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. 2016 Feb 18;14(8):1850–1857. doi: 10.1016/j.celrep.2016.01.071

Early Developmental Program Shapes Colony Morphology in Bacteria

Gideon Mamou 1, Ganesh Babu Malli Mohan 1, Alex Rouvinski 1,2, Alex Rosenberg 1, Sigal Ben-Yehuda 1,∗
PMCID: PMC4785774  PMID: 26904951

Summary

When grown on a solid surface, bacteria form highly organized colonies, yet little is known about the earliest stages of colony establishment. Following Bacillus subtilis colony development from a single progenitor cell, a sequence of highly ordered spatiotemporal events was revealed. Colony was initiated by the formation of leading-cell chains, deriving from the colony center and extending in multiple directions, typically in a “Y-shaped” structure. By eradicating particular cells during these early stages, we could influence the shape of the resulting colony and demonstrate that Y-arm extension defines colony size. A mutant in ymdB encoding a phosphodiesterase displayed unordered developmental patterns, indicating a role in guiding these initial events. Finally, we provide evidence that intercellular nanotubes contribute to proper colony formation. In summary, we reveal a “construction plan” for building a colony and provide the initial molecular basis for this process.

Graphical Abstract

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Highlights

  • •

    Highly ordered spatiotemporal events occur during bacterial colony development

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    Colony typically initiates by formation of leading-cell chains arranged in a Y shape

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    Y-arm extension defines the size and the shape of the future colony

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    A mutant in the phosphodiesterase ymdB displays aberrant developmental patterns

Mamou et al. show that a sequence of hierarchical spatiotemporal events occurs during colony initiation by the bacterium Bacillus subtilis. Utilizing laser-induced ablations, they demonstrate that the size and shape of the future colony relies on these primary deterministic events. Furthermore, they identify molecular cues directing this developmental process.

Introduction

During the course of colony formation, a single bacterium divides to create a colony composed of billions of its descendants organized in a remarkable ordered structure. These massive cell divisions are associated with secretion of various molecules, comprising the extracellular matrix, essential for establishing a proper colony structure (Ben-Jacob and Levine, 2006, Branda et al., 2005, Flemming et al., 2007). Bacterial colonies display distinct properties such as size, color, shape, and texture, which fundamentally vary among different species. These features were the basic means for identifying, classifying, and characterizing bacteria in the early days of microbiology (Kaufmann and Schaible, 2005) and are presently still exploited for clinical and research applications. Formation of bacterial colonies is a constructive survival strategy, enabling bacteria to utilize a greater variety of nutrients, endure rapid environmental changes, and resist antibiotic threats (Christensen et al., 2002, Lewis, 2001, Moons et al., 2009).

Colony formation and maintenance require coordinated communal activities to benefit colony members. Such remarkable coordination is exemplified by the Gram-negative bacterium Myxococcus xanthus, which employs social motility to swarm, predate, and build organ-like spore filled fruiting bodies (Kaiser, 2006, Mauriello and Zusman, 2007, Zhang et al., 2012). Similarly, cells of the Gram-positive bacterium Paenibacillus dendritiformis utilize swarming to develop complex colonial branching patterns. Interestingly, it has been demonstrated that secretion of a sibling lethal factor serves to direct colony growth and morphology of these elaborated structures (Be’er et al., 2009). In the soil bacterium Bacillus subtilis, mature colonies display a high degree of spatiotemporal organization that was found to be affected by genes involved in motility, matrix production, and sporulation, highlighting the complexity of colony architecture (Branda et al., 2001, Branda et al., 2004, Branda et al., 2006, Kearns et al., 2005).

Despite the fact that analysis of bacterial colonies began decades ago, relatively little is known about the earliest stages of colony formation. Furthermore, factors limiting colony size and expansion are largely unrevealed. Here, we show that a sequence of hierarchical spatiotemporal events occurs during colony initiation by B. subtilis. Moreover, by utilizing laser induced ablations, we demonstrate that the size and shape of the future colony relies on these primary deterministic events. Finally, we identified molecular cues directing this developmental process.

Results

Early Stages of Colony Development Exhibit Characteristic Morphological Patterns

To examine the initial stages of colony establishment, we designed a chamber (Figures S1A and S1B) to image a colony as it develops from a single progenitor cell into a three dimensional (3D) structure, utilizing confocal laser scanning microscopy (CLSM). Inspecting the early stages of colony formation revealed that subsequent to a lag phase, the progenitor cell divides to create elongated chains that are typically arranged in a “Y-like” shape (Figure 1A). A closer examination revealed that the founder cell initially generates a linear elongated chain of ∼10–15 cells. The cell chain then tends to split in the middle, such that the cells adjacent to the break point are pushed perpendicularly to the original chain layout. This breakage ultimately produces a third, double-chained Y arm (Figure 1C; Movie S1). Notably, similar initial stages were apparent when the colony developed from a single dormant spore (Figure S1C), implying that this pattern persists even when the establishing cell resides in an entirely different physiological state. Statistical analysis of colony initiation patterns (n = 272) revealed that ∼55% initiated with a Y shape (Figure 1D). The second common motif was a linear shape, in which the initial chain remained unbroken, and ∼10% of the colonies exhibited multibranched forms (Figures 1B and 1D). We focused our subsequent investigation on developing colonies exhibiting the most abundant Y-shape pattern.

Figure 1.

Figure 1

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Early Morphological Patterns of Developing Colonies

(A) Fluorescence images of typical Y-shaped structures observed at t = 3–4 hr during colony formation by AR16 (PrrnE-gfp) cells.

(B) Fluorescence images of linear (1, 1′) and multi-branched (2, 2′) structures, observed at t = 2.5 hr (1, 2) and subsequently at t = 4 hr (1′, 2′) during colony formation by AR16 cells.

(C) Fluorescence images of initial chain branching and Y-shape formation of AR16 developing colony at the indicated time points. White arrow points to the position of chain breakage, and yellow and red arrows indicate single- and double-chained Y arms, respectively.

(D) Frequency of each of the initial morphologies described above (A and B), as calculated from 11 separate experiments. The total colony number was 272.

Scale bars represent 5 μm.

By following the construction of a large number of colonies, we identified four characteristic morphological stages that occur in a defined temporal manner (Figure 2A; Movie S2). Stage I, Y-shape formation (0–4 hr); stage II, Y-shape elongation (4–5 hr), whereby the Y-shape morphology is rapidly expanded by further cell divisions, generating elongated chains that occupy a large area; stage III, central thickening (6–11 hr), in which numerous cell divisions take place at the center of the colony, while the Y arms start branching. These central divisions expand radially until the produced cells reach the tips of the Y arms. Stage IV, colony expansion (>11 hr), where the colony further expands radially to give rise to a full-sized colony. A similar developmental program was observed to be executed by the undomesticated B. subtilis strain 3610 (Figure S1D), which forms elaborate biofilm assemblies (Branda et al., 2001). Investigating the formation of colonies derived from the less abundant linear and multi-branched patterns revealed a similar series of stages, including initial chain elongation, central thickening, and expansion (Figure S1E). Collectively, the defined developmental stages monitored, indicate the existence of “construction rules” required for building up a colony.

Figure 2.

Figure 2

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Characterizing Stages of Colony Development

(A) Time-lapse fluorescence images of AR16 (PrrnE-gfp) developing colony at the defined stages and the indicated time points. Each stage was pseudo-colored and overlaid over previous stages. Color map corresponds to the different stages. Scale bars represent 40 μm.

(B and C) Cells (PY79) forming a Y shape were irradiated at the indicated position of the double-chained Y arm (blue frame). The irradiated cells were marked by the addition of PI (red), and their location (highlighted by arrows) was tracked at the indicated time points. Shown are overlaid transmitted light and fluorescence images. Representative experiments out of 13 independent biological repeats. Scale bars represent 20 μm.

Y-Arm Cells Guide Colony Development

To study the directionality of Y-arm extension, we employed laser irradiation to eradicate specific cells within the Y arm, and followed their position by the addition of the red fluorescent stain propidium iodide (PI), which preferentially penetrates dead cells. Initially, we irradiated cells located at mid-position within single- or double-chained Y arm. In both cases, the irradiated arms extended normally, but the change in position of the labeled cells indicated that the Y arm extends in an asymmetric fashion, with cells located at the tip of the arm mainly contributing to this directed extension (Figures 2B and S2A). In accord, damaging cells close to the tip of a Y arm severely impaired arm extension compared to the non-irradiated arms (Figures 2C and S2B). Furthermore, irradiation of the leading cells located at the tips of all three Y arms (Figure 3A; t = 4 hr) inflicted dramatic long-term consequences. Consistent with previous results, the reach of the Y arms was significantly reduced. At 2 hr post-irradiation (Figure 3A; t = 6 hr), the average length of the Y arms was 46 μm, while the Y arms of a similar non-irradiated colony reached an average of 136 μm. Moreover, hours later, the irradiated Y arms yielded a colony with a small diameter, occupying an area four times less than the non-irradiated colony (0.85 mm2 compared to 3.29 mm2) (Figure 3A; t = 8 hr). The size of the irradiated colony appeared to be constrained by the limited extension of the Y arms (Figure 3B). Remarkably, when an entire single Y arm was irradiated, the colony expanded in an asymmetric manner, failing to spread toward the area of the damaged Y arm (Figure 3C; Movie S3). These results emphasize the critical role of cells located at the arm’s tip for arm elongation and indicate that the Y arm cells are guiding cells, as their reach determines the size and the architecture of the future colony.

Figure 3.

Figure 3

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Early Stages of Colony Formation Determine Colony Morphology

(A) Fluorescence images overlaid with transmitted light images of AR16 (PrrnE-gfp) cells at the indicated time points during colony formation. Top: the leading cells of a developing colony at t = 4 hr were irradiated (red boxes). Bottom: an untreated colony followed in parallel, as a control. Circle marks the radial area occupied by a developing colony. Representative experiment out of four independent biological repeats.

(B) Different stages of the developing colonies in (A) were pseudo-colored and overlaid. Shown are irradiated (left) and non-irradiated (right) colonies. Color map corresponds to the different stages, with the blue circle representing the earliest time point. Note the blue and the white layers of Y-shape stages that mark the reach of the irradiated colony (left).

(C) An entire Y arm of a developing AR16 colony was irradiated (red box) and colony formation followed at the indicated time points by CLSM. White circle represents the position of the progenitor cell. Representative experiment out of 12 independent biological repeats. Scale bars represent 40 μm.

YmdB Plays a Key Role in Directing the Early Stages of Colony Development

To gain insight into the genetic basis of the initial events of colony construction, an assortment of strains, harboring mutations in genes known to be involved in colony structure, biofilm formation, or motility, were screened for defects in Y-shaped formation (Table S1). A mutant in ymdB, encoding a phosphodiesterase previously implicated in biofilm formation (Diethmaier et al., 2011), was severely deficient in producing the characteristic Y shape or the linear and multi-branched structures. Instead, morphologies, missing noticeable extending arms or any other obvious pattern, were formed (Figure 4A). The average radial area occupied by colony founders of the ΔymdB strain, at a time parallel to Y-shaped formation, was calculated to be 6,000 ± 1,950 μm2. At the same time, the average area captured by the same number of wild-type cells, forming the typical Y shape, was 15,100 ± 2,800 μm2. Consistently, even after 20 hr of incubation, ΔymdB strain formed significantly smaller colonies than wild-type (Figure 4D1 and 2). In line with this view, cells, harboring ymdB under an inducible promoter as the sole ymdB copy, produced aberrant morphologies at a time equivalent to Y-shape formation, when grown in the absence of the inducer (Figure 4B; t = 3 hr). Supplementing the inducer at this stage could no longer restore the typical Y arms but resulted in resumption of cell chain extension projected at multiple directions (Figure 4B). Furthermore, the induced cells generated a considerably larger colonies containing a higher number of progeny in comparison to the non-induced ones, suggesting that the formation of extending arms could overcome the lack of Y shape (Figures 4B and 4C). Thus, YmdB plays a key role in directing early stages of colony development.

Figure 4.

Figure 4

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ymdB Mediates Proper Colony Establishment

(A) Fluorescence images of wild-type (AR16) and ΔymdB (GB112) cells expressing PrrnE-gfp. Shown are images of a typical wild-type colony residing at stage I (top left, red frame; t = 3.5 hr) and five independent ΔymdB colonies at the equivalent stage of colony development (white frames; t = 3.5–4 hr). Scale bar, 10 μm.

(B) Fluorescence images (red) overlaid with transmitted light images (gray) of GB118 (ΔymdB, Phyper-spank-ymdB, rpsB-mCherry) cells. Developing colonies were initiated in the absence of isopropyl β-D-1-thiogalactopyranoside (IPTG), and at t = 3 hr, IPTG was added. Examples of two different colonies at the indicated time points (top and middle) (+IPTG). In parallel, cells were grown without IPTG throughout the experiment (bottom) (−IPTG). Arrows indicate cell chains projected at multiple directions. Scale bar, 40 μm.

(C) PY79 (wild-type) and GB115 (ΔymdB, Phyper-spank-ymdB) strains were grown on LB solid medium and incubated at 37°C for 20 hr. Strain GB115 was grown with or without IPTG as indicated. Shown are typical colonies photographed using a binocular. Scale bar, 0.5 mm.

(D) (1) The indicated strains were grown on LB solid medium and incubated at 37°C for 20 hr. Shown are typical colonies photographed using a binocular. Scale bar, 0.5 mm. (2) Average diameter of ∼100 colonies of each indicated strain grown as described in (D1). (3) Percentage of initial colony morphologies exhibiting a normal Y shape in comparison to wild-type (100%). For each strain, at least 100 developing colonies were examined. (4) Intracellular cAMP level of 1 ml cells (optical density 600 [OD600] 0.7 ∼5 × 108 cells/ml) of the indicated strains grown in liquid medium as measured by ELISA assay. Error bars represent three biological repeats (see Supplemental Experimental Procedures).

Intracellular cAMP Levels Correlate with Colony Shape

YmdB is a phosphodiesterase that hydrolyzes cyclic nucleotides, such as cyclic AMP (cAMP) (Shin et al., 2008). So far, cAMP has been detected in B. subtilis cells only under anaerobic conditions (Mach et al., 1984). Using highly sensitive ELISA assay, we were able to detect low levels of cAMP in extracts from wild-type growing cells (Figure 4D4). Furthermore, measuring the intracellular cAMP levels showed a large increase in YmdB mutant in comparison to wild-type (Figure 4D4). To investigate if YmdB catalytic activity is required for Y-shape formation, we mutated the YmdB catalytic site (YmdB-D8A) (Diethmaier et al., 2014). Accordingly, cAMP levels within this strain were increased (Figure 4D4). The mutant strain largely displayed aberrant colony morphologies at the time corresponding to Y-shape formation (Figures 4D3 and S3A) and generated a small colony after 20 hr (Figure 4D1 and 2). Thus, intracellular cAMP level likely affects the ability to form a Y shape and eventually to produce a full-sized colony. We next tested whether adenylate cyclase (AC), which is known to synthesize cAMP (Danchin, 1993, Gancedo, 2013), influences colony development. Searching the B. subtilis genome for a potential AC did not yield an obvious candidate; nevertheless, an uncharacterized protein, YjbK, was found to harbor a potential AC catalytic site (Iyer and Aravind, 2002) (Figure S3B). Deletion of yjbK had no significant effect on colony size, Y-shape formation, or cAMP levels (Figures S3C–S3F). Furthermore, introducing ΔyjbK mutation into the ΔymdB strain did not restore colony size (Figures S3C and S3D). However, we found that YmdB levels corresponded to YjbK production, as overexpression of YjbK was associated with increased level of YmdB (Figure S3G), suggesting that the expression of these two proteins, potentially having opposing activities, is coordinated.

Evidence that Intercellular Nanotubes Contribute to Colony Formation

We have recently found that YmdB is required for efficient formation of intercellular nanotubes that bridge neighboring cells to allow molecular exchange (Dubey and Ben-Yehuda, 2011, Dubey et al., 2016, Pande et al., 2015). To investigate if such communication might be involved in colony establishment, Y-shaped developing colonies were examined for the presence of nanotubes. Cells were followed utilizing CLSM and then imaged using high-resolution scanning electron microscopy (HR-SEM). This analysis revealed the existence of extensive intercellular tubular networks emanating from bacteria and occupying the space between the Y arms (Figure 5A), similar in appearance to the nanotube webs we observed at low cell density (Dubey et al., 2016). Yet, they were generally more challenging to detect, since they appeared to be partially buried within the relatively soft agarose surface. Importantly, these structures were clearly less abundant in the aberrant developing colonies produced by the ΔymdB mutant (Figures 5B and S4A). To examine the potential impact of the extracellular structures on colony formation, we attempted to damage them by particularly irradiating the area surrounding the cells. Correlative CLSM-HR-SEM analysis confirmed that treated areas were depleted from the nanotubular structures in comparison to non-treated proximal regions, while the cells appeared intact (Figures S4B, S4C, and S5A). Notably, we cannot exclude the possibility that other extracellular structures were damaged by this procedure. Nevertheless, when we precisely irradiated the intercellular area between the Y arms, we observed a significant developmental delay followed by an atypical colony morphology. Such treatment had little effect on Y-arm extension; however, the majority of the newly dividing cells branched from the Y arms instead of displaying the characteristic thickening at the center (Figures 5C and 5D). As a control, we irradiated intercellular regions slightly away from the extending Y arms; however, such treatment did not significantly affect colony development (Figure S5B). Additionally, fluorescence recovery after photobleaching experiments of small molecules within the agar revealed the recovery time to be very short (≤1.5 min), implying that if any toxic molecules were formed due to the treatment they were likely to diffuse rapidly (Figure S5C). We have previously shown that the presence of SDS perturbs nanotube formation (Dubey and Ben-Yehuda, 2011). Consistently, colony development on SDS containing agar resulted in the production of aberrant patterns of developing colonies (Figure S5D; Movie S4). Taken together, these results support the view that nanotubes contribute to colony formation by facilitating communication among colony founders.

Figure 5.

Figure 5

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Evidence that Intercellular Nanotubes Contribute to Colony Development

(A) AR16 (PrrnE-gfp) cells were followed by CLSM. At t = 3 hr, the cells were fixed, washed, gold coated, and observed with HR-SEM: (1) Fluorescence image of stage I colony. (2) HR-SEM image of the same colony at low magnification. Notably, cell position was slightly shifted during fixation. (3) Magnification of the boxed region in 2. Red arrows highlight intercellular tubular structures.

(B) GB61 (ΔymdB) cells were treated as in (A). (1) Low-magnification HR-SEM image. (2) Magnification of the boxed region in 1.

(C and D) Time-lapse fluorescence images of AR16 developing colonies.

(C) The intercellular region between the three Y arms (t = 4 hr) was irradiated, and colony was photographed at t = 7 hr.

(D) Untreated colony was followed in parallel. Scale bars represent 20 μm.

Discussion

Based on our analysis, we propose the following model for colony establishment in B. subtilis. A single progenitor cell undergoes initial divisions creating a chain of progeny cells, which tends to break early on to produce arms, typically extending in a Y-shaped structure. The cells located at the tips of the Y arms propagate away from the colony center to define the borders of the future colony, marking a large radial area in clock needle-like fashion. Nanotube networks formed among the Y arms facilitate their coordination to enable the subsequent stage of central thickening. During this phase, the cells divide extensively mainly at the colony center until reaching the borders marked by the leader cells. Success or failure to properly perform these primary stages has a deterministic impact on the size and morphology of the future colony and, most importantly, on the efficiency of progeny production. Intriguingly, we found that similar to B. subtilis, Bacillus cereus occupies a large territory prior to an extensive division phase that fills up the entire marked area (Movie S5), suggesting that this approach is not restricted to B. subtilis and likely maximizes bacterial propagation in natural habitats.

The multi-directional growth of the leading cells early during development is most likely guided by cAMP levels, as a clear correlation was observed between Y-shape formation, intracellular cAMP levels, and the final colony size (Figure 4D). YmdB acts to control the internal cAMP level, which in turn determines colony fate. Other phosphodiesterases and opposing ACs, such as YjbK, could be additional determinants, affecting the overall cellular cAMP levels. It still remains to be explored how cAMP levels modulate colony growth; however, the finding that the mRNA levels of >800 genes change in the absence of YmdB (Diethmaier et al., 2014) suggests large amount of cAMP-responsive proteins. In light of this view, cAMP is a key molecule in chemotaxis, motility, and multicellular assemblies in several microorganisms, such as Dictyostelium, Trypanosoma, Vibrio, and Pseudomonas (Firtel and Meili, 2000, Huynh et al., 2012, Liang et al., 2007, Lopez et al., 2015). Furthermore, cAMP was found to induce switching in turning direction of nerve growth cones (Song et al., 1997), highlighting its importance in guiding cell growth directionality in nature.

Experimental Procedures

Bacterial Strains and Plasmids

Bacterial strains used in this study are listed in Table S2, plasmid constructions are described in Table S3, and primers are listed in Table S4.

Live Imaging of Developing Colonies

A custom-designed construct was used to grow bacterial colonies under the microscope. A 40-mm metal ring was filled with LB agarose (1.5%) and assembled as described in Figures S1A and S1B. Bacterial cells were spotted at a concentration of one cell per microliter. When indicated, cells were stained by adding FM1-43, FM4-64 (Invitrogen) fluorescent membrane dyes, or propidium iodide (PI) (Sigma) to the solid LB medium at a final concentration of 1 μg/ml (FM1-43, FM4-64) and 5 μg/ml (PI). Colony growth construct was covered with a 35-mm cultFoil membrane (Pecon) to reduce agar dehydration and incubated in Lab-Tek S1 heating insert (Pecon) placed inside an incubator XL-LSM 710 S1 (Pecon). Developing colonies were visualized and photographed by CLSM LSM700 (Zeiss) with Plan-Apochromat ×10/NA0.45 or LD-Plan Neufloar ×20/NA0.4 lenses (Zeiss), and single cells were photographed with a Plan-Apochromat ×100/NA1.4 lens (Zeiss). Cells expressing GFP or stained with FM1-43 were irradiated using 488-nm laser beam, while cells expressing mCherry or stained with FM4-64 or PI were irradiated using a 555-nm laser beam. For each experiment, both transmitted and reflected light were collected. For intervening with colony development by irradiating specific cells or cell surrounding areas, regions were defined and marked as regions for bleaching. Bleaching was performed using the 405-nm laser beam at full power (5 mW) and the regions were repeatedly bleached, 25–50 iterations. Similar conditions were used when the sample was further analyzed by HR-SEM for correlative microscopy (see below). System control and image processing were carried out using Zen software version 8.0 (Zeiss). In order to create overlay images of stages of colony development, each stage was pseusocolored differentially and the images were overlaid as separate layers with the earliest time point on top.

Correlative CLSM-HR-SEM Procedure

For sample preparations, LB agarose (3%) was poured onto a 25 mm round cover glass and gently pressed with a slide to form a uniform thin layer of solid medium. Exponentially growing B. subtilis cells were diluted, spread on the agarose surface and incubated under the microscope until early stages of colony development were visible (2-4 hr). Next, cells were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (Na (CH3)2 AsO2 ⋅ 3H2O [pH 7.2]) for 2 hr at 25°C and then washed three times with 0.1 M sodium cacodylate buffer. Specimens were coated with gold-palladium (∼8 nm cluster size) with Quorum Technologies SC7640 Sputter Coater, and cells were observed with a FEG EHR-SEM (Magellan 400L [FEI]).

Additional procedures including general methods, measuring the radial area occupied by a developing colony, testing diffusion rates of small molecules on agarose pads, and cAMP ELISA testing are described in the Supplemental Experimental Procedures.

Author Contributions

Conceptualization, G.M., G.B.M.M., A. Rouvinski, and S.B.-Y.; Methodology, G.M., A. Rouvinski, and S.B.-Y.; Investigation, G.M., G.B.M.M., and S.B.-Y.; Writing – Original Draft, G.M and S.B.-Y.; Writing –Review & Editing, G.M., G.B.M.M., A. Rouvinski, A. Rosenberg, and S.B.-Y; Funding Acquisition, S.B.-Y; Resources, A. Rosenberg.

Acknowledgments

We thank E. Blayvas (Hebrew University) for support during EM analysis the and National BioResource Project National Institute of Genetics, Japan for B. subtilis mutant strains. We are grateful to members of the S.B.-Y. laboratory and G. Bachrach, I. Rosenshine, and A. Taraboulos (Hebrew University) for valuable comments. This work was supported by a European Research Council Advance Grant (339984) and by the Israel Science Foundation (327/11) awarded to S.B.-Y. G.B.M.M. was supported by the Israeli Council for Higher Education PBC-outstanding postdoctoral fellowship.

Published: February 18, 2016

Footnotes

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Supplemental Information includes Supplemental Experimental Procedures, five figures, four tables, and five movies and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2016.01.071.

Supplemental Information

Document S1. Supplemental Experimental Procedures, Figures S1–S5, and Tables S1–S4
mmc1.pdf (16.2MB, pdf)
Movie S1. Y-Shape Formation, Related to Figure 1

Time lapse images of Y shape formation. Cells expressing PrrnE-gfp (AR16) were diluted, plated on LB solid medium and incubated at 37°C in a 2D construct. Fluorescence images were taken by CLSM using 100X immersion lens at 12 min intervals in the course of 2 hrs.

mmc2.jpg (81.6KB, jpg)
Movie S2. B. subtilis Colony Development, Related to Figure 2

Time lapse images of colony development. Cells expressing PrrnE-gfp (AR16) were diluted, plated on LB solid medium and incubated at 37°C. Fluorescence images were taken by CLSM at 15 min intervals in the course of 8 hrs.

mmc3.jpg (90.7KB, jpg)
Movie S3. Irradiating One Arm Leads to Asymmetric Colony Growth, Related to Figure 3

Time lapse images of colony development following Y arm eradication. Cells expressing PrrnE-gfp (AR16) were diluted, plated on LB solid medium and incubated at 37°C. At t= 3 hrs one arm of the developing colony was irradiated using 405 nm laser. Fluorescence images were taken by CLSM at 20 min intervals in the course of 6 hrs.

mmc4.jpg (102.1KB, jpg)
Movie S4. B. subtilis Colony Development on SDS-Containing Medium, Related to Figure 5

Time lapse images of colony development. Cells expressing PrrnE-gfp (AR16) were diluted, plated on LB solid medium supplemented with 0.007% SDS and incubated at 37°C. Fluorescence images were taken by CLSM at 20 min intervals in the course of 3.5 hrs.

mmc5.jpg (400.3KB, jpg)
Movie S5. B. cereus Colony Development, Related to Figure 2

Time lapse images of B. cereus colony development. B. cereus cells were diluted, plated on LB solid medium and incubated at 37°C. Transmitted light microscopy images were taken by CLSM at 20 min intervals in the course of 6 hrs.

mmc6.jpg (573.8KB, jpg)
Document S2. Article plus Supplemental Information
mmc7.pdf (19MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Supplemental Experimental Procedures, Figures S1–S5, and Tables S1–S4
mmc1.pdf (16.2MB, pdf)
Movie S1. Y-Shape Formation, Related to Figure 1

Time lapse images of Y shape formation. Cells expressing PrrnE-gfp (AR16) were diluted, plated on LB solid medium and incubated at 37°C in a 2D construct. Fluorescence images were taken by CLSM using 100X immersion lens at 12 min intervals in the course of 2 hrs.

mmc2.jpg (81.6KB, jpg)
Movie S2. B. subtilis Colony Development, Related to Figure 2

Time lapse images of colony development. Cells expressing PrrnE-gfp (AR16) were diluted, plated on LB solid medium and incubated at 37°C. Fluorescence images were taken by CLSM at 15 min intervals in the course of 8 hrs.

mmc3.jpg (90.7KB, jpg)
Movie S3. Irradiating One Arm Leads to Asymmetric Colony Growth, Related to Figure 3

Time lapse images of colony development following Y arm eradication. Cells expressing PrrnE-gfp (AR16) were diluted, plated on LB solid medium and incubated at 37°C. At t= 3 hrs one arm of the developing colony was irradiated using 405 nm laser. Fluorescence images were taken by CLSM at 20 min intervals in the course of 6 hrs.

mmc4.jpg (102.1KB, jpg)
Movie S4. B. subtilis Colony Development on SDS-Containing Medium, Related to Figure 5

Time lapse images of colony development. Cells expressing PrrnE-gfp (AR16) were diluted, plated on LB solid medium supplemented with 0.007% SDS and incubated at 37°C. Fluorescence images were taken by CLSM at 20 min intervals in the course of 3.5 hrs.

mmc5.jpg (400.3KB, jpg)
Movie S5. B. cereus Colony Development, Related to Figure 2

Time lapse images of B. cereus colony development. B. cereus cells were diluted, plated on LB solid medium and incubated at 37°C. Transmitted light microscopy images were taken by CLSM at 20 min intervals in the course of 6 hrs.

mmc6.jpg (573.8KB, jpg)
Document S2. Article plus Supplemental Information
mmc7.pdf (19MB, pdf)

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