Margo G. Haygood

Vertical Transmission of Diverse Microbes in the Tropical Sponge Corticium sp.

ABSTRACT Sponges are host to extremely diverse bacterial communities, some of which appear to be spatiotemporally stable, though how these consistent associations are assembled and maintained from one sponge generation to the next is not well understood. Here we report that a diverse group of microbes, including both bacteria and archaea, is consistently present in aggregates within embryos of the tropical sponge Corticium sp. The major taxonomic groups represented in bacterial 16S rRNA sequences amplified from the embryos are similar to those previously described in a variety of marine sponges. Three selected bacterial taxa, representing proteobacteria, actinobacteria, and a clade including recently described sponge-associated bacteria, were tested and found to be present in all adult samples tested over a 3-year period and in the embryos throughout development. Specific probes were used in fluorescence in situ hybridization to localize cells of the three types in the embryos and mesohyl. This study confirms the vertical transmission of multiple, phylogenetically diverse microorganisms in a marine sponge, and our findings lay the foundation for future work on exploring vertical transmission of specific, yet diverse, microbial assemblages in marine sponges.

Structure of Trichamide, a Cyclic Peptide from the Bloom-Forming Cyanobacterium Trichodesmium erythraeum, Predicted from the Genome Sequence

ABSTRACT A gene cluster for the biosynthesis of a new small cyclic peptide, dubbed trichamide, was discovered in the genome of the global, bloom-forming marine cyanobacterium Trichodesmium erythraeum ISM101 because of striking similarities to the previously characterized patellamide biosynthesis cluster. The tri cluster consists of a precursor peptide gene containing the amino acid sequence for mature trichamide, a putative heterocyclization gene, an oxidase, two proteases, and hypothetical genes. Based upon detailed sequence analysis, a structure was predicted for trichamide and confirmed by Fourier transform mass spectrometry. Trichamide consists of 11 amino acids, including two cysteine-derived thiazole groups, and is cyclized by an N—C terminal amide bond. As the first natural product reported from T. erythraeum, trichamide shows the power of genome mining in the prediction and discovery of new natural products.

Complex microbiome underlying secondary and primary metabolism in the tunicate-Prochloron symbiosis

The relationship between tunicates and the uncultivated cyanobacterium Prochloron didemni has long provided a model symbiosis. P. didemni is required for survival of animals such as Lissoclinum patella and also makes secondary metabolites of pharmaceutical interest. Here, we present the metagenomes, chemistry, and microbiomes of four related L. patella tunicate samples from a wide geographical range of the tropical Pacific. The remarkably similar P. didemni genomes are the most complex so far assembled from uncultivated organisms. Although P. didemni has not been stably cultivated and comprises a single strain in each sample, a complete set of metabolic genes indicates that the bacteria are likely capable of reproducing outside the host. The sequences reveal notable peculiarities of the photosynthetic apparatus and explain the basis of nutrient exchange underlying the symbiosis. P. didemni likely profoundly influences the lipid composition of the animals by synthesizing sterols and an unusual lipid with biofuel potential. In addition, L. patella also harbors a great variety of other bacterial groups that contribute nutritional and secondary metabolic products to the symbiosis. These bacteria possess an enormous genetic potential to synthesize new secondary metabolites. For example, an antitumor candidate molecule, patellazole, is not encoded in the genome of Prochloron and was linked to other bacteria from the microbiome. This study unveils the complex L. patella microbiome and its impact on primary and secondary metabolism, revealing a remarkable versatility in creating and exchanging small molecules.

Bryostatins: biological context and biotechnological prospects

Bryostatins are a family of protein kinase C modulators that have potential applications in biomedicine. Found in miniscule quantities in a small marine invertebrate, lack of supply has hampered their development. In recent years, bryostatins have been shown to have potent bioactivity in the central nervous system, an uncultivated marine bacterial symbiont has been shown to be the likely natural source of the bryostatins, the bryostatin biosynthetic genes have been identified and characterized, and bryostatin analogues with promising biological activity have been developed and tested. Challenges in the development of bryostatins for biomedical and biotechnological application include the cultivation of the bacterial symbiont and heterologous expression of bryostatin biosynthesis genes. Continued exploration of the biology as well as the symbiotic origin of the bryostatins presents promising opportunities for discovery of additional bryostatins, and new functions for bryostatins.

Genome streamlining and chemical defense in a coral reef symbiosis

Secondary metabolites are ubiquitous in bacteria, but by definition, they are thought to be nonessential. Highly toxic secondary metabolites such as patellazoles have been isolated from marine tunicates, where their exceptional potency and abundance implies a role in chemical defense, but their biological source is unknown. Here, we describe the association of the tunicate Lissoclinum patella with a symbiotic α-proteobacterium, Candidatus Endolissoclinum faulkneri, and present chemical and biological evidence that the bacterium synthesizes patellazoles. We sequenced and assembled the complete Ca. E. faulkneri genome, directly from metagenomic DNA obtained from the tunicate, where it accounted for 0.6% of sequence data. We show that the large patellazoles biosynthetic pathway is maintained, whereas the remainder of the genome is undergoing extensive streamlining to eliminate unneeded genes. The preservation of this pathway in streamlined bacteria demonstrates that secondary metabolism is an essential component of the symbiotic interaction.

The complete genome of Teredinibacter turnerae T7901: An intracellular endosymbiont of marine wood-boring bivalves (shipworms)

Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms). This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the hosts nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2–40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (>100). However, unlike S. degradans, which degrades a broad spectrum (>10 classes) of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism) and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection). These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production of cellulose-derived biofuels.

In Vivo and In Vitro Trans-Acylation by BryP, the Putative Bryostatin Pathway Acyltransferase Derived from an Uncultured Marine Symbiont

The putative modular polyketide synthase (PKS) that prescribes biosynthesis of the bryostatin natural products from the uncultured bacterial symbiont of the marine bryozoan Bugula neritina possesses a discrete open reading frame (ORF) (bryP) that encodes a protein containing tandem acyltransferase (AT) domains upstream of the PKS ORFs. BryP is hypothesized to catalyze in trans acylation of the PKS modules for polyketide chain elongation. To verify conservation of function, bryP was introduced into AT-deletion mutant strains of a heterologous host containing a PKS cluster with similar architecture, and polyketide production was partially rescued. Biochemical characterization demonstrated that BryP catalyzes selective malonyl-CoA acylation of native and heterologous acyl carrier proteins and complete PKS modules in vitro. The results support the hypothesis that BryP loads malonyl-CoA onto Bry PKS modules, and provide the first biochemical evidence of the functionality of the bry cluster.

Localization of 'Candidatus Endobugula sertula' and the bryostatins throughout the life cycle of the bryozoan Bugula neritina.

‘Candidatus Endobugula sertula,’ the uncultivated γ-proteobacterial symbiont of the marine bryozoan Bugula neritina, synthesizes bryostatins, complex polyketides that render B. neritina larvae unpalatable to predators. Although the symbiosis is well described, little is known about the locations of ‘E. sertula’ or the bryostatins throughout larval settlement, metamorphosis and early development. In this study, we simultaneously localized ‘E. sertula’ and the bryostatins in multiple stages of the B. neritina life cycle, using a novel bryostatin detection method based on its known ability to bind mammalian protein kinase C. Our results suggest that the bryostatins are deposited onto the exterior of B. neritina larvae during embryonic development, persist on the larval surface throughout metamorphosis and are shed prior to cuticle formation. During metamorphosis, ‘E. sertula’ remains adhered to the larval pallial epithelium and is incorporated into the preancestrula cystid tissue layer, which ultimately develops into a bud and gives rise to the next zooid in the colony. Colocalization of bryostatin signal with aggregates of ‘E. sertula’ in buds of ancestrulae suggested new synthesis of bryostatins in ancestrulae. In adult B. neritina colonies, symbiont microcolonies were observed in the funicular cords of rhizoids, which likely result in asexual transmission of ‘E. sertula’ to regenerated colonies. Furthermore, bryostatin signal was detected on the surface of the rhizoids of adult B. neritina colonies. Through simultaneous localization of the bryostatins and the ‘E. sertula,’ we determined how ‘E. sertula’ is transmitted, and identified shifts in bryostatin localization throughout the life cycle of the host B. neritina.

Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills

Shipworms are marine wood-boring bivalve mollusks (family Teredinidae) that harbor a community of closely related Gammaproteobacteria as intracellular endosymbionts in their gills. These symbionts have been proposed to assist the shipworm host in cellulose digestion and have been shown to play a role in nitrogen fixation. The genome of one strain of Teredinibacter turnerae, the first shipworm symbiont to be cultivated, was sequenced, revealing potential as a rich source of polyketides and nonribosomal peptides. Bioassay-guided fractionation led to the isolation and identification of two macrodioloide polyketides belonging to the tartrolon class. Both compounds were found to possess antibacterial properties, and the major compound was found to inhibit other shipworm symbiont strains and various pathogenic bacteria. The gene cluster responsible for the synthesis of these compounds was identified and characterized, and the ketosynthase domains were analyzed phylogenetically. Reverse-transcription PCR in addition to liquid chromatography and high-resolution mass spectrometry and tandem mass spectrometry revealed the transcription of these genes and the presence of the compounds in the shipworm, suggesting that the gene cluster is expressed in vivo and that the compounds may fulfill a specific function for the shipworm host. This study reports tartrolon polyketides from a shipworm symbiont and unveils the biosynthetic gene cluster of a member of this class of compounds, which might reveal the mechanism by which these bioactive metabolites are biosynthesized.

A Bacterial Source for Mollusk Pyrone Polyketides

In the oceans, secondary metabolites often protect otherwise poorly defended invertebrates, such as shell-less mollusks, from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites because of reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, which are synthesized by symbiotic bacteria. The bacteria, Nocardiopsis alba CR167, are related to widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are unknown, but they are active in neurological assays, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity.