All animals, including humans, evolved in a world filled with bacteria. Although bacteria are most familiar as pathogens, some bacteria produce small molecules that are essential for the biology of animals and other eukaryotes, although the details of the ways in which these bacterial molecules are beneficial are not well understood.

The choanoflagellates are water-dwelling organisms that use their whip-like flagella to move around, feeding on bacteria. They can exist as one cell or a colony of multiple cells and, perhaps surprisingly, are the closest known living relatives of animals. This means that experiments on these organisms have the potential to improve our understanding of animal development and the transition from egg to embryo to adult.

Alegado et al. have explored how the morphology of Salpingoeca rosetta, a colony-forming choanoflagellate, is influenced by its interactions with various species of bacteria. In particular, they find that the development of multicellularity in S. rosetta is triggered by the presence of the bacterium Algoriphagus machipongonensis as well as its close relatives. They also identify the signaling molecule produced by the bacteria to be C₃₂H₆₄NO₇S; this lipid molecule is an obscure relative of the sphingolipid molecules that have important roles in signal transmission in animals, plants, and fungi. Moreover, Alegado et al. show that S. rosetta can respond to this molecule – which they call rosette-inducing factor (RIF-1) – over a wide range of concentrations, including concentrations as low as 10⁻¹⁷ M.

The work of Alegado et al. suggests that interactions between S. rosetta and Algoriphagus bacteria could be a productive model system for studying the influences of bacteria on animal cell biology, and for investigating the mechanisms of signal delivery and reception. Moreover, the molecular mechanisms revealed by this work leave open the possibility that bacteria might have contributed to the evolution of multicellularity in animals.

Eukaryotes 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 (McFall-Ngai 1999; Koropatnick et al. 2004; Mazmanian et al. 2005; Falkow 2006; Hughes and Sperandio 2008; Desbrosses and Stougaard 2011) and may have exerted critical influences on animal evolution. Choanoflagellates, microscopic bacteria-eating eukaryotes that are the closest living relatives of animals (James-Clark 1868; Saville Kent 1880; Hibberd 1975; Carr et al. 2008; King et al. 2008; Ruiz-Trillo et al. 2008), 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 (Leadbeater 1983; Karpov and Coupe 1998; Dayel et al. 2011) and understanding the environmental cues that regulate choanoflagellate colony formation could provide a molecular model for animal multicellularity.

In 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 (Fairclough et al. 2010; Dayel et al. 2011). Although the original stock of S. rosetta (ATCC50818) was established from a rosette colony (Dayel et al. 2011), 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.

Figure 1 with 1 supplement

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To 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; Alegado et al. 2012), 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.

What 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.

Figure 2

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Although Bacteroidetes bacteria regulate morphogenetic processes in such diverse lineages as animals, red algae, and green algae (Provasoli and Pintner 1980; Matsuo et al. 2005; Mazmanian et al. 2005), 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).

The bacterial cell envelope components lipopolysaccharide (LPS) and peptidoglycan (PGN) from Gram-negative bacteria have long been known to affect host biology (Cohn and Morse 1960; Hoffmann et al. 1999; Kopp and Medzhitov 1999; Takeuchi et al. 1999; Medzhitov and Janeway 2000; Kimbrell and Beutler 2001; Koropatnick et al. 2004), 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 (Prieschl and Baumruker 2000; Pyne and Pyne 2000; Spiegel and Milstien 2000; Hannun et al. 2001; Merrill et al. 2001; Herr et al. 2003). Moreover, sphingolipids serve essential functions both as structural components of cell membranes and as signaling molecules in diverse eukaryotes (Hannich et al. 2011). In contrast, the phylogenetic distribution of sphingolipids in bacteria is largely limited to Bacteroidetes and Sphingomonas, where their endogenous functions are poorly understood (Olsen and Jantzen 2001; An et al. 2011).

Figure 3 with 15 supplements

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Figure 3—figure supplement 15

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Figure 3—figure supplement 2

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Figure 3—figure supplement 1

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To 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.

Sulfonolipids 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.

Finally, 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.

Figure 4 with 3 supplements

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Figure 4—figure supplement 3

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Figure 4—figure supplement 1

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These 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 (Schaefer et al. 1996) and the tripeptide pheromones of the Caribbean spiny lobster (Ziegler and Forward 2007)]. 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 (Butenandt et al. 1961; Agosta 1992; Roelofs 1995). 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 (Godchaux and Leadbetter 1980, 1983, 1984, 1988; Drijber and McGill 1994; Kamiyama et al. 1995a, 1995b; Kobayashi et al. 1995) 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 (Kreft 2010). In analogy to the chemotaxis system of bacteria (Falke et al. 1997), 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 (Zhou et al. 1998; Møller et al. 2005), 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.

The 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 (Falkow 2006; Mazmanian et al. 2008; Lee and Mazmanian 2010; An et al. 2011). 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 (Kamiyama et al. 1995a; Kobayashi et al. 1995). The pervasiveness of interactions between Bacteroidetes and animals (Webster et al. 2004; Wexler 2007), coupled with the close evolutionary relationship between choanoflagellates and animals (King and Carroll 2001; King 2004; King et al. 2008; Ruiz-Trillo et al. 2008), raise the possibility that the connection between Bacteroidetes and animal development has deep evolutionary roots (McFall-Ngai 1999). 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.

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Author details

1. Rosanna A Alegado

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   RA: Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Laura W Brown

   Competing interests

   No competing interests declared.

2. Laura W Brown

   Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   LB: Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Rosanna A Alegado

   Competing interests

   No competing interests declared.

3. Shugeng Cao

   Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   SC: Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

4. Renee K Dermenjian

   Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   RD: Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

5. Richard Zuzow

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   RZ: Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

6. Stephen R Fairclough

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   SF: Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

7. Jon Clardy

   Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   JC: Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   jon_clardy@hms.harvard.edu

   Competing interests

   JC: Reviewing Editor, eLife.

8. Nicole King

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   NK: Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   nking@berkeley.edu

   Competing interests

   No competing interests declared.

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