Evaluation of secondary metabolites from algae and epibiotic bacteria as allelochemicals and/or inducers of coral larval settlement

In the marine environment chemical signals play critical roles at every organisational level. One emerging area of interest in the field of Marine Chemical Ecology is the role of bacterially derived chemical signals, in particular for the induction of larval settlement of marine invertebrates. Although bacteria have long been recognised to induce larval settlement, to date no inducer of bacterial origin has been fully characterised. In Chapter 2, I describe the isolation of the bacterium Pseudoalteromonas J010 from the surface of crustose coralline algae, which trigger the larval settlement of acroporid corals. In a bioassay-guided fashion, I characterised the metamorphic inducer in this bacterium as tetrabromopyrrole (TBP). TBP rapidly induces larval metamorphosis, however the larvae fail to attach to the substratum when exposed to TBP. The toxic nature of this compound suggests that larval metamorphosis to TBP is a stress response, rather than an evolved response to a habitat specific cue. The production of TBP may provide Pseudoalteromonas J010 with an advantage to persist in the highly competitive biofilm environment. To further explore this hypothesis, I screened the allelochemical profile of this strain and characterised further bioactive metabolites, including novel korormicin derivatives and a polybrominated pyrrole with anti-larval, anti-bacterial, anti-fungal and anti-protozoal properties (Chapter 3). Because, TBP did not explain the inductive properties of CCA on coral larval settlement, I addressed the origin and characteristics of inductive cues from CCA (Chapter 4). This resulted in the isolation of purified fractions that readily induced complete larval settlement, including i) low molecular weight organic-soluble compounds identified as glycoglycerolipids and ii) high molecular weight polymeric aqueous-soluble cue(s). These common algal metabolites may explain the highly inductive properties of CCA on acroporid coral larval settlement. I further demonstrated that these settlement inducers can be immobilised, enabling controlled settlement of coral larvae on target surfaces. Targeted larval settlement on substrata is common practise in reef rehabilitation, particularly for techniques based on sexual reproduction of corals. To improve current reef rehabilitation techniques, I explored methods to enhance post settlement survival of settled coral spat (Chapter 5)

In Silico Modeling of Spirolides and Gymnodimines: Determination of S Configuration at Butenolide Ring Carbon C-4

Only few naturally occurring cyclic imines have been fully structurally elucidated or synthesized to date. The configuration at the C-4 carbon plays a pivotal role in the neurotoxicity of many of these metabolites, for example, gymnodomines (GYMs) and spirolides (SPXs). However, the stereochemistry at this position is not accessible by nuclear Overhauser effect—nuclear magnetic resonance spectroscopy (NOE-NMR) due to unconstrained rotation of the single carbon bond between C-4 and C-5. Consequently, the relative configuration of GYMs and SPXs at C-4 and its role in protein binding remains elusive. Here, we determined the stereochemical configuration at carbon C-4 in the butenolide ring of spirolide- and gymnodimine-phycotoxins by comparison of measured 13C NMR shifts with values obtained in silico using force field, semiempirical and density functional theory methods. This comparison demonstrated that modeled data support S configuration at C-4 for all studied SPXs and GYMs, suggesting a biosynthetically conserved relative configuration at carbon C-4 among these toxins

Siderophore purification with titanium dioxide nanoparticle solid phase extraction

Siderophores are metal chelators produced by microorganisms to facilitate binding and uptake of iron. The isolation and characterization of siderophores are impeded by typically low siderophore yields and the complexity of siderophore-containing extracts generated with traditional purification methods. We investigated titanium dioxide nanoparticle solid-phase extraction (TiO2 NP SPE) as a technique to selectively concentrate and purify siderophores from complex matrices for subsequent LC-MS detection and identification. TiO2 NP SPE showed a high binding capacity (15.7 ± 0.2 μmol mg−1 TiO2) for the model siderophore desferrioxamine B (DFOB) and proved robust to pH changes and the presence of EDTA. These are significant advances in comparison to immobilized metal affinity chromatography (IMAC). The TiO2 NP SPE was highly selective and recovered 77.6 ± 6.2% of DFOB spiked to a compositionally complex bacterial culture supernatant. The simple clean-up procedure removed the majority of contaminants and allowed direct detection of siderophores from the LC-MS base peak chromatogram. The ‘untargeted’ purification and analysis of an untreated supernatant of iron-deprived bacterial culture allowed for the direct identification of two known and three novel ferrioxamines. Thus, TiO2 NP SPE in combination with LC-MS offers great potential as a discovery platform for the purification and subsequent quantification or identification of novel siderophores of microbial origin

Niche-based assembly of bacterial consortia on the diatom Thalassiosira rotula is stable and reproducible

With each cell division, phytoplankton create new space for primary colonization by marine bacteria. Although this surface microenvironment is available to all planktonic bacterial colonizers, we show the assembly of bacterial consortia on a cosmopolitan marine diatom to be highly specific and reproducible. While phytoplankton–bacteria interactions play fundamental roles in marine ecosystems, namely primary production and the carbon cycle, the ecological paradigm behind epiphytic microbiome assembly remains poorly understood. In a replicated and repeated primary colonization experiment, we exposed the axenic diatom Thalassiosira rotula to several complex and compositionally different bacterial inocula derived from phytoplankton species of varying degrees of relatedness to the axenic Thalassiosira host or natural seawater. This revealed a convergent assembly of diverse and compositionally different bacterial inocula, containing up to 2071 operational taxonomic units (OTUs), towards a stable and reproducible core community. Four of these OTUs already accounted for a cumulative abundance of 60%. This core community was dominated by Rhodobacteraceae (30.5%), Alteromonadaceae (27.7%), and Oceanospirillales (18.5%) which was qualitatively and quantitatively most similar to its conspecific original. These findings reject a lottery assembly model of bacterial colonization and suggest selective microhabitat filtering. This is likely due to diatom host traits such as surface properties and different levels of specialization resulting in reciprocal stable-state associations

Selective purification of catecholate, hydroxamate and α-hydroxycarboxylate siderophores with titanium dioxide affinity chromatography

Siderophores, high affinity iron chelators, play a key role in the uptake of iron by microorganisms and regulate many biological functions. Siderophores are categorized by their chelating group, e.g., catecholates, hydroxamates, α-hydroxycarboxylates. Natural concentrations of siderophores are often either too low or sample matrices are too complex for direct analysis by, e.g., liquid chromatography – mass spectrometry. Therefore, both concentration and purification are prerequisite for reliable analyses. However, a chromatographic technique that is selective for all siderophore classes and affords high levels of purification is lacking. We developed a titanium dioxide affinity chromatography (TDAC) solid-phase extraction (SPE) that affords the selective purification of these siderophore classes from complex sample matrices with recoveries up to 82%. The one-step purification removed most non-ligand sample ‘contaminants’, therefore, affording the straightforward identification of siderophore peaks in base peak chromatograms. As a proof of concept, the bioinformatic processing, dereplication of known features and selection of significant features in the TDAC eluates afforded a fast identification of six novel siderophores (woodybactines) from bacterial supernatants. We propose TDAC SPE as a fast and cost-effective methodology to screen for known or discover novel siderophores in natural samples in combination with untargeted bioinformatic processing by, e.g., XCMS. The method is scalable and yielded large amounts of highly purified siderophores from bacterial culture supernatants, providing an effective quantitative sample clean-up for, e.g., NMR structure elucidation

Antioxidant Activity and Phenolic Content of Marine Dissolved Organic Matter and Their Relation to Molecular Composition

The potential of marine dissolved organic matter (DOM) for free radical scavenging has been extensively evaluated, however, the quantitative assessment of the antioxidant potential has been recently measured for the first time. The linkage of the DOM antioxidant potential to its molecular composition has not yet been examined. Following this line, this article takes a step forward by assessing, throughout a polarity-mediated fractionation, (1) the antioxidant capacity and phenolic content and (2) the molecular characterization of DOM in a more exhaustive manner. (3) The DOM antioxidant potential and phenolic content was linked to the molecular composition of DOM, which was molecularly characterized using ultrahigh resolution Fourier transform Ion Cyclotron Resonance mass spectrometry (FT-ICR MS). Antioxidant activity and phenolic content were quantified by the free radical 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS⋅) and the Folin-Ciocalteu methods, respectively. We considered three types of different natural DOM samples: the deep North Pacific Ocean, the oligotrophic surface of the North Pacific Ocean and porewater from the sulfidic tidal flats of the Wadden Sea. Bulk porewater and its individual polarity fractions presented the highest antioxidant activity and phenolic content. DOM from the water column samples had lower antioxidant activity and phenolic content than porewater, but exceeded what it is commonly found in macroalgae, microalgae, fruits and vegetables with cosmeceutical purposes. Our values were similar to published values for terrestrial DOM. The variations in bioactivity were dependent on polarity and molecular composition. The high resolution and high mass accuracy used to determine the molecular composition of marine DOM and the chemometric and multistatistical analyses employed have allowed to distinguish molecular categories that are related to the bioactive potential. As a future perspective, we performed cytotoxicity tests with human cells and propose marine DOM as a natural ingredient for the development of cosmeceutical products

LC-MS/MS Method Development for the Discovery and Identification of Amphidinols Produced by Amphidinium

Amphidinols are polyketides produced by dinoflagellates suspected of causing fish kills. Here, we demonstrate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of amphidinols (AM). Novel AM were detected by neutral loss (NL) scan and then quantified together with known AM by selection reaction monitoring (SRM). With the new method, AM were detected in four of eight analyzed strains with a maximum of 3680 fg toxin content per cell. In total, sixteen novel AM were detected by NL scan and characterized via their fragmentation patterns. Of these, two substances are glycosylated forms. This is the first detection of glycosylated AM

Crystalline iron oxides stimulate methanogenic benzoate degradation in marine sediment- derived enrichment cultures

Elevated dissolved iron concentrations in the methanic zone are typical geochemical signatures of rapidly accumulating marine sediments. These sediments are often characterized by co-burial of iron oxides with recalcitrant aromatic organic matter of terrigenous origin. Thus far, iron oxides are predicted to either impede organic matter degradation, aiding its preservation, or identified to enhance organic carbon oxidation via direct electron transfer. Here, we investigated the effect of various iron oxide phases with differing crystallinity (magnetite, hematite, and lepidocrocite) during microbial degradation of the aromatic model compound benzoate in methanic sediments. In slurry incubations with magnetite or hematite, concurrent iron reduction, and methanogenesis were stimulated during accelerated benzoate degradation with methanogenesis as the dominant electron sink. In contrast, with lepidocrocite, benzoate degradation, and methanogenesis were inhibited. These observations were reproducible in sediment-free enrichments, even after five successive transfers. Genes involved in the complete degradation of benzoate were identified in multiple metagenome assembled genomes. Four previously unknown benzoate degraders of the genera Thermincola (Peptococcaceae, Firmicutes), Dethiobacter (Syntrophomonadaceae, Firmicutes), Deltaproteobacteria bacteria SG8_13 (Desulfosarcinaceae, Deltaproteobacteria), and Melioribacter (Melioribacteraceae, Chlorobi) were identified from the marine sediment-derived enrichments. Scanning electron microscopy (SEM) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) images showed the ability of microorganisms to colonize and concurrently reduce magnetite likely stimulated by the observed methanogenic benzoate degradation. These findings explain the possible contribution of organoclastic reduction of iron oxides to the elevated dissolved Fe2+ pool typically observed in methanic zones of rapidly accumulating coastal and continental margin sediments

Anti-Staphylococcal Calopins from Fruiting Bodies of Caloboletus radicans

Three new and seven known calopins were isolated from Caloboletus radicans. The structures of the new cyclocalopins, 8-deacetylcyclocalopin B (1), cyclocalopin A-15-ol (2), and 12,15-dimethoxycyclocalopin A (3), were mainly elucidated by NMR and MS data analysis. The stereochemistry of 1–3 was assigned based on ROE correlations, coupling constants and by comparison of their CD spectra with those of similar known calopins. While 1–10 were inactive against two cancer cell lines, they displayed antistaphylococcal activity against methicillin-resistant Staphylococcus aureus strains (MRSA) with MIC values of 16−256 μg/mL. Moreover, some calopins were active against the fish pathogen Enterococcus faecalis F1B1

Identification of the Antibacterial Compound Produced by the Marine Epiphytic Bacterium Pseudovibrio sp. D323 and Related Sponge-Associated Bacteria

Surface-associated marine bacteria often produce secondary metabolites with antagonistic activities. In this study, tropodithietic acid (TDA) was identified to be responsible for the antibacterial activity of the marine epiphytic bacterium Pseudovibrio sp. D323 and related strains. Phenol was also produced by these bacteria but was not directly related to the antibacterial activity. TDA was shown to effectively inhibit a range of marine bacteria from various phylogenetic groups. However TDA-producers themselves were resistant and are likely to possess resistance mechanism preventing autoinhibition. We propose that TDA in isolate D323 and related eukaryote-associated bacteria plays a role in defending the host organism against unwanted microbial colonisation and, possibly, bacterial pathogens