A bacterial sulfonolipid triggers multicellular development in the closest living relatives of animals

1Eukaryotes evolved in a world filled with bacteria and throughout their shared history these two branches of life have developed a complex set of ways to compete and cooperate with each other. While research on these interactions has historically emphasized bacterial pathogens, bacteria also regulate the biology of eukaryotes in many other ways and may have exerted critical influences on animal evolution. Choanoflagellates, microscopic bacteria-eating eukaryotes that are the closest living relatives of animals, could provide particularly important insights into the mechanisms underlying bacterial influences on animal biology and evolution. Moreover, some choanoflagellates have both solitary and multicellular stages in their life histories and understanding the environmental cues that regulate choanoflagellate colony formation could provide a molecular model for animal multicellularity.

2In the choanoflagellate Salpingoeca rosetta, rosette-shaped multicellular colonies develop when a single founder cell undergoes multiple rounds of incomplete cytokinesis, leaving neighboring cells physically attached by fine intercellular bridges. Although the original stock of S. rosetta (ATCC50818) was established from a rosette colony, laboratory cultures consistently produced single cells, with small numbers of rosette colonies forming only sporadically (Figure 1A, Figure 1—figure supplement 1). Serendipitously, we discovered that the bacterial community influences rosette colony development. Treatment of the ATCC50818 culture with an antibiotic cocktail resulted in a culture of S. rosetta cells that proliferated robustly by feeding on the remaining antibiotic-resistant bacteria but never formed rosette colonies, even upon removal of antibiotics (Figure 1B). This culture line is hereafter referred to as RCA (for ‘Rosette Colonies Absent’). Supplementation of RCA cultures with bacteria from ATCC50818 restored rosette colony development, revealing that S. rosetta cells in the RCA culture remained competent to form colonies and would do so when stimulated by the original community of environmental bacteria.

3To determine which co-isolated bacterial species stimulate rosette colony development in S. rosetta, the RCA cell line was supplemented with 64 independent bacterial isolates from ATCC50818 and monitored for the appearance of rosette colonies. Only one bacterial species from ATCC50818, the previously undescribed Algoriphagus machipongonensis (phylum Bacteroidetes; ), induced rosette colony development in the RCA cell line (Figure 1C). S. rosetta cultures fed solely with A. machipongonensis yielded high percentages of rosette colonies (Figure 1—figure supplement 1), demonstrating that no other co-isolated bacterial species is required to stimulate rosette colony development.

4What was not clear was whether other bacteria might also be competent to induce rosette colony development. Therefore, representative Bacteroidetes and non-Bacteroidetes bacteria were grown and fed to RCA cultures (Figure 2, Table 1). None of the non-Bacteroidetes species tested, including members of the γ-proteobacteria, α-proteobacteria, and Gram-positive bacteria, were competent to induce rosette colony development. In contrast, all 15 Algoriphagus species tested induced rosette colony development, as did six of 16 other closely related species tested in the Bacteroidetes phylum (Table 1). Therefore, the ability to induce rosette colony development is enriched in Algoriphagus bacteria and their relatives.

5Although Bacteroidetes bacteria regulate morphogenetic processes in such diverse lineages as animals, red algae, and green algae, the bacterially produced chemical cues that regulate most of these partnerships remain obscure. The limited phylogenetic distribution of bacteria capable of inducing rosette colony development suggested that A. machipongonensis and its close relatives may produce a characteristic molecule that could be identified biochemically. The complete absence of rosette colonies in RCA cultures provided the basis for a robust bioassay that we developed to identify the rosette-inducing molecule(s), which we named RIFs (Rosette-Inducing Factors), from A. machipongonensis cultures. Preliminary studies demonstrated that RIF activity was present in conditioned medium from A. machipongonensis, even when grown in the absence of choanoflagellates. Furthermore, the activity was also found in the A. machipongonensis cell envelope and was heat, protease, and nuclease resistant, revealing that the compound is not a protein, RNA, or DNA (Table 2).

6The bacterial cell envelope components lipopolysaccharide (LPS) and peptidoglycan (PGN) from Gram-negative bacteria have long been known to affect host biology, but neither LPS nor PGN from A. machipongonensis triggered rosette development, alone or in combination (Figure 3A). Instead, we found that A. machipongonensis crude lipid extracts enriched in sphingolipids robustly induced rosette development (Figure 3A). In animals, sphingolipid signaling pathways regulate developmental processes such as cell death, survival, differentiation, and migration. Moreover, sphingolipids serve essential functions both as structural components of cell membranes and as signaling molecules in diverse eukaryotes. In contrast, the phylogenetic distribution of sphingolipids in bacteria is largely limited to Bacteroidetes and Sphingomonas, where their endogenous functions are poorly understood.

7To isolate and characterize the molecule(s) underlying RIF activity, we focused on the fraction enriched in sphingolipids. Lipids isolated from 160 L of A. machipongonensis culture were separated using preparative liquid chromatography–mass spectrometry (LC-MS) and the activity of each fraction was measured using the rosette colony induction bioassay. RIF activity tracked with a single fraction, which was further purified by several rounds of preparative thin-layer chromatography (Figure 3—figure supplement 1) to yield approximately 700 µg of active compound (RIF-1) with sufficient purity for structural analysis. RIF-1 represents only 0.015% of the A. machipogonensis sphingolipid pool. Based on high-resolution mass spectrometry, RIF-1 has a molecular formula of C32H64NO7S (M-H: exptl. 606.44027, calcd. 606.44035, Figure 3—figure supplement 2). Detailed analysis of one- and two-dimensional (COSY, HMBC, TOCSY, Figure 3—figure supplements 3–15) nuclear magnetic resonance (NMR, 600 MHz, Table 3) spectra revealed the planar structure of RIF-1, an unusual sulfonolipid shown in Figure 3B.

8Sulfonolipids like RIF-1 resemble sphingolipids, but there are important differences between the two. In sphingolipids, a sphingoid base (1,3-dihydroxy-2-aminoalkane) is linked through an amide bond to a fatty acid. The long alkyl chains of both the sphingoid base and fatty acid vary in length, branching, number of double bonds, and placement of hydroxyl substituents. In RIF-1, a capnoid base (2-amino-3-hydroxy-15-methyl-1-sulfonic acid) replaces the sphingoid base, and the sphingolipid hydroxyl, which is the attachment point for the major diversifying elements of the sphingolipid family, is replaced by a sulfonic acid. While members of the sphingolipid family, such as the ceramides, glycosphingolipids, sphingomyelins, and gangliosides, differ by the groups attached to the hydroxyl, the sulfonic acid function in sulfonolipids like RIF-1 has no reported diversifying modifications. In this sense sulfonolipid diversity appears more limited than sphingolipid diversity. Significantly, commercial sphingolipids (sphingomyelin, monosiloganglioside, galactocerebroside, and N-palmitoyl-DL-dihydrolacto cerebroside; Table 2) failed to show any activity in our assay system. To our knowledge, RIF-1 is the first sulfonolipid demonstrated to influence developmental processes in eukaryotes.

9Finally, we investigated the potency of RIF-1 and its ability to induce colony development under plausible environmental conditions. Purified RIF-1 induces rosette formation with a bell-shaped dose-response curve over a broad range of concentrations, from 10−2 to 107 fM or some nine orders of magnitude (Figure 4). No observable effects were seen below 10−5 fM, and RIF-1 appears to be inactive above 108 fM. A. machipongonensis conditioned medium contains 104 fM RIF-1 (Figure 4—figure supplements 1–3), and even if this conditioned medium measurement exaggerates natural concentrations by a factor of 106, S. rosetta could still respond to its presence. The shape of the dose-response curve and the potency of RIF-1 suggest that S. rosetta perceives RIF-1 in a manner consistent with a receptor-ligand interaction, albeit a receptor of exquisite sensitivity and remarkable dynamic range. While RIF-1 is the only molecule detected with rosette-inducing activity, its maximal activity (5.6 ± 0.5% colonial cells/total cells) differs from that of the sphingolipid-enriched lipid fraction (19.2 ± 4.6% colonial cells/total cells; Figure 4). This difference may be due to delivery issues of the purified and highly hydrophobic molecule, which in nature resides in membranes and potentially in membrane vesicles. Alternatively, the full potency of RIF-1 as an inducer of colony development may require additional A. machipongonensis molecules not identified in this study.

10These data reveal that RIF-1, a sulfonolipid produced by the prey bacterium A. machipongonensis, regulates morphogenesis in its predator, S. rosetta. The ecological relevance of this signaling interaction is indicated both by the coexistence of S. rosetta and A. machipongonensis in nature and by the fact that the activity of RIF-1 at femtomolar concentrations makes it markedly more potent than other marine signaling molecules [e.g., Vibrio autoinducer and the tripeptide pheromones of the Caribbean spiny lobster]. The potency of RIF-1 signaling compares favorably with that of silkworm moth sex pheromone signaling, in which vapors from an ∼4 fM solution of bombykol, the sex pheromone of the silkworm moth, induce a pronounced wing fluttering response in males. While it is formally possible that RIF-1-dependent rosette colony development is a promiscuous response to sphingolipid-type molecules, only a handful of sulfonolipids like RIF-1 have been reported and no other A. machipongonensis lipid tested in this study induced rosette colony development. Therefore we favor a model in which A. machipongonensis cell density, as revealed by RIF-1 concentration, provides S. rosetta with an indication of conditions under which rosette colony development would be advantageous, for instance by promoting more efficient capture of planktonic bacteria. In analogy to the chemotaxis system of bacteria, the ability of S. rosetta to respond to increasing bacterial cell density likely requires the hypothesized RIF-1 receptor to become less sensitive at higher concentrations. The high concentration cutoff in the dose-response curve reflects a complete loss of sensitivity at high, but non-physiological, RIF-1 concentrations. Although the presence of RIF-1 in the A. machipongonensis cell envelope suggests that it may be encountered by S. rosetta during phagocytosis, it can also function at a distance. We hypothesize that RIF-1 may be released into the environment in membrane vesicles, which have been described in Gram-negative bacteria such as Bacteroidetes, and that additional membrane constituents might be required for the full potency of RIF-1. In the future, elucidating RIF-1 delivery, along with determining the three-dimensional structure of RIF-1 and characterizing sulfonolipids from other Bacteroidetes will begin to provide the needed foundation for a molecular understanding of how S. rosetta perceives RIFs.

11The morphogenetic interaction described here between S. rosetta and A. machipongonensis raises the possibility that bacterially-produced sphingolipids in general, and sulfonolipids in particular, may be essential for the chemical signaling that allows Bacteroidetes to influence cell differentiation and morphogenesis in diverse animals. Sulfonolipids like RIF-1 have been reported to have therapeutic activities, but their endogenous functions are not known. Sulfobacins A and B, which were isolated from the culture broth of a Chryseobacterium sp. were reported as von Willebrand factor receptor antagonists, and flavocristamide A, from a related bacterial species, was reported as a DNA polymerase α inhibitor. The pervasiveness of interactions between Bacteroidetes and animals, coupled with the close evolutionary relationship between choanoflagellates and animals, raise the possibility that the connection between Bacteroidetes and animal development has deep evolutionary roots. The discovery of RIF-1 and its biological activity toward S. rosetta provides both the molecular basis and model organism for further understanding a new and potentially important class of small molecule information transfer.

12The environmental isolate of Salpingoeca rosetta is deposited at the American Tissue Culture Collection (ATCC) under the designation ATCC50818. The Rosette Colonies Absent (RCA) culture line was produced from ATCC50818 by serial treatment with chloramphenicol (68 μg mL−1), ampicillin (50 μg mL−1), streptomycin (50 μg mL−1), and erythromycin (50 μg mL−1). A monoxenic line of S. rosetta (Px1) was generated by treating ATCC 50818 with a combination of ofloxacin (10 μg mL−1), kanamycin (50 μg mL−1), and streptomycin (50 μg mL−1) antibiotics to kill the undefined environmental bacteria. Following several rounds of serial dilution, a single cell was isolated by FACS and supplemented with A. machipongonensis. All three S. rosetta cell lines (ATCC 50818, RCA, and Px1) were grown in cereal grass infused seawater at 25°C and maintained by splitting cultures 1:10 into fresh medium every 3 days. Live cells were imaged with a Leica DMI6000B microscope equipped with a DFC350 FX camera.

13Under laboratory conditions, S. rosetta differentiates into a variety of cell types including attached thecate cells, solitary swimmers, rosette colonies, chain colonies and loose, disorganized associations of cells attached to one another at the collar or to bacterial biofilms. S. rosetta rosette colonies can be distinguished from other cell types in that they contain clusters of at least four closely associated cells with organized polarity; each cell oriented with its flagellum pointing outward from a central focus. In the qualitative bioassay, RCA cultures were diluted in fresh medium to a density of approximately 104–105 cells mL−1, aliquoted into 24-well flat bottom culture dishes (Costar, Corning, NY, USA), supplemented with various treatments, and scored for the presence or absence of rosette colonies after 48 hr. For quantitative measurements, RCA cultures were diluted as before into six-well flat bottom culture dishes. To measure the percentage of cells within rosette colonies, each well was scraped to detach thecate cells and the total number of cells and the total number of cells in each rosette colony were counted with a Bright-Line hemacytometer (Hausser Scientific, Horsham, PA, USA).

14A partial representation of the bacterial flora from ATCC50818 was isolated by standard dilution-plating technique on modified Zobell medium agar at 25°C. Individual isolates were tested for their morphogenic activity by supplementing RCA cultures with a single colony of each isolate. Of 64 isolates tested, the only one that restored rosette colony development to the RCA cell line was a species that formed pink-pigmented colonies (designated strain PR1). Strain PR1 was used to inoculate liquid modified Zobell medium at 25°C and grown with aeration overnight. PR1 cells were harvested by centrifugation, and genomic DNA was isolated using a Bacterial Genomic DNA Mini-prep Kit (Bay Gene, Burlingame, CA, USA) according to the manufacturer’s specifications. The 16S rRNA gene was amplified using universal primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACCTTGTTACGRCTT-3′); comparison of the PR1 16S rRNA sequence to the Greengenes 16S rRNA database revealed strain PR1 to be most closely related to members of the Algoriphagus genus within the Bacteroidetes phylum. PR1 was subsequently named Algoriphagus machipongonensis.

15To investigate whether the ability to trigger rosette colony development was specific to A. machipongonensis, we tested three classes of bacterial species for their morphogenic capacity: 15 species in the Algoriphagus genus, 16 non-Algoriphagus members of the Bacteroidetes phylum, and eight species representing three additional major clades within Bacteria. Each species was screened for morphogenic activity using the bioassay for rosette colony development. Live cells from individual colonies grown from solid agar plates were added directly to RCA cultures and scored for the presence or absence of rosette colonies 48 hr after inoculation. Each bacterial species was tested three times.

16To determine the phylogenetic distribution of morphogenic activity in the bacterial species tested (Table 1), a sequence alignment of 16S rDNA genes from each species was generated by iterative pairwise comparisons using FSA. Poorly aligned regions were removed by Gblocks version 0.91b using default block parameters. A distance matrix (distance options according to the Kimura two-parameter model), including clustering with the maximum likelihood algorithm, was calculated using Phylip version 3.67. Support for the resulting tree topology was estimated using bootstrap analysis (1000 replicates).

17To determine the biochemical nature of RIF-1, A. machipongonensis cell fractions and conditioned medium were subjected to a battery of treatments. The results of these tests are summarized in Table 2. Conditioned medium (CM) was generated by pelleting either choanoflagellates grown in cereal grass infused with seawater or A. machipongonensis cultures grown in seawater complete medium and filtering the culture supernatant through a 0.22 μm pore filter (Millipore) to remove live bacteria. To test whether RIF-1 activity required live bacteria, A. machipongonensis was grown overnight at 25°C, centrifuged at 16,000×g for 1 min to pellet cells, and heated for 30 min at 80°C to kill viable bacteria. To test whether RIF-1 activity might be heat labile (e.g., a polypeptide), A. machipongonensis CM was boiled for 10 min. To test whether RIF-1 was a protein, A. machipongonensis CM was incubated with 200 μg mL−1 proteinase K (New England Biolabs, Ipswich, MA, USA) for 2 hr at 37°C. To test whether RIF-1 was a nucleic acid, 25 mL of A. machipongonensis CM was lyophilized, resuspended in 2.5 mL of water, and extracted with 100% ethanol to a final concentration of 80% (vol/vol) for 2 hr at −20 °C and the precipitate was collected by centrifugation for 30 min at 4000×g at 4°C. The precipitate was dissolved in 0.01 M PBS (containing 10 mM MgCl2 and 1 mM CaCl2) and incubated with either RNase A (100 μg mL−1; Sigma) or DNase I (100 μg mL−1; Sigma) for 2.5 hr at 37°C. To test whether RIF-1 activity was in the methanolic extract, A. machipongonensis cell pellet and CM were lyophilized and vortexed with methanol. Each suspension was centrifuged at 8000 rpm for 5 min and the methanol layer recovered and dried.

18To test whether RIF-1 was a lipid, A. machipongonensis cell pellet was extracted according to the Bligh–Dyer method. Briefly, the cell pellet was resuspended in 3 vol of 1:2 (vol/vol) CHCl3:MeOH and vortexed. One volume of CHCl3 was added, and the mixture vortexed. One volume of distilled water was then added, and the mixture vortexed. The same was then centrifuged at 1000 rpm for 5 min and the bottom layer recovered and dried.

19To test whether RIF-1 was a component of lipopolysaccharide (LPS), A. machipongonensis LPS was isolated using a method from. Lyophilized A. machipongonensis cells were ground with a mortar and pestle and suspended in 10 mM Tris–Cl buffer (pH 8.0), containing 2% sodium dodecyl sulfate (SDS), 4% 2-mercaptoethanol, and 2 mM MgCl2. The mixture was vortexed and incubated at 65°C until solubilized. Proteinase K (20 mg mL−1) was added to the mixture, and incubated at 65°C for an additional hour, followed by 37°C incubation overnight. 3 M sodium acetate was then added and the sample mixed. Following addition of cold absolute ethanol to the cell suspension, the sample was incubated overnight at –20°C to allow precipitate to form. The mixture was centrifuged at 4000×g for 15 min, and the supernatant discarded. The precipitate was suspended in distilled water. 3 M sodium acetate was added, and the mixture vortexed. Following addition of cold absolute ethanol, the mixture was vortexed again, and the suspension again allowed to precipitate overnight at –20°C. After the centrifugation, the precipitate was suspended in 10 mM Tris–Cl (pH 7.4), and DNase I (100 μg mL−1; NEB) and RNase (25 μg mL−1; NEB) added. The mixture was incubated at 37°C for 4 hr, then placed in a 65°C water bath for 30 min. Ninety percent phenol preheated to 65°C was added, and allow to set at 65°C for 15 min. The mixture was placed in an ice bath to cool, and then centrifuged at 6000×g for 15 min. The top aqueous layer was removed, and the phenolic layer re-extracted with an equal volume of distilled water. This sample was again incubated at 65°C for 15 min and then placed in ice water. After centrifugation at 6000×g for 15 min, the two aqueous layers were combined and dialyzed against multiple changes of distilled water over 2 days.

20To test whether RIF-1 was a peptidoglycan, A. machipongonensis peptidoglycan was isolated using a method adapted from, A. machipongonensis cell pellet was washed with 0.8% NaCl. The cells were resuspended in hot 4% SDS, boiled for 30 min, and then incubated at room temperate overnight. The sample was boiled for an additional 10 min and then centrifuged at 15,000×g for 15 min at room temperature. The pellet was washed four times with water and resuspended in water. The sample was digested for mutanolysin (10 μg mL−1; Sigma) overnight at 37°C. The enzyme was inactivated by incubation at 80°C for 20 min.

21A. machipongonensis was cultured in seawater complete medium (16×1 L) at 30°C for 2 days. The cells were harvested by centrifugation and extracted with CHCl3:MeOH (2:1, 4 L). The organic extract was filtered, dried over sodium sulfate (Na2SO4), and concentrated to give approximately 4 g crude lipid extract. The crude extract was dissolved in a minimum amount of CHCl3:MeOH (2:1), and purified by preparative high performance liquid chromatography (HPLC). All solvents were purchased from Fisher Scientific unless otherwise noted. Preparative reversed phase HPLC (RP-HPLC) was performed on an Agilent Technologies 1200 Series HPLC using a Phenomenex Luna 5 µm C8(2) 100 Å 250×21.2 mm column. Isolation of RIF-1 continued with a crude fractionation in which compounds were eluted at 10 mL min−1 in a gradient of solvents A (0.1% NH4OH in water) and B (0.1% NH4OH in methanol): 65% B increasing to 100% B over 30 min, isocratic at 100% B for 1 min. before returning to 65% B and re-equilibrating over 10 min. Fractions were analyzed by low-resolution mass spectrometry (LC-MS) on an Agilent 6130 LC/MS using a Phenomenex Gemini-NX 5 µm C18 110 Å 100×2 mm column. The next stage involved a higher resolution separation in which compounds were eluted at 0.5 mL min−1 in a gradient of solvents A (0.1% NH4OH in water) and B (0.1% NH4OH in methanol): 65% B increasing to 100% B over 30 min, isocratic at 100% B for 1 min before returning to 65% B and re-equilibrating over 3 min and those which contained a mass peak corresponding to RIF-1 ([M-H]=606.4) were combined and concentrated. This material was then purified by preparative TLC (1 mm, silica gel 60), eluted with CHCl3:MeOH:AcOH:H2O (100:20:12:5, Rf=0.5). RIF-1 was visualized by staining with ammonium molybdate in 10% H2SO4. The portion of the plate (Fraction F; Figure 3—figure supplement 1) that induced colony formation and contained RIF-1 (LC/MS: [M-H] 606) was scraped off after RIF-1 was visualized by staining with ammonium molybdate in 10% H2SO4, and the silica was extracted with CHCl3:MeOH (5:1). This material was further purified by preparative TLC on a 250 μm TLC plate (silica gel 60), eluted with CHCl3:MeOH:AcOH:H2O (100:20:12:5). From 16 L of A. machipongonensis culture, approximately 50 μg RIF-1 was obtained in sufficient purity. The entire process, from growth of the cells to isolation of pure RIF-1, was repeated nine times in order to obtain approximately 0.7 mg RIF-1 from a total of 160 L of A. machipongonensis culture.

22High Resolution Mass Spectrometry (HRMS) was carried out by Ted Voss at the WM Keck Foundation Biotechnology Resource Laboratory at Yale University on a Bruker 9.4T FT-ICR MS. RIF-1 was dissolved in 200 μL DMSO-d6 and transferred into a 3 mm NMR tube. 1H, TOCSY, gCOSY and dqfCOSY were recorded on a Varian Inova 600 spectrometer. HMQC and gHMBC experiments were performed on a Bruker Advance (sgu) 900 MHz and Varian Unity Inova 600 MHz equipped with a cryoprobe, respectively. Chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance resulting from incomplete deuteration as the internal standard (DMSO: δ 2.50). Data are reported in Table 3 as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants, and integration. Optical rotation was measured on a Jasco P-2000 digital polarimeter with a sodium lamp at 21.4°C. Unless otherwise noted, all solvents and reagents were purchased from VWR or Fisher and used without further purification.

231H NMR (600 MHz, DMSO) and 13C NMR (600 MHz, DMSO): see Table 3. Optical rotation: +6.4 (c=0.07, MeOH). HRMS m/z calcd for C32H64NO7S (M-H): 606.44035. Found: 606.44027 (M-H)−. MS/MS analysis: A major fragment derived from m/z=606 (M-H) in the MS/MS spectrum of RIF-1 corresponds to amino-sulfonic acid, Figure 3—figure supplement 2. HRMS/MS m/z calculated for C17H36NO5S (M-H): 366.23142. Found: 366.2310 (M-H)−.

24Conditioned medium was prepared from A. machipongonensis grown in seawater complete medium (750 mL) at 30°C for 2 days. The conditioned medium was lyophilized and extracted with CHCl3:MeOH (2:1; 78 mL). The organic extract was filtered, further extracted with CHCl3 (60 mL×2), and filtrates were combined and concentrated to dryness under vacuum. The crude extract was suspended in 5 mL 50% MeOH:H2O and was passed through a C-18 SPE (1 g) column. The open column was then washed with 10 mL 90% MeOH:CHCl3. The organic eluate was concentrated and dissolved in 3 mL CHCl3:MeOH (2:1) for LC/MS analysis. The chromatography was carried out using an Agilent 6130 Quadrupole LC/MS system with a C18 reverse-phase column (4.6×100 mm; Phenomenex Luna; 5 μ) for 30 min in a linear gradient from solvent A (60% methanol/water with 0.1% ammonium hydroxide) to solvent B (100% methanol with 0.1% ammonium hydroxide). The RIF-1 was detected in the conditioned medium at a concentration of 80 ng L−1. The purified RIF-1 was used as the standard (Figure 4—figure supplements 1–3).

25The potency of pure RIF-1 was determined using the quantitative bioassay for rosette colony development. Briefly, 100 ug of pure RIF-1 was solubilized in 100 μL DMSO and this 1 g L−1 stock was stored at -80°C. For each experiment, serial dilutions ranging from 10−1 g L−1 down to 10−17 g L−1 were made in DMSO. 2 μL of each dilution was premixed with 1 mL of fresh cereal grass infused seawater to avoid precipitation of RIF-1 and the premixed RIF-1 dilution was then added to 1 mL RCA cultures to yield final concentrations ranging from 10−3 to 10−20 g L−1, equivalent to 1.6×109 fM down to 1.6×10−8 fM. The percentage of rosette colonial cells was determined as described above in three independent cell lines in triplicate. From the percent rosette colony development, a bell-shaped dose-response model was determined to be the nonlinear regression curve of best fit determined using GraphPad Prism 5 statistical software.