Christopher N. Battershill

Shotgun Cloning and Heterologous Expression of the Patellamide Gene Cluster as a Strategy to Achieving Sustained Metabolite Production

Shotgun cloning into E. coli of genomic DNA from Prochloron sp., symbiont of the seasquirt Lissoclinum patella, resulted in the heterologous expression of the patellamide gene cluster and subsequent production of patellamide D (1) and ascidiacyclamide (2) at levels of 80?100 ng?mL?1.

The distribution of brominated long-chain fatty acids in sponge and symbiont cell types from the tropical marine sponge Amphimedon terpenensis

The tropical marine spongeAmphimedon terpenensis (family Niphatidae, order Haplosclerida) has previously been shown to possess unusual lipids, including unusual fatty acids. The biosynthetic origin of these fatty acids is of interest as the sponge supports a significant population of eubacterial and cyanobacterial symbionts. The total fatty acid composition of the sponge was analyzed by gas chromatography/mass spectrometry of the methyl esters. Among the most abundant of the fatty acids in intact tissue were 16∶0, 18∶0 and 3,7,11,15-tetramethylhexadecanoic (phytanic) acid. In addition, three brominated fatty acids, (5E,9Z)-6-bromo-5,9-tetracosadienoic acid (24∶2Br), (5E,9Z)-6-bromo-5,9-pentacosadienoic acid (25∶2Br) and (5E,9Z)-6-bromo-5,9-hexacosadienoic acid (26∶2Br) were also present. The three brominated fatty acids, together with phytanic acid, were isolated from both ectosomoal (superficial) and choanosomal (internal) regions of the sponge. Analysis of extracts prepared from sponge/symbiont cells, partitioned by density gradient centrifugation on Ficoll, indicated that phytanic acid and the three brominated fatty acids were associated with sponge cells only. Further, a fatty acid methyl ester sample from intact tissue ofA. terpenensis was partitioned according to phospholipid class, and the brominated fatty acids were shown to be associated with the phosphatidylserine and phosphatidylethanolamine fractions that are commonly present in marine sponge lipids. The phosphatidylcholine and phosphatidylglycerol fractions were rich in the relatively shorter chain fatty acids (16∶0 and 18∶0). The association of brominated long-chain fatty acids (LCFA) with sponge cells has been confirmed. The findings allow comment on the use of fatty acid profiles in chemotaxonomy and permit further interpretation of LCFA biosynthetic pathways in sponges. The assignment of the sponge studied, which is currently placed asA. terpenensis, is being supported to some extent, but the species is unusual in having C25 fatty acids as the major constituent in this group. Other factors, such as season or microenvironmental conditions, may influence observed fatty acid composition which tends to reduce the usefulness of fatty acid profiles as markers in sponge chemotaxonomy.

TERPENES IN SPONGE CELL-MEMBRANES - CELL-SEPARATION AND MEMBRANE FRACTIONATION STUDIES WITH THE TROPICAL MARINE SPONGE AMPHIMEDON-SP

The differing sponge and symbiotic microbial cell types in the tropical marine spongeAmphimedon sp. were fractionated according to density, investigated by electron microscopy, and analyzed by high-performance liquid chromatography and nuclear magnetic resonance for the presence of the terpene metabolite diisocyanoadociane (1) and Δ5,7-sterols (2–7). A sample of whole sponge was dissected into superficial ectosome and deeper choanosome. The superficial tissue was found to be enriched in sterol relative to choanosome; however, extracts from both tissues contained terpene. Dissociation of whole sponge followed by Ficoll density gradient fractionation showed that there are two chemically distinct types of sponge cells inAmphimedon sp.—small non-nucleolated cells of low density contain terpene 1 together with sterols, while larger nucleolated cells contain significant levels of terpene, but only traces of sterol. Membrane fractionation studies were undertaken to establish whether the terpene components were located specifically in the cell membranes of these two cell types. A membrane vesicle pellet spun down at 100,000×g from small sponge cells contained sterols, but only traces of terpene, whereas the membrane vesicle preparation from heavier cells contained both terpenes and sterols. Subsequently, the presence of terpenes together with sterols was demonstrated in a membrane vesicle preparation of purity >90% prepared from bacteria-free sponge cells. These results provide the first experimental evidence that terpenes are associated with sponge cell membranes, where they may function as structural components.

The distribution and abundance of dictyoceratid sponges in relation to hydrodynamic features: identifying candidates and environmental conditions for sponge aquaculture

The distribution and abundance of dictyoceratid sponges was surveyed to a depth of 20 m at eleven locations within the Palm Island Group, Great Barrier Reef (GBR), Australia. These surveys were related to prevailing hydrodynamic conditions to identify candidates and environmental conditions for sponge aquaculture. Locations were classified as sheltered, intermediate and exposed using quantitative wave exposure and current force models. The species richness of dictyoceratid sponges was high with ten taxa, but the abundance of most species was low with patchy distributions. Two species, Coscinoderma sp. and Rhopaloeides odorabile, were abundant, and detailed surveys of these species were conducted at seven locations representing common habitats within the Palm Island Group. Coscinoderma sp. was present at all locations and although abundances differed significantly across locations, this was not related to hydrodynamic conditions. In contrast, R. odorabile was only present at exposed locations with low abundance. The higher abundance and broad distribution of Coscinoderma sp. supports its selection as an aquaculture candidate. In contrast, R. odorabile was less abundant and was restricted to high-energy environments making aquaculture more problematic. The present study demonstrates the importance of ecological data in the decision-making process for new species aquaculture.

Isolation of brominated long-chain fatty acids from the phospholipids of the tropical marine spongeAmphimedon terpenensis

Preliminary investigation of the phospholipid fatty acid composition of the tropical marine spongeAmphimedon terpenensis by gas chromatography/mass spectrometry revealed the presence of some novel brominated fatty acids. Two new brominated fatty acids, (5E, 9Z)-6-bromo-5,9-tetracosadienoic acid (2a) and (5E, 9Z)-6-bromo-5,9-pentacosadienoic acid (3a) were subsequently isolated from a chloroform/methanol (3∶1, vol/vol) extract of the sponge and characterized as their methyl esters 2b and 3b. The known brominated fatty acid (5E, 9Z)-6-bromo-5,9-hexacosadienoic acid (4a) was also isolated. The new fatty acid methyl esters were confirmed as brominated δ5,9 acid derivatives by chemical ionization mass spectrometry. The position of the bromine substituent was determined to be C-6 by nuclear magnetic resonance techniques while the stereochemistry of the two double bonds was deduced by nuclear Overhauser enhancement difference spectroscopy. The biosynthetic implications of the co-occurrence of the three brominated acids are discussed.

New methods for medicinal chemistry - Universal gene cloning and expression systems for production of marine bioactive metabolites

Natural products from symbiotic or commensal associations between marine invertebrate and microbial organisms show exceptional promise as pharmaceuticals in many therapeutic areas. An economic and sustainable global market supply due to difficulty of synthesis is cited as the main obstacle for exploitation of these otherwise exciting marine bioactive compounds. Different strategies have been evoked to overcome this impediment as long-term harvesting of wild stocks from the environment is considered unsound, and other modes of production based on biosynthesis, such as aquaculture, have not yet been proven as reliable. One option is to clone the genes encoding the biosynthetic expression of a lead metabolite into a surrogate host suitable for industrial-scale fermentation. To facilitate this goal we are developing a universal system to clone and express genes responsible for biosynthesis of natural products from both eukaryotic and prokaryotic partners of marine symbioses. The ability to harness the complete meta-transcriptome of entire biosynthetic pathways is particularly valuable where the biogenesis of a target natural product occurring within a complex symbiotic association is unclear.

Demographic variability and long-term change in a coral reef sponge along a cross-shelf gradient of the Great Barrier Reef

Effects of anthropogenic inputs on corals are well documented in regard to the ecology of coral reefs. However, responses to anthropogenic changes by sponge assemblages, also a key component of coral reefs, have received less attention. This paper quantifies differences in abundance, size and distribution of the sponge Rhopaloeides odorabile across a cross-shelf reef system on the central Great Barrier Reef (GBR) from neritic to outer reef oligotrophic waters. Benthic surveys were conducted in reef habitats spatially separated across the continental shelf. The mean abundance of R. odorabile increased significantly with increasing distance from coastal habitats, with 3.5 times more individuals offshore than inshore. In contrast, the mean size (volume) of R. odorabile individuals did not differ significantly across the cross-shelf reef system. On inshore reefs, R. odorabile was restricted to depths <10 m, with a preference for shallower depths (5–6 m). On offshore reefs, R. odorabile was found as deep as 15 m and predominantly between 9 and 10 m. These demographic changes in R. odorabile populations, together with a general population size reduction from surveys made decades prior, suggest a response to anthropogenic impacts across the continental shelf of the central GBR.

Phylogeny drives large scale patterns in Australian marine bioactivity and provides a new chemical ecology rationale for future biodiscovery.

Twenty-five years of Australian marine bioresources collecting and research by the Australian Institute of Marine Science (AIMS) has explored the breadth of latitudinally and longitudinally diverse marine habitats that comprise Australia’s ocean territory. The resulting AIMS Bioresources Library and associated relational database integrate biodiversity with bioactivity data, and these resources were mined to retrospectively assess biogeographic, taxonomic and phylogenetic patterns in cytotoxic, antimicrobial, and central nervous system (CNS)-protective bioactivity. While the bioassays used were originally chosen to be indicative of pharmaceutically relevant bioactivity, the results have qualified ecological relevance regarding secondary metabolism. In general, metazoan phyla along the deuterostome phylogenetic pathway (eg to Chordata) and their ancestors (eg Porifera and Cnidaria) had higher percentages of bioactive samples in the assays examined. While taxonomy at the phylum level and higher-order phylogeny groupings helped account for observed trends, taxonomy to genus did not resolve the trends any further. In addition, the results did not identify any biogeographic bioactivity hotspots that correlated with biodiversity hotspots. We conclude with a hypothesis that high-level phylogeny, and therefore the metabolic machinery available to an organism, is a major determinant of bioactivity, while habitat diversity and ecological circumstance are possible drivers in the activation of this machinery and bioactive secondary metabolism. This study supports the strategy of targeting phyla from the deuterostome lineage (including ancestral phyla) from biodiverse marine habitats and ecological niches, in future biodiscovery, at least that which is focused on vertebrate (including human) health.

Marine Metabolites and Metal Ion Chelation: Intact Recovery and Identification of an Iron(II) Complex in the Extract of the Ascidian Eudistoma gilboviride

Sequestering sea squirts: Liquid chromatography with parallel inductively coupled plasma mass spectrometry/electrospray mass spectrometry was used to discover and identify novel lipophilic metal complexes in the solvent extract of the ascidian Eudistoma gilboviride. Using these tools it is now possible to examine whether such complexes play an integral part in an organisms physiology.

Taxonomic revision of the genus Latrunculia Du Bocage (Porifera: Demospongiae: Latrunculiidae) in New Zealand

Abstract The genus Latrunculia in New Zealand was believed to be represented by at least three species: L. brevis Ridley & Dendy and two undescribed species. However, results from recent studies indicate that the genus contained a complex of at least eight genetically distinct species. In this paper we revise the taxonomy of Latrunculia in New Zealand and describe nine new species. Eight of these were previously identified using genetic techniques (i.e., L. kaakaariki sp. nov., L. duckworthi sp. nov., L. procumbens sp. nov., L. wellingtonensis sp. nov., L. kaikoura sp. nov., L. triverticillata sp. nov., L. fiordensis sp. nov., and L. millerae sp. nov.) and one (L. oxydiscorhabda sp. nov.) was distinguished based only on morphological differences. The species described fall into two morphological groups, spinispiraefera and triverticillata, which can be diagnosed by the number of whorls present along the discorhabd axis. The status of L. brevis, and other subantarctic species (i.e., Latrunculia bocagei, L. antarctica, L. lendenfeldi, L. spinispiraefera) is discussed; all of these species are considered to be valid but not conspecific with any of the species described here.