A microbial factory for defensive kahalalides in a tripartite marine symbiosis – Science Magazine

A microbial factory for defensive kahalalides in a tripartite marine symbiosis – Science Magazine

A little help from a friend

The Hawaiian sea slug Elysia rufescens grazes on an alga called Bryopsis sp. The alga defends itself from predators using peptide toxins decorated with fatty acids, called kahalalides. Zan et al. wondered if a third party was involved in toxin production (see the Perspective by Mascuch and Kubanek). Within the alga, a species of bacterium with a very reduced genome was discovered to be a factory for the nonribosomal assembly of a family of kahalalides. The authors elucidated the pathways for generating this chemical diversity. It seems that the sea slug not only tolerates the toxins but, to protect itself from being eaten by fish, grazes on the alga to accumulate kahalalide.

Science, this issue p. eaaw6732; see also p. 1034

Structured Abstract


Chemical defense strategies, in which organisms use toxic molecules for protection against pathogens or predators, are widespread in the marine environment. In some cases, the same defensive molecules are shared by taxonomically distant organisms, raising questions about their molecular origin. The actual source of these molecules may be the organism itself, as observed in marine algae; a microbial symbiont, as commonly seen in marine sponges and tunicates; or diet, as in several marine mollusks. Elucidating the molecular basis of toxin production in chemically defended organisms is important for a complete understanding of their ecological interactions.


In this work, we studied toxin production in the Hawaiian marine alga Bryopsis sp. and its predator, the mollusk Elysia rufescens. Both organisms are chemically defended against predators by a diverse library of lipopeptide toxins, the kahalalides, but the details of kahalalide production and diversification are unknown (see the figure). One of these molecules, kahalalide F, is a potent cytotoxin and has been evaluated clinically as an anticancer agent. The molecular structures of the kahalalides show several features of microbial biosynthesis: They are fatty acid–cyclic peptide hybrids with several d- and nonproteinogenic amino acids, thus motivating us to hypothesize that the kahalalides are produced by a bacterial or fungal symbiont of Bryopsis sp. or of both Bryopsis sp. and E. rufescens. We combined metagenomic, metatranscriptomic, and chemical analyses with microbial cultivation, fluorescence microscopy, and evolutionary genomics to determine the molecular bases of kahalalide production and evolution in this tripartite marine symbiosis.


Using metagenomic analyses, we discovered a bacterium—termed “Candidatus Endobryopsis kahalalidefaciens”—that has no cultured close relatives, and we show that it lives in symbiosis with the alga Bryopsis sp. (see the figure). Using fluorescence microscopy, bacterial cultivation, and comparative genomics, we show that “Ca. E. kahalalidefaciens” is an intracellular, obligate, genome-reduced symbiont that has lost essential functions for free living (e.g., amino acid biosynthesis). Despite this reduced metabolic capacity, 20% of the “Ca. E. kahalalidefaciens” genome encodes a diverse set of 20 nonribosomal peptide synthetase pathways. We link nine of these pathways to nine structurally diverse kahalalides (including kahalalide F, the main defensive toxin of both Bryopsis sp. and E. rufescens), which we then chemically identify in the same sample of Bryopsis sp.

None of the amino acid substrates that make up the kahalalides can be produced by “Ca. E. kahalalidefaciens” itself; therefore, these substances are mostly provided by the autotrophic Bryopsis sp., highlighting an unusual strategy of collaborative biosynthesis between a symbiotic bacterium and its host. Moreover, using metagenomic analysis and fluorescence microscopy, we show that “Ca. E. kahalalidefaciens” is not a symbiont of the mollusk E. rufescens, establishing chemical sequestration as the means by which this animal indirectly acquires bacterially produced kahalalides from its algal diet.

Detailed analysis of the “Ca. E. kahalalidefaciens” genome reveals a high level of plasticity and a distinctive model of diversifying evolution that is independent of horizontal gene transfer, consistent with the intracellular lifestyle of this symbiont. In this model, new nonribosomal peptide synthetase pathways arise through duplication and divergence, accompanied by extensive interpathway recombination events. Finally, metatranscriptomic analysis reveals that 26% of the transcriptional activity of “Ca. E. kahalalidefaciens” is dedicated to kahalalide biosynthesis and that biosynthetic pathways for different kahalalides vary widely in their expression levels, further emphasizing the importance of these molecules in maintaining a successful symbiosis.


In a chemically defended, tripartite marine symbiosis, we show that an obligate bacterial symbiont of a marine alga produces a library of defensive molecules that protect the host from predation, and that the same molecules are in turn hijacked by a predatory mollusk and used for its own defense. Living intracellularly in algal cells, the symbiont acts as a microbial factory for the biosynthesis of complex defensive molecules from simple host-derived substrates. Symbiont-derived production of defensive molecules in marine algae and indirect acquisition of microbial products by predatory mollusks may thus be important yet rarely studied phenomena in marine ecosystems.

Chemical defense in a tripartite marine symbiosis.

The bacterial symbiont “Ca. E. kahalalidefaciens,” which lives intracellularly in the marine alga Bryopsis sp., produces a diverse library of toxins (the kahalalides) that protect the host from predation. The mollusk E. rufescens sequesters the same toxins from its algal diet and employs them for its own defense.


Chemical defense against predators is widespread in natural ecosystems. Occasionally, taxonomically distant organisms share the same defense chemical. Here, we describe an unusual tripartite marine symbiosis, in which an intracellular bacterial symbiont (“Candidatus Endobryopsis kahalalidefaciens”) uses a diverse array of biosynthetic enzymes to convert simple substrates into a library of complex molecules (the kahalalides) for chemical defense of the host, the alga Bryopsis sp., against predation. The kahalalides are subsequently hijacked by a third partner, the herbivorous mollusk Elysia rufescens, and employed similarly for defense. “Ca. E. kahalalidefaciens” has lost many essential traits for free living and acts as a factory for kahalalide production. This interaction between a bacterium, an alga, and an animal highlights the importance of chemical defense in the evolution of complex symbioses.

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