Metabolite plasticity drives carbon-nitrogen resource budgeting to enable division of labor in a clonal community

Previously, we discovered that in glucose-limited yeast colonies, metabolic constraints drive cells into groups exhibiting gluconeogenic and glycolytic metabolic states. Here, threshold amounts of trehalose - a limiting, produced resource, controls the emergence and self-organization of the cells exhibiting the glycolytic state, by acting as a carbon source to fuel these metabolic demands (Varahan et al., 2019). We now discover that the plasticity of use of a non-limiting resource, aspartate, controls both resource production and the emergence of heterogeneous cell states, based on differential cellular metabolic budgeting. In gluconeogenic cells, aspartate provides carbon for trehalose production, while in glycolytic cells using trehalose for carbon, aspartate supplies nitrogen to drive nucleotide synthesis. This metabolic plasticity of aspartate enables carbon-nitrogen budgeting, thereby driving the biochemical self-organization of distinct cell states. Through this organization, cells in each state exhibit true division of labor, providing bet-hedging and growth/survival advantages for the whole community.


Introduction: 23
During the development of microbial communities, groups of cells come together and exhibit 24 heterogeneity within spatial organization (Ackermann 2015). As the community develops, cells can 25 present specialization of function, which allows the community as a whole to perform various tasks 26 including the acquisition of food, defense against competing microorganisms, or more efficient growth 27 (Newman, 2016;Niklas, 2014;West and Cooper, 2016). This division of labor allows breakdown of 28 complex biological processes into simpler steps, eliminating the need for individual cells to perform 29 several tasks simultaneously, thereby enhancing the overall efficiency with which cells in the community Due to these advantages, division of labor is widely prevalent across diverse microbial communities and 32 can be found at different levels of biological organization (Gordon, 2016; Kirk, 2003;Tarnita et al., 2013). 33 However, the underlying rules that enable division of labor within cell populations remain to be 34 deciphered. 35 In particular, microbial community development is commonly triggered by nutrient limitation 36 (Ackermann, 2015;Hoehler and Jørgensen, 2013;Johnson et al., 2012). Clearly, an optimal allocation of 37 resources is critical for maximizing overall fitness within a microbial community, especially when the 38 availability of nutrients is limiting. One strategy by which the community can manage the requirement 39 of different resources is by sharing metabolic products, and this is employed by many microbial 40 communities (D'Souza et al., 2018; Liu et al., 2015). Since resources can often be insufficient, the sharing 41 of such resources might incur a cost to the cell. Hence, different cells of the community exhibit 42 metabolic interdependencies, presumably to balance out trade-offs arising from resource sharing. While 43 this concept has been demonstrated for example, in synthetically engineered systems, where required 44 metabolic dependencies are created between non-isogenic cells (Campbell et al., 2016(Campbell et al., , 2015, this has 45 been exceptionally challenging to demonstrate within a clonal community of cells. We recently 46 enables a self-organizing system based on non-limiting and limiting resources, which creates organized 70 phenotypic heterogeneity in cells. 71 72

Results: 73
Amino acid driven gluconeogenesis is critical for emergence of metabolic heterogeneity: 74 In a previous study (Varahan et al., 2019), we discovered that trehalose controls the emergence of 75 spatially organized, metabolically heterogeneous groups of cells within a yeast colony growing in low 76 glucose. Within this colony were cells with high gluconeogenic activity, and other cells showing high 77 glycolytic/pentose phosphate pathway (PPP) activity ( Figure 1A). The high glycolytic/PPP activity cells 78 could be distinguished as 'light' cells, and the highly gluconeogenic cells as 'dark', based purely on 79 optical density as observed by brightfield microscopy, as shown in Figure 1A (Varahan et al., 2019). In 80 this system, cells start in a gluconeogenic state, and these cells (dark) produce trehalose. When a 81 threshold concentration of external trehalose is reached, a subpopulation of cells switch to trehalose 82 consumption that drives a glycolytic state, and these cells continue to proliferate as light cells ( Figure  83 1A). Trehalose is a limiting resource since it is not freely available in the glucose limited external 84 environment, and must be synthesized via gluconeogenesis (François et al., 1991). We therefore first 85 asked how the loss of gluconeogenesis affects the emergence of metabolically specialized light cells. For 86 this, we genetically generated mutants that lack two key gluconeogenic enzymes (PCK1 and FBP1). 87 These gluconeogenic mutants (Δpck1 and Δfbp1) expectedly formed smooth colonies completely lacking 88 structured morphology (which correlates with the absence of metabolic heterogeneity)( Figure 1B and 89 Figure 1-figure supplement 1A). Further, these mutants had essentially undetectable cells with high PPP 90 activity (light cells), based on the fluorescence-signal of a PPP reporter, as compared to a wild-type 91 colony, although the total number of viable cells in all the colonies were comparable (Figure 1C and 92 Figure 1-figure supplement 1B). This confirms that gluconeogenesis is critical for the emergence and 93 maintenance of metabolic heterogeneity in the colony. 94 Trehalose, the produced resource controlling the switch to the light state (Varahan et al., 2019), 95 is a disaccharide made up of two molecules of glucose and is produced via gluconeogenesis. This two-96 state community of cells requires a continuous supply of trehalose to sustain itself. Therefore, in order 97 to address how dark cells maintained threshold concentrations of trehalose, we asked how this resource 98 itself is produced. Notably, the media conditions under which these colonies develop essentially have 99 non-limiting amounts of amino acid resources (2% yeast extract and 2% peptone). We therefore 100 hypothesized that amino acids (available in non-limiting levels) could act as carbon sources (via possible 101 anaplerotic processes) to fuel trehalose production in dark cells. We tested this by growing wild-type 102 cells in media devoid of free amino acids, but with sufficient ammonium sulfate (Minimal media). Wild-103 type colonies failed to develop structured colonies (which correlates with the lack of metabolic 104 heterogeneity) in the absence of free amino acids, and this could be rescued by adding back amino acids 105 to this media ( Figure 1D). Expectedly, this amino acid dependent rescue of colony morphology 106 depended on gluconeogenesis, since a Δpck1 strain failed to develop morphology even after the 107 addition of amino acids to the medium ( Figure 1D). This shows that non-limiting amino acids promote 108 the development of structured colonies exhibiting metabolic heterogeneity, in a gluconeogenesis 109 dependent manner. Interestingly this amino acid dependent effect is very specific. In add-back 110 experiments in minimal medium, amongst all amino acids tested, aspartate supplementation strongly 111 promoted the development of structured colonies exhibiting metabolic heterogeneity, more robustly 112 than the addition of any other amino acids individually or in combination ( Figure 1D and Figure 1-figure  113 supplement 1C). This was validated by experiments wherein wild-type colonies that developed in 114 minimal media, supplemented either with all amino acids, or only aspartate alone, exhibited spatially 115 restricted metabolic heterogeneity comparable to the wild-type colonies grown in rich media. The light 116 cell population was estimated using the fluorescent PPP reporter, which serves as an excellent proxy for 117 light cells (Varahan et al., 2019) (Figure 1E and Figure 1-figure supplement 1B). Collectively, these 118 results reveal that aspartate is essential for the development of metabolically specialized colonies in a 119 gluconeogenesis-dependent manner. 120 121 Aspartate promotes light cell emergence by directly fueling trehalose synthesis: 122 In contrast to their canonical roles as nitrogen sources, amino acids can also act as carbon donors for 123 several metabolic processes (Boyle, 2005). While amino acids can enter the tricarboxylic acid (TCA) cycle 124 via anaplerosis, and TCA intermediates in turn can enter gluconeogenesis, aspartate is unique. It is the 125 only amino acid that can directly enter gluconeogenesis, without feeding into the TCA cycle. This is by 126 conversion of aspartate into oxaloacetate directly in the cytosol. All the other amino acids have to be 127 first transported to the mitochondria and enter the TCA cycle, and these TCA intermediates must then 128 be transported back to the cytosol to enter gluconeogenesis (Brunengraber and Roe, 2006). Since the 129 addition of aspartate alone to minimal media was sufficient for light cells to emerge, we tested if 130 aspartate is a direct carbon source required for trehalose production within the colony, since trehalose 131 is a pre-requisite for light cell emergence. Wild-type colonies were grown in minimal media 132 supplemented with all amino acids, or aspartate alone, or all amino acids without aspartate (aspartate 133 dropout) and total trehalose levels in the 7-day old colonies were measured. As controls, trehalose 134 levels in the Δpck1 colonies (gluconeogenesis defective) and Δtps1 colonies (trehalose synthesis 135 defective) were measured. Compared to colonies grown in minimal medium, colonies grown in minimal 136 medium supplemented with all amino acids, or aspartate alone, had significantly higher amounts of 137 trehalose (Figure 2A). Notably, the level of trehalose in wild-type colonies grown in aspartate dropout 138 minimal medium was significantly lower compared to colonies grown in minimal media supplemented 139 with all amino acids or just aspartate, demonstrating that aspartate can be the primary carbon 140 contributor towards trehalose synthesis (Figure 2A). As expected, Δpck1 colonies (gluconeogenesis 141 defective) and Δtps1 (trehalose synthesis defective) had background levels of trehalose (Figure 2A). 142 Furthermore, colonies grown on aspartate dropout medium had fewer light cells (quantified using the 143 PPP reporter activity) compared to colonies grown in minimal media supplemented with all amino acids 144 or just aspartate ( Figure 2B and Figure 2-figure supplement 1). This shows that aspartate enables 145 trehalose production, which in turn controls the emergence of metabolic heterogeneity in these clonal 146 colonies (Figure 2A & 2B). To demonstrate that aspartate directly provides the carbon backbone of 147 trehalose, we grew colonies in minimal medium (low glucose) supplemented with 13 C-labeled aspartate, 148 and measured intracellular levels of 13 C-labeled gluconeogenic intermediates or end-products directly 149 by targeted mass spectrometric methods described earlier (Vengayil et al., 2019)( Figure 2C). Cells in 150 wild-type colonies accumulated 13 C-labeled 3-phosphoglycerate (3-PG) and 13 C-labeled trehalose, while 151 these labeled metabolites were undetectable in a gluconeogenic mutant (Δpck1) (Figure 2D). 152 Collectively, these data show that aspartate provides the carbon skeleton for trehalose production via 153 gluconeogenesis, and this turn is essential for the emergence of spatially restricted metabolic 154 heterogeneity. 155

156
An agent-based model suggests how differential aspartate utilization drives the emergence of self-157 organized, metabolically heterogeneous states: 158 We had previously noted that the light cells had higher rates of nucleotide synthesis (Varahan et al.,159 2019). Synthesis of the nucleotide backbone requires an assimilation of carbon (typically from glucose 160 derived metabolites, notably pentose sugars from the PPP), as well as nitrogen that comes from amino 161 acids (primarily glutamine and aspartate) (Boyle, 2005). Indeed, this donation of nitrogen by aspartate 162 towards nucleotide synthesis is considered a primary role of this amino acid. Interestingly, within the 163 dark cells of the colony, aspartate is used as a carbon source for the synthesis of trehalose ( Figure 2D). 164 We therefore hypothesized that distinct cells in the colony might differentially utilize aspartate 165 predominantly as either a carbon or a nitrogen source. This raises the central idea of molecular 166 budgeting: how is the utilization of aspartate as a carbon/nitrogen source managed in different types of 167 cells? To theoretically address this question, we refined our originally coarse-grained mathematical 168 model from (Varahan et al., 2019). In the original model that simulates the development of the colony 169 with dark and light cells, the resource driving the emergence of light cells was featureless and could only 170 be used to drive hypothetically opposite metabolism (Varahan et al., 2019). In our new model, we now 171 build-in molecule specificity. Based on experimental data, we incorporate aspartate utilization for the 172 emergence of metabolic subpopulations, as well as differential growth rates, and self-organization 173 within the colony. The processes now included in the model are explained below (See Materials and 174 Methods for a detailed description): 175 Both dark and light cells utilize externally available resources to synthesize and accumulate the 176 metabolites needed for growth. We can now assign two specific categories for these accumulating 177 metabolites: carbon (C) and nitrogen (N). The dark cells utilize a single resource, aspartate, to serve both 178 C and N requirements. Aspartate itself is a molecule that is in excess in the environment (non-limiting). 179 We propose that the dark cells budget the aspartate flux for both these requirements, and some of the 180 accumulated C (as trehalose) becomes available in the extracellular environment. From our earlier 181 findings (Varahan et al., 2019), we know that the extracellular trehalose controls when some dark cells 182 switch to being light cells. The light cells utilize the available trehalose for their C needs (driving 183 glycolysis and the PPP). However, aspartate remains readily available for their N requirements, which 184 includes nucleotide synthesis (this is illustrated in the model schematic and sample colony in Figure 3A). (iii) The aspartate to C conversion requires a yield coefficient, Y, because aspartate is a 4-carbon 194 molecule and trehalose (from gluconeogenesis) is a larger molecule (12-carbon). Three molecules of 195 aspartate will therefore be required to make one molecule of trehalose. 196 (iv) A fraction, Pf, of this accumulated C inside dark cell blocks is secreted into the extracellular 197 environment as trehalose. Thus, we can couple the trehalose production by dark cells to their aspartate 198 consumption and utilization. Additionally, there will be an imposed upper limit to this secreted amount, 199 but for our simulation this extra constraint is not limiting to cells (see  Table  206 3 for values of these parameters and see By varying the two main parameters in this study, the model makes the following predictions: 209

More of the aspartate taken up by dark cells is allocated for carbon metabolism and trehalose 210
synthesis 211 We vary the fraction of the aspartate flux allocated to nitrogen, f, from 0.0-1.0 (0% -100%) only in the 212 dark cells. The colonies formed from some selected values are shown in Figure 3B to show the general 213 trend. Low values of 'f' generate virtual colonies which are similar to experimental ones. As the value of 214 f increases, enough resource cannot be allocated to fulfil carbon requirements for light cells to divide. 215

Aspartate uptake rate by both types of cells is higher than the rate of uptake of trehalose by light cells 216
The parameter AspU dictates the relative rate of aspartate uptake compared to trehalose uptake rate by 217 light cells. Dark cells take up aspartate at the same rate as light cells. However, in dark cells, aspartate is 218 responsible for carbon metabolism and trehalose generated in the system. Varying this parameter as 219 shown in Figure 3C, we see that if the rate of uptake for aspartate is the same as the uptake rate for 220 trehalose (AspU = 1.0), the colonies cannot grow like the wild-type colony ( Figure 3B). This can be 221

Aspartate allows differential carbon/nitrogen budgeting in light and dark cells of the colony in vivo: 231
In our model, we now observe that carbon/nitrogen budgeting of aspartate by the dark cells is critical 232 for the emergence of light cells. We previously showed that light cells exhibit high PPP activity and 233 nucleotide biosynthesis, using carbon precursors derived from the trehalose, provided by the dark cells 234 (Varahan et al., 2019). As mentioned earlier, aspartate serves a nitrogen donor in the synthesis of purine 235 and pyrimidine nucleotides and serves as a carbon donor in the synthesis of trehalose ( Figure 4A) 236 (Jones, 1980). Based both on theory and our model simulations, can we now experimentally test if 237 aspartate predominantly serves as a carbon source in dark cells to fuel trehalose production, while 238 primarily providing nitrogen for nucleotide biosynthesis in light cells? We decided to investigate this 239 directly, by using a stable-isotope based metabolic-flux approach. We grew wild-type colonies in 240 minimal media containing 13 C-labeled aspartate, and collected light and dark cells by rapid micro-241 dissection of the ~1 cm colonies, followed by immediate quenching of the cells and metabolite 242 extraction (see Materials and methods), and measured the amounts of 13 C-labeled gluconeogenic 243 metabolites (3PG and 13 C-trehalose), respectively in dark and light cells by LC-MS/MS. Dark cells 244 accumulated significantly higher levels of 13 C-labeled 3-PG and 13 C-labeled trehalose as compared to the 245 light cells (Figure 4B). Using a similar experimental approach with 15 N-labeled aspartate provided, we 246 next measured the relative nitrogen-label incorporation into nucleotides in light and dark cells. Here, in 247 stark contrast to the earlier results for carbon, the light cells accumulated substantially higher levels of 248 15 N-labeled nucleotides compared to dark cells ( Figure 4C). Collectively, we experimentally demonstrate 249 differential C/N budgeting in light and dark cells, based on aspartate utilization. 250 Thus, aspartate exhibits metabolite plasticity within the cells of a colony. The gluconeogenic 251 dark cells utilize this amino acid primarily as a carbon source (for trehalose production), while the light 252 cells (with high PPP activity) primarily utilize aspartate as a nitrogen donor for nucleotide biosynthesis. also hypothesized that these colonies are compromised at colony expansion as well. To test this, wild-293 type, ∆nth1 and ∆pck1 were spotted as colonies and colony expansion was monitored over time (7 days 294 and 21 days). At 21 days, the Δnth1 and Δpck1 colonies had significantly reduced expansion compared to 295 wild-type colonies. This reiterates that the light cells are important for the effective long-term expansion 296 of the colony (Figure 5D & 5E). This also suggests the possibility that colonies lacking light cells may not 297 be able to expand towards suitable nutrients. To contextualize this with the localized availability of high-298 quality nutrients, we designed an experiment where an external source of glucose was added to the 299 plate at some distance from the colony, and the expansion of colonies towards this glucose source was 300 estimated ( Figure 5F). Strikingly, the light cells from wild-type colonies showed rapid, directional 301 proliferation towards the glucose source. Notably, both the Δnth1 cells (trehalose-breakdown deficient, 302 no light cells), and the Δpck1 cells (no trehalose production) showed markedly reduced directional 303 movement towards the glucose source ( Figure 5F). This was quantified using an expansion factor (the 304 ratio of the colony area of the half of the colony growing towards the glucose source/ colony area of the 305 other half of the colony) ( Figure 5F). These data conclusively show that light cells are essential for the 306 outward expansion and foraging response of the colony. Together, the presence of dark and light cells 307 allows greater colony survival, resistance to stress, and the ability to expand towards preferred nutrient 308 sources. 309 310

Discussion: 311
We present a model illustrating how plasticity in the use of a non-limiting resource, aspartate, is critical 312 for the emergence and maintenance of spatially organized, distinct metabolic states of groups of cells. 313 Aspartate is required for gluconeogenic cells to achieve threshold concentrations of a limiting resource, 314 trehalose, which in turn drives specialization in these clonal microbial communities (Figure 6). In low 315 glucose conditions, cells expectedly perform gluconeogenesis to replenish glucose reserves. During this 316 process, cells utilize aspartate predominantly as a carbon source that drives gluconeogenesis. One 317 eventual metabolic outcome of gluconeogenesis is trehalose synthesis, and cells accumulate synthesized 318 trehalose. Trehalose also directly benefits gluconeogenic cells, allowing them to survive environmental 319 stresses including desiccation and repeated freeze/thaw cycles. As threshold concentrations of trehalose 320 available externally are reached, some cells stochastically take up and consume trehalose, breaking it 321 down to glucose. This uptake and consumption of trehalose switches the metabolic state of these cells 322 to that of high PPP/Glycolysis. In this complimentary metabolic state, cells now utilize aspartate as a 323 nitrogen source. The combination of available glucose (from trehalose) combined with the use of 324 aspartate as a nitrogen source allows light cells to synthesize end point molecules like nucleotides, 325 which enable rapid proliferation, and efficient expansion and foraging for nutrients. 326 Our previous study showed how trehalose availability can create a self-organized system, where some 327 cells will switch a new (glycolytic) metabolic state, and these cells will themselves be sustained by the 328 cells in the original (gluconeogenic) metabolic state that produce trehalose (Varahan et al., 2019). Such 329 an idea of threshold amounts of sentinel metabolites that can control cell states is an emerging area of 330 interest (Cai and Tu, 2011; Krishna and Laxman, 2018). In this study, we take a step back, to discover 331 how such a self-organizing system can emerge by using a metabolically plastic resource, aspartate. In 332 order for cells to achieve threshold levels of the limiting, controlling resource, trehalose, cells utilize a 333 non-limiting resource (aspartate) to fuel trehalose biosynthesis. Conventionally, aspartate is only 334 thought of as a 'nitrogen' source since it is required for nucleotide metabolism (Boyle, 2005). However, 335 as we observe in this study, aspartate serves as an effective carbon source to synthesize trehalose via 336 gluconeogenesis in dark cells. Notably, in light cells, when carbon becomes non-limiting (via trehalose 337 utilization), aspartate can go back to its 'conventional' role as a nitrogen donor for nucleotide synthesis. 338 This differential use of a single metabolite to meet distinct carbon and nitrogen demands of cell in 339 opposite metabolic states is a remarkable example of metabolic budgeting within spatially organized 340 cells. This plastic ability of aspartate, combined with non-limiting amounts at which it is available makes 341 it the driver of phenotypic heterogeneity in this system. 342 The principles emerging from this two-state system in a yeast colony are pertinent to the emergence of 343 complexity from relatively simple processes. In an elegant theoretical framework, Cornish-Bowden and 344 Cardenas formulated how in a living system, self-organizing processes can maintain themselves 345 indefinitely, and how they can be modified across generations (Cornish-Bowden and Cárdenas, 2008). In 346 their study, they extend the original idea of 'metabolism-replacement systems' (M-R systems), and the 347 importance of metabolic closure (Rosen, 1972(Rosen, , 1966(Rosen, , 1965. A living M-R system, as conceptualized 348 (Cornish-Bowden and Cárdenas, 2008), requires a few specific properties: (1) some molecules are 349 available in unlimited quantities from the environment, (2) a partition must be present to separate the 350 system from its environment, (3) these molecules can enter in and out of the partition, (4) the chemistry 351 of these molecules enable them to participate in biochemical cycles, (5) these molecules/reactions will 352 not participate in processes that interfere with these biochemical cycles, and (6)  This yeast colony, with its self-organized system of cells in opposite metabolic states, appears to satisfy 371 these criteria for division of labor. The result is a community of clonal cells where each 372 metabolic/phenotypic state has individual advantages (greater survival or greater proliferation), enables 373 the colony to bet-hedge the best condition for growth and survive adversity, and also provides an 374 increased growth advantage and capability to forage for new nutrients.  For observing colony morphology, colonies were imaged using SZX-16 stereo microscope (Olympus) 396 wherein the light source was above the colony. Bright-field imaging of 7-day old colonies were done 397 using SZX-16 stereo microscope (Olympus) wherein the light source was below the colony. 398 Epifluorescence microscopy imaging of 7-day old gluconeogenesis reporter colonies (pPCK1-mCherry), 399 pentose phosphate pathway (PPP) reporter colonies (pTKL1-mCherry) and HXK1 reporter colonies 400 (pHXK1-mCherry) were imaged using the red filter (excitation of 587 nm, emission of 610 nm) of SZX-16 401 stereo microscope (Olympus). 402

Biochemical estimation of trehalose/glycogen levels: 403
Trehalose and glycogen from yeast samples were quantified as described previously, with minor 404 modifications (Gupta and Laxman, 2020). 10 OD 600 of light cells and dark cells from 7-day old wild-type 405 colonies (rich medium, 0.1% glucose) were collected. After re-suspension in water, 0.5 ml of cell 406 suspension was transferred to 4 tubes (2 tubes for glycogen assay and the other 2 tubes for trehalose 407 assay). When sample collections were complete, cell samples (in 0.25 M sodium carbonate) were boiled 408 at 95-98°C for 4 hr, and processed as described earlier (Gupta and Laxman, 2020) to estimate steady 409 state trehalose amounts, based on glucose release. Assays were done using a 96-well plate format. 410 Samples were added into each well with appropriate dilution within the dynamic range of the assay (20-411 80 µg/ml glucose). For the measurement of extracellular trehalose measurement, a single wild-type 412 colony (1-day to 7-day old colony) was re-suspended in 100 microliters of water and centrifuged at 413 20000g for 5 min. The supernatant was collected and buffered to a pH of 5.4 (optimal for trehalase 414 activity) using sodium acetate buffer (pH 5.0), and subsequently trehalose was estimated using the same 415 protocol. 416 Freeze-thaw survival assay: 417 Light cells and dark cells were isolated from 7-day old wild-type colonies and washed twice with water. 418 Subsequently cells were resuspended at an OD 600 of 0.1. These were subjected to rapid freezing by 419 plunging tubes into liquid nitrogen, followed by thawing at room temperature, for multiple cycles. 5 µl 420 from each of these samples were spotted onto rich medium plates. Cells were allowed to grow for 18 421 hours before imaging the plates and estimating survival. Wild-type, ∆nth1 and ∆pck1 cells were grown overnight and 5 µl were spotted onto rich, low glucose 435 medium. A small paper disc was soaked in 50% glucose solution overnight and placed at a distance of 436 2cm from the colony spots. Colonies were allowed to develop at 30˚C for 7 days and imaged. As a 437 control, strains were spotted on a plate containing paper disc soaked in PBS. The immediate neighborhood is the set of locations {(x-1,y), (x + 1,y), (x,y-1), (x,y + 1)}. 508 6. If there is at least one empty space, preferably divide into an empty location that has more 509 occupied neighbors; if not, pick randomly an empty location to divide into. After division, the 510 two daughter blocks are each assigned half the internal C & N reserves of the original mother 511 cell block. 512 513 C) After every time step, update the trehalose concentrations on the grid by implementing 2D 514 diffusion using the FTCS scheme identical to the one used in (Varahan et al., 2019). 515

516
The above algorithm and parameter values simulate a wild type colony as seen in Figure 3A

Model parameters 521
The new parameters introduced in the current model are chosen to reliably reproduce patterns similar 522 to the experimental WT colony (both the final form, as well as at different stages of its growth). 523 1. The parameter 'f' is the fraction of aspartate that a dark cell block allocates towards nitrogen 524 needs. Its default value is 0.125. 525 2. The parameter 'AspU' controls the relative influx of aspartate compared to the influx of 526 trehalose. We argue that since aspartate is a much smaller molecule, it has a higher flux 527 compared to trehalose for the light cells. Both light and dark cell blocks take up aspartate at this 528 same rate. Its default value is 4.0. 529 3. Conversion of aspartate to carbon necessitates a yield factor, 'Y'. We use a value of 0.31 as it 530 gives us a better pattern. 531 4. This model links aspartate consumption to trehalose production. The aspartate is converted and 532 adds to a growing internal C pool. A small fraction of this pool is secreted/leaked into the 533 extracellular environment. This fraction 'Pf' is 0.049. 534 5. In addition, we put an upper limit on the absolute amount of trehalose secreted by a dark cell 535 block in a time step. This is set at 0.12 units. This was inserted to prevent a large amount of 536 trehalose being secreted by a dark cell block if it had not divided for several time steps. 537 However, in our simulations, we find that only a negligible fraction of the cell blocks are 538 operating at this limit (see Figure 3-