for wild-type spores with
that of GFP-inducible spores (complements Fig. 3d). The GFP-inducible spores (Fig. 3a), aside from GFP, has additional genes (selection markers such as amino-acid biosynthesis genes) that we inserted during the yeast transformations (i.e., ADE2, TRP1, URA3). To see how these selection markers, as some of them pertain to amino-acid biosynthesis, might affect the percentage of spore bags that germinate for a given glucose-concentration, we gave different concentrations of glucose to the GFP-inducible spores (without any doxycycline, shown as blue data points) and compared the percentage of these spore bags that germinated with the percentage of wild-type spore bags that germinated (red data points) for the same glucoseconcentration. As shown here, more GFP-inducible spores germinate than the wild-type spores for the same glucose-concentration but the overall trend is the same for both types of spores. n = 3; error bars are s.e.m.
Supplementary Figure 15 | Inducing GFP production does not appreciably alter the average time taken by spore bags to germinate (complements Fig. 3d). Same experiments and data as in Fig. 3d but now showing the average time taken by the GFP-inducible spore bags to germinate for low glucose-concentrations. Orange data points are for the GFP-inducible spores ( Fig. 3a) that we first incubated with doxycycline for ~24 hours prior to receiving glucose (thus these spores have steady-state GFP levels prior to receiving glucose) and the black data points are for the GFP-inducible spores that did not receive any doxycyline (thus these spores have not produced GFP prior to receiving glucose). There is virtually no difference between the two conditions. Only for the lowest glucose concentration shown here, we see some differences in the average time taken for germination between the two conditions. This is due to small-number fluctuations (i.e., almost no spore bag germinates at such a low glucose concentration; we observed at most one or two spore bag germinating, if any, out of hundreds (c.f. Supplementary   Fig. 1H)). Although not shown here, inducing GFP expression also does not appreciably alter the average time taken to germinate at much higher glucose concentrations than shown here (up to 2%-glucose) and it also does not appreciably alter the percentage of spore bags that germinate at each glucose concentration (shown in Fig. 3d). n = 3; error bars are s.e.m. 3e). As liquid cultures inside rotating tubes, we incubated the GFP-inducible spores (Fig. 3a) in PBS first with 100 µg/ml of doxycycline for 22 hours. The spore bags' GFP levels reached steadystate values which we measured after transferring the spores onto microscope-imaging wells.
After measuring the GFP levels at the end of the 22-hour incubation in this way, we removed the PBS containing doxycycline and then replaced it with a minimal medium that contained a relatively low concentration of glucose (indicated above each panel). We then measured, for each glucose concentration, how many of the spore bags that had similar GFP levels (i.e., GFP levels that fall within a binning range shown in the histograms above) germinated. replicates. We used all three biological replicates to construct the germination landscape in Fig.   3f. We used the procedure outlined here to measure all germination landscapes ( Fig. 3f and in Fig. 4). We observed that spores could achieve higher GFP levels for the same doxycycline concentration if we incubated them in a continuously mixing liquid medium for 22 hours, as we did here, rather than in a stationary liquid medium inside a microscope well for 22 hours ( Supplementary Fig. 12), due to the difference in culturing conditions. To be consistent, we used the method shown here (rotating liquid cultures) to obtain all germination landscapes and all the main conclusions in our study (e.g., probability of germinating as a function of GFP). We only cultured the GFP-inducible spores in microscope-imaging wells and continuously imaged them for 22 hours to show the kinetics of GFP-expression over time (only for Supplementary Figs. 10, 12, and 13 and Fig. 3c) but not for deriving the probabilities of germinating as a function of GFP levels. Importantly, our study's main conclusions, such as those expressing more GFP are more likely to germinate, are unaffected by the method of culturing GFP-inducible spores since these conclusions rely on relative levels of GFP rather than on the absolute levels of GFP.
point represents a single spore bag. For each spore bag, whose steady-state GFP-level we measured before adding glucose, we assigned it a "1" if it germinated or a "0" if it did not germinate after receiving the specified concentration of glucose. We then plot these grey data points (1 or 0) as a function of the GFP-level of each spore bag in the panels shown above (A-F). Afterwards, we performed a logistic regression on the grey data points, for each glucose-concentration, by fitting a logistic function, ( ) = & &'( )(* + ,* -.) , for the probability ( ) that a spore bag with a steadystate GFP-level of germinates with a specified glucose-concentration. We used MATLAB's builtin "mnrfit" script to perform the logistic-regression fits (colored curves for each panel (A-F)). With the logistic function ( ), testing a statistical link -that is, showing that there is a positive correlation between the GFP-level of a spore bag and its probability to germinate for a given glucose-concentration -is equivalent to testing whether the x (GFP-level before the spore bag receives glucose) is a sufficient predictor of the observed probability to germinate. We have done this by computing the p-value associated with the Wald test on the fit parameter & which multiplies the x in ( ). For every glucose-concentration, we found that the p-values were either below or equal to 0.01, meaning that the steady-state GFP-level of a spore bag indeed is a sufficient predictor for that spore bag's probability of germinating at the given glucoseconcentration. Specifically, we found: (A) for a 0.003%-glucose: p-value ≈ 0.01, & = -0.00063 ± 0.00049, n = 118 grey data points (92% germinated); (B) for a 0.0025%-glucose, p ≈ 0.002; & = -0.0006 ± 0.00037, n = 113 grey data points (89% germinated); (C) for a 0.002%-glucose, pvalue ≈ 3 x 10 -5 ; & = -0.00054 ± 0.00026, n = 125 grey data points (77% germinated); (D) for a 0.0015%-glucose, p-value ≈ 4 x 10 -7 ; & = -0.00066 ± 0.00025, n = 131 grey data points (61% germinated); (E) for a 0.001%-glucose, p-value ≈ 2 x 10 -5 ; & = -0.00077 ± 0.00035, n = 118 grey data points (14% germinated); and (F) for a 0.005%-glucose, we did not observe any germinations in this data set. We have shown data from just one biological replicate here as a representative data set.

Supplementary Figure 18 | Average time taken to germinate depends weakly on the steady-
state GFP-levels that spore bags have without any nutrients (complements Fig. 3f). Each color represents the average time taken by a spore bag to germinate as a function of the glucoseconcentration that it encounters and its steady-state GFP-level before it receives any glucose (result of GFP-induction with 100 µg/ml of doxycycline for 22 hours in PBS). Each color represents an average from three different populations of the GFP-inducible spores (from the same three biological replicate-populations as in Fig. 3f). For the lowest row, which represents a 0.0005%glucose, the average time taken by a spore bag to germinate is undefined because we did not observe any spores germinating with this very low glucose-concentration. The lack of any dramatic changes in the shading of the colors across the pixels indicates that the average time taken by a spore bag to germinate depends weakly on the glucose concentration -a result that mirrors our earlier observation that the average time taken to germinate by the wild-type spores is also nearly independent of the glucose concentration (Fig. 1d). Crucially, we see here that the average time taken to germinate is nearly independent of a spore bag's steady-state GFP-level in PBS (as indicated by the absence of any clear changes in the shading of the colors across the pixels within a given row).

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Supplementary Figure 19 | Three-step procedure shown here establishes that the minimum glucose-concentration that is required for guaranteeing that a spore bag will germinate (i.e., probability of germinating ~ 0.99) decreases as the spore bag's ability to express a generic gene without nutrients increases (complements Fig. 3f). (A) The germination landscape (copy of Fig. 3f) groups the GFP-levels of spore bags into bins (columns of the heat map), with each bin (column) thus representing a defined range of GFP-levels. For each column of the germination landscape, we read-off the probability to germinate from each pixel (by moving 30 up within the red box as shown in the figure). (B) We plot the values that we read-off from each pixel as a function of the glucose-concentration (red data points). We then fit a logistic function that has the same mathematical form as in Supplementary Fig. 17 but now with a different meaning: ( ) = & &'( )(* + ,* -.) . Here, ( ) is the probability that a spore bag with the GFP-level specified in (A) germinates after encountering a glucose-concentration equal to (green curve)note that the in Fig. S17 represented a spore bag's steady-state GFP-level. Here we chose the logistic function for its simplicity. From the fitted logistic function (green curve), we can extract the value of (blue point) for which ( ) ≈ 0.99 (i.e., the glucose-concentration for which the probability of germinating is 0.99). We chose 0.99 because choosing "1" will yield an artificially high value of given that the logistic function ( ) asymptotically approaches 1 without ever reaching it. (C) Repeated the procedure in (A) and (B) for each column of the germination landscape yields the plot shown here: the minimum glucose-concentration that is required to guarantee that a spore bag will germinate (i.e., probability of germination ~ 0.99) if the spore bag's steady-state GFP-level in PBS is as specified in (A). The data points here are averages from three biological replicates (n = 3) and the error bars are s.e.m. Supplementary Fig. 22, which distinguishes dormant from dead spores by measuring their GFP levels without nutrients on different days of incubation in water without any nutrients at 30 O C (complements Fig. 4c-f). Protocol for measuring how the ability to express a generic gene (GFP) without nutrients changes over time in spore bags incubated in water for many days. The procedure shown here allows us to prove that spore bags lose their ability to express GFP (express a generic gene) and that as a result, the spore bag dies (i.e., once the ability-level reaches "zero"). There is a subtle procedural detail that we did not explain in the main text. By solely measuring the GFP-levels of individual spore bags after inducing them with doxycycline, we cannot distinguish between the values that correspond to dead or non-dead (still dormant) spores. This ambiguity is problematic because we want to demonstrate a cause-and-effect: the ability to express genes decreases before death.

Supplementary Figure 21 | Protocol for
Since the GFP-level of dead spores is always close to zero (as defined by the relationship in Fig.   4b), by not excluding the GFP-level of dead spores in our analysis, we would then see that the mean GFP-level of the population would decrease over time, simply because spores are dying over time (Fig. 4a). Thus, after inducing GFP expression with doxycycline without nutrients, we levels) (complements Fig. 4g). The procedure outlined here shows how the mathematical analysis (based on equation 1), which uses solely measured quantities, determines the lifespans of individual spore bags based on their current GFP-levels (i.e., gene-expressing abilities). For brevity, we denote a spore bag's GFP-level by " ". (A) To determine t ⁄ , we assumed it to be a function f( ) that is linear in and is parametrized by two values: t ⁄ ( NOP ) and t ⁄ ( NQR ).

(B)
On each day of incubation in water without nutrients (as in Supplementary Fig. 21), we used f( ) to simulate histograms of GFP levels that we would observe for dormant spore bags (same as the yellow bars in Supplementary Fig. 22 but simulated). We then computed the "distance" between the simulated histograms and the experimentally measured histograms ( Supplementary   Fig. 22). Specifically, if ℎ SOTO ( Q ) represents a simulated histogram with Q being the simulated GFP-levels, then at each time t, the distance between the simulated and measured histograms is T = ∑ |ℎ SOTO Q ( Q ) − ℎ NXS(Y ( Q )| . The total distance over time, as a normalized sum, is then = (∑ T )/ R T\& , with being the number of time points that we used. With this definition, being close to zero means that the proposed f( ) almost perfectly reproduces the experimental histograms shown in Supplementary Fig. 22 whereas being close to one means that the proposed f( ) fails to reproduce the data in Supplementary Fig. 22. We computed the for a wide range of two-tuple values ( t ⁄ ( NOP ), t ⁄ ( NQR )), which we represent as a heat map shown here. From this heat map, we determined the value ( t ⁄ ( NOP ), t ⁄ ( NQR )) that minimizes the distance (brown point on the heatmap, d = 0.23). (C) The choice of f( ) that most closely reproduces the data (i.e., histograms in Supplementary Fig. 22), determined by steps 1 and 2 (A and B). (D) Inferred a(t) -the gene-expressing ability decreasing over time -for spore bags with three different starting GFP-levels (from bottom to top green curve: ( = 0)= 2000, 6000, 10000, and 13000). We obtained these curves by integrating t ⁄ that is shown in (C) over time. Spore bag is pronounced "dead" when ( ) reaches zero (blue points). (E) A spore bag's inferred lifespan as a function of its initial GFP level ( ( = 0)), determined by extracting the time t at which ( ) = 0.