Evidence for the biosynthesis of bryostatins by the bacterial symbiont "Candidatus Endobugula sertula" of the bryozoan Bugula neritina

The marine bryozoan, Bugula neritina, is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont "Candidatus Endobugula sertula." In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-"E. sertula" association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from B. neritina directly and use these to produce bryostatins in heterologous host bacteria.     

Isolation of two polyketide synthase gene fragments from the uncultured microbial symbiont of the marine bryozoan Bugula neritina

"Candidatus Endobugula sertula," the uncultured microbial symbiont of the bryozoan Bugula neritina, produces ecologically and biomedically important polyketide metabolites called bryostatins. We isolated two gene fragments from B. neritina larvae that have high levels of similarity to polyketide synthase genes. These gene fragments are clearly associated with the symbiont and not with the host.     

Cryptic species,cryptic endosymbionts,and geographical variation in chemical defences in the bryozoan Bugula neritina

Molecular markers often offer the only means to discriminate between species and to elucidate the specificity of many community interactions, both of which are key to the understanding of ecological patterns. Western Atlantic populations of the bryozoan Bugula neritina vary in the palatability of their larvae to predators: individuals south of Cape Hatteras produce chemical deterrents to fish predators that are absent in more northern individuals. We use mitochondrial cytochrome oxidase c subunit I (COI) sequences to show that the differences in palatability between populations correlate with the geographical distributions of two cryptic species within B. neritina. Furthermore, these cryptic species differ in their associations with bacteria that may confer chemical resistance to predation. Small subunit rRNA primers specific to a subset of gamma-proteobacteria amplified only the bacterium Endobugula sertula from the southern cryptic species. Endobugula sertula produces a family of chemical compounds (bryostatins) that may deter predators of its animal host. In contrast, the same primers amplified an array of gamma-proteobacteria from the unprotected northern cryptic bryozoan species, but never E. sertula. In combination, these findings suggest that the geographical variation in palatability observed in the larvae of B. neritina is not the result of local adaptation of a single species to regions of differing predation pressure, but rather results from the comparison of cryptic species that differ in the presence or absence of a bacterium that may provide protection against predators. The ability to identify the cryptic Bugula species and their differing relationships with bacterial associates provides an example of the important role molecular techniques may play in addressing ecological questions.     

"Candidatus Endobugula glebosa," a specific bacterial symbiont of the marine bryozoan Bugula simplex

The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, "Candidatus Endobugula sertula." "Candidatus E. sertula" has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with "Candidatus E. sertula." In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status "Candidatus Endobugula glebosa" is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.     

Evidence for the Biosynthesis of Bryostatins by the Bacterial Symbiont “Candidatus Endobugula sertula” of the Bryozoan Bugula neritina

The marine bryozoan, Bugula neritina, is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont “Candidatus Endobugula sertula.” In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-“E. sertula” association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from B. neritina directly and use these to produce bryostatins in heterologous host bacteria.     

Microbial symbionts of marine invertebrates: opportunities for microbial biotechnology

Marine invertebrates are sources of a diverse array of bioactive metabolites with great potential for development as drugs and research tools. In many cases, microorganisms are known or suspected to be the biosynthetic source of marine invertebrate natural products. The application of molecular microbiology to the study of these relationships will contribute to basic biological knowledge and facilitate biotechnological development of these valuable resources. The bryostatin-producing bryozoan B. neritina and its specific symbiont "Candidatus Endobugula sertula" constitute one promising model system. Another fertile subject for investigation is the listhistid sponges that contain numerous bioactive metabolites, some of which originate from bacterial symbionts.     

Isolation of Two Polyketide Synthase Gene Fragments from the Uncultured Microbial Symbiont of the Marine Bryozoan Bugula neritina

“Candidatus Endobugula sertula,” the uncultured microbial symbiont of the bryozoan Bugula neritina, produces ecologically and biomedically important polyketide metabolites called bryostatins. We isolated two gene fragments from B. neritina larvae that have high levels of similarity to polyketide synthase genes. These gene fragments are clearly associated with the symbiont and not with the host.     

Aquaculture of three phyla of marine invertebrates to yield bioactive metabolites: process developments and economics

Large-scale, renewable supplies of chemical constituents derived from marine invertebrates have limited development of potential new natural product drugs. This paper describes the development of two in-sea aquaculture systems designed and engineered for production of large quantities of biomass for two species of marine invertebrates desired for their natural product chemical constituents. The two invertebrates and their products were: (1) the cosmopolitan, arborescent bryozoan Bugula neritina (Phylum Bryozoa) for its anticancer chemical constituent bryostatin 1; and (2) Ecteinascidia turbinate (Phylum Tunicata) the source of anticancer ecteinascidin 743. For the third invertebrate Phylum Porifera, and its representative sponge Acanthella cavernosa (desired for its anti-parasitic and anti-infective kalihinols) in-sea systems were not developed in favor of controlled environment tank aquaculture systems. For the bryozoan and tunicate, projected economics for commercial-scale in-sea production proved cost effective. This was in contrast to the controlled environment sponge culture tank system, which did not prove to be economical due to inherent slow growth and low natural product yields of the sponge in culture. A non-destructive method for "milking" natural product chemicals from sponges was tested and is described.     

Molecular and phenotypic descriptions of Stachybotrys chlorohalonata sp. nov. and two chemotypes of Stachybotrys chartarum found in water-damaged buildings

Twenty-five Stachybotrys isolates from two previous studies have been examined and compared, using morphological, chemical and phylogenetic methods. The results show that S. chartarum sensu lato can be segregated into two chemotypes and one new species. The new species, S. chlorohalonata, differs morphologically from S. chartarum by having smooth conidia, being more restricted in growth and producing a green extracellular pigment on the medium CYA. S. chlorohalonata and S. chartarum also have different tri5, chs1 and tub1 gene fragment sequences. The two chemotypes of S. chartarum, chemotype S and chemotype A, have similar morphology but differ in production of metabolites. Chemotype S produces macrocyclic trichothecenes, satratoxins and roridins, while chemotype A produces atranones and dolabellanes. There is no difference between the two chemotypes in the tub1 gene fragment, but there is a one nucleotide difference in each of the tri5 and the chs1 gene fragments.     

“Candidatus Endobugula glebosa,” a Specific Bacterial Symbiont of the Marine Bryozoan Bugula simplex

The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gamma-proteobacterium, “Candidatus Endobugula sertula.” “Candidatus E. sertula” has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with “Candidatus E. sertula.” In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status “Candidatus Endobugula glebosa” is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.     

Alkaloid chemotypes of Glaucium flavum (Papaveraceae) from Bulgaria

The alkaloid pattern of Glaucium flavum Crantz. from four natural populations in Bulgaria were investigated and six aporphine, two protopine and one morphinane alkaloids were determined. In accordance with the particular content and composition of alkaloids and the different localities of the species three alkaloid chemotypes were suggested. The first one contains aporphines and protopines with a main alkaloid glaucine. The second chemotype contains again aporphine and protopine alkaloids but the main alkaloid is isocorydine and the third chemotype contains besides these two types of alkaloids and the morphinane alkaloid salutaridine.     

GENE FLOW IN LICHENS

Experimental studies of the evolutionary biology of lichen fungi have been hampered by massive difficulties of in vitro culture and artificial crosses are still not possible. Gene flow in these organisms is demonstrated here for the first time by the analysis of secondary products in the progeny of individuals from natural populations of mixed chemotypes of the Cladonia chlorophaea complex. All of the chemotypes in this study have been interpreted as distinct sibling species. In the Appalachian Mountains of North Carolina, however, the grayi and merochlorophaea chemotypes are found to belong to a single interbreeding populations that is reproductively isolated from the cryptochlorophaea chemotype. In the Coastal Plain, the cryptochlorophaea chemotype hybridizes with the local endemic perlomera chemotype. This study has major impact for species concept in lichens because consideration of neither morphologial tendencies nor biogenetic relationships of the secondary products could have predicted its result.     

Chemical and morphological diversity in wild populations of Mentha longifolia in Israel

Populations of Mentha longifolia, an endangered species in Israel, were tested for essential oil composition and conservational ability. In 2002-2003, 25 wild populations country-wide were tested, indicating population divergence into two chemotypes. Chemotype A was characterized by high levels of menthone and pulegone, and chemotype B by high levels of piperitenone oxide and piperitone oxide. Chemotype A was more abundant (22 of 25 populations) than chemotype B (11 of 25 populations). However, a chemotype/population interaction was not recorded (P?>?0.05). In spring 2003, seven of the 25 wild populations were resampled, propagated, and cultivated at the Newe Ya'ar campus. Then, in 2004, the propagated plants were tested for essential oil composition. The propagated plants maintained the essential oil composition as well as the chemotype-frequency distribution of the original wild population from which they were obtained. Since a chemotype/population interaction was not recorded, and the cultivated plants displayed the wild population essential oil composition, it can be concluded that i) the chemotype diversity is genetically based, and ii) the M. longifolia populations sampled can be horticulturally conserved.     

Host-plant choice and larval growth in the cinnabar moth: do pyrrolizidine alkaloids play a role?

Witte et al. (1992) described two distinct chemotypes of Senecio jacobaea L. Asteraceae, a chemotype with jacobine as one of the major pyrrolizidine alkaloids (PAs) and a chemotype with erucifoline as one of the major PAs. We hypothesized that the presence of erucifoline might be the factor responsible for the lack of success of the cinnabar moth on Senecio erucifolius L. Asteraceae and the S. jacobaea erucifoline chemotype. We performed a survey of the distribution of the two chemotypes in the Netherlands and compared this with the distribution map of Tyria jacobaeae L. Lepidoptera, Arctiidae. The distribution of the two chemotypes in the Netherlands is poorly correlated with the distribution of the cinnabar moth. The jacobine chemotype occurs along the coast and the erucifoline chemotype predominantly inward.An oviposition experiment showed that the cinnabar moth did not discriminate between the two chemotypes of S. jacobaea and S. erucifolius. Larval performance did not differ between the two chemotypes and species. Although the distribution of S. jacobaea jacobine chemotype is loosely associated with the abundance of the cinnabar moth the oviposition and growth experiments indicate that other factors than the presence of erucifoline play a role in this association.The absence of recordings of S. erucifolius as a foodplant for the cinnabar moth might be explained by the phenology of the foodplant. Ovipositing females of the univoltine cinnabar moth prefer flowering plants for oviposition. S. erucifolius starts flowering about 1–2 month later than S. jacobaea just after the peak density of moths.     

Cryptic species in the cosmopolitan Bugula neritina complex (Bryozoa,Cheilostomata)

Previous analyses of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) and γ‐proteobacterial endosymbiont diversity have suggested that the marine bryozoan Bugula neritina is a complex of three cryptic species, namely Types S, D and N. Types D and N were previously reported to have restricted distributions along California (western USA) and Delaware and Connecticut (eastern USA), respectively, whereas Type S is considered widespread in tropical, subtropical and temperate regions due to anthropogenic transport. Here, Bayesian species delimitation analysis of a data set composed of two mitochondrial (COI and large ribosomal RNA subunit [16S]) and two nuclear genes (dynein light chain roadblock type‐2 protein [DYN] and voltage‐dependent anion‐selective channel protein [VDAC]) demonstrated that Types S, D and N correspond to three biological species. This finding was significantly supported, in spite of the combinations of priors applied for ancestral population size and root age. Furthermore, COI sequences were used to assess the introduction patterns of the cosmopolitan Type S species. Two COI haplotypes of Type S (S1a and S1d) were found occurring at a global scale. Mantel tests showed correlation between these haplotypes and local sea surface temperature tolerance. Accordingly, the distributions of Type S haplotypes may reflect intraspecific temperature tolerance variation, in addition to the role of introduction vectors. Finally, we show that the Type N may also have been introduced widely, as this species was found for the first time in Central California and north‐eastern Australia.     

Potent cytotoxins produced by a microbial symbiont protect host larvae from predation

Larvae of the sessile marine invertebrate Bugula neritina (Bryozoa) are protected by an effective chemical defense. From the larvae, we isolated three bryostatin-class macrocyclic polyketides, including the novel bryostatin 20, that deterred feeding by a common planktivorous fish that co-occurs with B. neritina. A unique bacterial symbiont of B. neritina, Endobugula sertula, was hypothesized as the putative source of the bryostatins. We show that: (1) bryostatins are concentrated in B. neritina larvae and protect them against predation by fish; (2) the adults are not defended by bryostatins; and (3) E. sertula produces bryostatins. This study represents the first example from the marine environment of a microbial symbiont producing an anti-predator defense for its host and, in this case, specifically for the hosts larval stage, which is exceptionally vulnerable to predators.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae)

The composition and yield of oil in 615 trees representing the natural populations of Melaleuca alternifolia, or tea tree, was investigated. A sixth distinct oil chemotype was identified. Of the six chemotypes, one chemotype is dominated by terpinen-4-ol, one by 1,8-cineole, one by terpinolene and the remaining three chemotypes are all dominated by 1,8-cineole and differ in either terpinen-4-ol or terpinolene content. Whilst most chemotypes are present throughout the distribution range, a definite correspondence of oil types with geographic location was found. Terpinen-4-ol types predominate in and around the Bungawalbin basin in the Casino area of northern New South Wales (NSW), high 1,8-cineole types predominate toward the southern end of the distribution around Grafton and terpinolene types predominate in southern Queensland. Preliminary formulae have been developed to allow comparisons of oil data obtained by steam distillation with a static headspace gas chromatography method.     

Three activators of protein kinase C, bryostatins, dioleins, and phorbol esters, show differing specificities of action on GH4 pituitary cells

Phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol acetate (TPA) activate the calcium- and phospholipid-dependent protein kinase C and enhance three biological responses (prolactin release, prolactin synthesis, and cell stretching) in GH4C5 rat pituitary cells. We have examined several actions on GH4C5 cells of TPA and two other classes of protein kinase C activators, synthetic cell permeant dioleins and bryostatins isolated from the marine bryozoan Bugula neritina. Bryostatins 1 and 2 (B1 and B2, respectively) competed for [3H]phorbol 12,13-dibutyrate binding to the protein kinase C complex in intact cells nearly equipotently with TPA. B1 and B2, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoylglycerol (Di8) as well as TPA each activated partially purified protein kinase C from GH4C5 cells. B1, B2, and TPA each enhanced the acute release of prolactin from GH4C5 cells to a similar maximal extent. B1, B2, and TPA also enhanced prolactin synthesis. However, B1 and B2 were only partial agonists because they enhanced prolactin synthesis to a lesser maximal extent than did TPA and, given in combination, they reduced TPA-enhanced prolactin synthesis. OAG and Di8 stimulated prolactin release (to a lesser maximal extent than TPA) and did not stimulate prolactin synthesis. Pretreatment with OAG did not reduce TPA-stimulated prolactin release or synthesis. B2 and TPA induced cell stretching in GH4C5 cells, whereas B1, OAG, and Di8 induced little if any stretching. B1, but not B2, given in combination with TPA antagonized TPA-induced stretching but did not reduce thyrotropin-releasing hormone- or epidermal growth factor-induced stretching. We conclude that the bryostatins, phorbol esters, and dioleins bind to the same site on the protein kinase C complex to activate the enzyme, but they alter three biological responses in GH4C5 cells with selectivities and efficacies that differ. We propose that different activators of protein kinase C (such as bryostatins, dioleins, and phorbol esters) may elicit different cellular responses by altering the substrate specificity or activating multiple forms of the kinase.     

Chemical variation in the leaf essential oil of Melaleuca quinquenervia (Cav.) S.T. Blake

An examination of the leaf oils of Melaleuca quinquenervia over its geographical range in Australia and Papua New Guinea has shown wide variation in chemical composition but only two major chemotypes. Chemotype 1 is comprised of E-nerolidol (74–95%) and linalool (14–30%) and is found from Sydney, north along the east coast of Australia to Selection Flat, New South Wales, with an isolated occurrence near Maryborough, Queensland. Two divisions occur in this chemotype which are based on the presence or absence of significant proportions of linalool (14–40%). Chemotype 2 contains 1,8-cineole (10–75%), viridiflorol (13–66%), α-terpineol (0.5–14%) and β-caryophyllene (0.5–28%) in varying proportions and order of dominance in the oils. It is found throughout the distribution of the species, from Sydney to Papua New Guinea and New Caledonia. Within chemotype 2 there appears to be a continuous spread of oil composition without formation of any further discrete divisions as in chemotype 1.Analyses have shown that M. quinquenervia trees that occur at latitudes south of 25°S have high oil yields (1–3% w/w%, fresh leaves) and comprise chemotypes 1 and 2. North of 25°S, however, chemotype 1 does not occur and oil yields amongst the Australian populations are uniformly low (0.1–0.2%).