Center for Marine Biodiversity and Conservation

A structurally novel metabolite, fatuamide A (1), was discovered from a laboratory cultured strain of the marine cyanobacterium Leptolyngbya sp., collected from Faga'itua Bay, American Samoa. A bioassay-guided approach using NCI-H460 human lung cancer cells directed the isolation of fatuamide A, which was obtained from the most cytotoxic fraction. The planar structure of fatuamide A was elucidated by integrated NMR and MS/MS analysis, and a combination of bioinformatic and computational approaches was used to deduce the absolute configuration at its eight stereocenters. A putative hybrid PKS/NRPS biosynthetic gene cluster responsible for fatuamide A production was identified from the sequenced genomic DNA of the cultured cyanobacterium. The biosynthetic gene cluster possessed elements that suggested fatuamide A binds metals, and this metallophore property was demonstrated by native metabolomics and indicated a preference for binding copper. The producing strain was found to be highly resistant to toxicity from elevated copper concentrations in culture media.

The diversity of the marine cyanobacterium Synechococcus can be broadly separated into clades, with clade II typically present in warm oligotrophic water, and clades I and IV found in cooler coastal water. We found amplicon sequence variants (ASVs) belonging to clade II in the nutrient-replete waters of San Diego Bay (SDB). Using the 16S rRNA gene, 18S rRNA gene and internal transcribed spacer region sequencing, we analysed multiple locations in SDB monthly for over a year, with additional samples dating back to 2015. Synechococcus community composition differed from the nearby coast into SDB in terms of dominant clade and ASVs. Specific clade II ASVs became relatively more abundant towards the back of the bay and showed seasonality, with higher relative abundance in the warm months. Select ASVs group phylogenetically and show similar seasonal and spatial distribution patterns, indicating these ASVs have adapted to SDB. Isolates matching clade II ASVs from SDB show pigment composition that is better adapted to the green light available in SDB, further supporting our findings. Other microbial taxa also show SDB enrichment, providing evidence that SDB is a chemostat-like environment where circulation, temperature, light and other environmental conditions create a zone for microbial evolution and diversification.

Drylands cover one-third of the Earths surface and are one of the largest terrestrial sinks for methane. Understanding the structure-function interplay between members of arid biomes can provide critical insights into mechanisms of resilience toward anthropogenic and climate-change-driven environmental stressors-water scarcity, heatwaves, and increased atmospheric greenhouse gases. This study integrates in situ measurements with culture-independent and enrichment-based investigations of methane-consuming microbiomes inhabiting soil in the Anza-Borrego Desert, a model arid ecosystem in Southern California, United States. The atmospheric methane consumption ranged between 2.26 and 12.73 μmol m2 h-1, peaking during the daytime at vegetated sites. Metagenomic studies revealed similar soil-microbiome compositions at vegetated and unvegetated sites, with Methylocaldum being the major methanotrophic clade. Eighty-four metagenome-assembled genomes were recovered, six represented by methanotrophic bacteria (three Methylocaldum, two Methylobacter, and uncultivated Methylococcaceae). The prevalence of copper-containing methane monooxygenases in metagenomic datasets suggests a diverse potential for methane oxidation in canonical methanotrophs and uncultivated Gammaproteobacteria. Five pure cultures of methanotrophic bacteria were obtained, including four Methylocaldum. Genomic analysis of Methylocaldum isolates and metagenome-assembled genomes revealed the presence of multiple stand-alone methane monooxygenase subunit C paralogs, which may have functions beyond methane oxidation. Furthermore, these methanotrophs have genetic signatures typically linked to symbiotic interactions with plants, including tryptophan synthesis and indole-3-acetic acid production. Based on in situ fluxes and soil microbiome compositions, we propose the existence of arid-soil reverse chimneys, an empowered methane sink represented by yet-to-be-defined cooperation between desert vegetation and methane-consuming microbiomes.

Pseudopeptides are attractive agents for protease inhibition due to their structural similarities to the natural substrates of these enzymes, as well as their enhanced stability and resistance to enzymatic degradation. We report three new ketomemicin pseudopeptides (1-3) from extracts of the marine actinomycete Salinispora pacifica strain CNY-498. Their constitution and relative configuration were elucidated using NMR, mass spectrometry, and quantum chemical calculations. Using GNPS molecular networking and publicly available Salinispora LCMS datasets, five additional ketomemicin analogs (4-8) were identified with ketomemicin production detected broadly across Salinispora species. The ketomemicin biosynthetic gene cluster (ktm) is highly conserved in Salinispora, occurring in 79 of 118 public genome sequences, including eight of the nine named species. Outside Salinispora, ktm homologs were detected in various genera of the phylum Actinomycetota that might encode novel ketomemicin analogs. Ketomemicins 1-3 were tested against a panel of eleven proteases, with 2 displaying moderate inhibitory activity. This study describes the first report of ketomemicin production by Salinispora cultures, the distribution of the corresponding biosynthetic gene cluster, and the protease inhibitory activity of new ketomemicin derivatives.

Pollution can negatively impact aquatic ecosystems, aquaculture operations, and recreational water quality. Many aquatic microbes can sequester or degrade pollutants and have been utilized for bioremediation. While planktonic and benthic microbes are well-studied, host-associated microbes likely play an important role in mitigating the negative impacts of aquatic pollution and represent an unrealized source of microbial potential. For example, aquatic organisms that thrive in highly polluted environments or concentrate pollutants may have microbiomes adapted to these selective pressures. Understanding microbe-pollutant interactions in sensitive and valuable species could help protect human well-being and improve ecosystem resilience. Investigating these interactions using appropriate experimental systems and overcoming methodological challenges will present novel opportunities to protect and improve aquatic systems. In this perspective, we review examples of how microbes could mitigate negative impacts of aquatic pollution, outline target study systems, discuss challenges of advancing this field, and outline implications in the face of global changes.

Mollusca is a morphologically diverse phylum, exhibiting an immense variety of calcium carbonate structures. Proteomic studies of adult shells often report high levels of rapidly-evolving, novel shell matrix proteins (SMPs), which are hypothesized to drive shell diversification. However, relatively little is known about the phylogenetic distribution of SMPs, or about the function of individual SMPs in shell construction. To understand how SMPs contribute to shell diversification a thorough characterization of SMPs is required. Here, we build tools and a foundational understanding of SMPs in the marine gastropod species Crepidula fornicata and Crepidula atrasolea because they are genetically-enabled mollusc model organisms. First, we established a staging system of shell development in C. atrasolea for the first time. Next, we leveraged previous findings in C. fornicata combined with phylogenomic analyses of 95 metazoan species to determine the evolutionary lineage of its adult SMP repertoire. We found that 55% of C. fornicatas SMPs belong to molluscan orthogroups, with 27% restricted to Gastropoda, and only 5% restricted at the species level. The low percentage of species-restricted SMPs underscores the importance of broad-taxon sampling and orthology inference approaches when determining homology of SMPs. From our transcriptome analysis, we found that the majority of C. fornicata SMPs that were found conserved in C. atrasolea were expressed in both larval and adult stages. We then selected a subset of SMPs of varying evolutionary ages for spatial-temporal analysis using in situ hybridization chain reaction (HCR) during larval shell development in C. atrasolea. Out of the 18 SMPs analyzed, 12 were detected in the larval shell field. These results suggest overlapping larval vs. adult SMP repertoires. Using multiplexed HCR, we observed five SMP expression patterns and three distinct cell populations within the shell field. These patterns support the idea that modular expression of SMPs could facilitate divergence of shell morphological characteristics. Collectively, these data establish an evolutionary and developmental framework in Crepidula that enables future comparisons of molluscan biomineralization to reveal mechanisms of shell diversification.

Prymnesium parvum are harmful haptophyte algae that cause massive environmental fish kills. Their polyketide polyether toxins, the prymnesins, are among the largest nonpolymeric compounds in nature and have biosynthetic origins that have remained enigmatic for more than 40 years. In this work, we report the "PKZILLAs," massive P. parvum polyketide synthase (PKS) genes that have evaded previous detection. PKZILLA-1 and -2 encode giant protein products of 4.7 and 3.2 megadaltons that have 140 and 99 enzyme domains. Their predicted polyene product matches the proposed pre-prymnesin precursor of the 90-carbon-backbone A-type prymnesins. We further characterize the variant PKZILLA-B1, which is responsible for the shorter B-type analog prymnesin-B1, from P. parvum RCC3426 and thus establish a general model of haptophyte polyether biosynthetic logic. This work expands expectations of genetic and enzymatic size limits in biology.

Several animal species use tools for foraging; however, very few manufacture and/or modify those tools. Humpback whales, which manufacture bubble-net tools while foraging, are among these rare species. Using animal-borne tag and unoccupied aerial system technologies, we examine bubble-nets manufactured by solitary humpback whales (Megaptera novaeangliae) in Southeast Alaska while feeding on krill. We demonstrate that the nets consist of internally tangential rings and suggest that whales actively control the number of rings in a net, net size and depth and the horizontal spacing between neighbouring bubbles. We argue that whales regulate these net structural elements to increase per-lunge prey intake by, on average, sevenfold. We measured breath rate and swimming and lunge kinematics to show that the resulting increase in prey density does not increase energetic expenditure. Our results provide a novel insight into how bubble-net tools manufactured by solitary foraging humpback whales act to increase foraging efficiency.

Metals are important cofactors in the metabolic processes of cyanobacteria, including photosynthesis, cellular respiration, DNA replication, and the biosynthesis of primary and secondary metabolites. In adaptation to the marine environment, cyanobacteria use metallophores to acquire trace metals when necessary as well as to reduce potential toxicity from excessive metal concentrations. Leptochelins A-C were identified as structurally novel metallophores from three geographically dispersed cyanobacteria of the genus Leptothoe. Determination of the complex structures of these metabolites presented numerous challenges, but they were ultimately solved using integrated data from NMR, mass spectrometry and deductions from the biosynthetic gene cluster. The leptochelins are comprised of halogenated linear NRPS-PKS hybrid products with multiple heterocycles that have potential for hexadentate and tetradentate coordination with metal ions. The genomes of the three leptochelin producers were sequenced, and retrobiosynthetic analysis revealed one candidate biosynthetic gene cluster (BGC) consistent with the structure of leptochelin. The putative BGC is highly homologous in all three Leptothoe strains, and all possess genetic signatures associated with metallophores. Postcolumn infusion of metals using an LC-MS metabolomics workflow performed with leptochelins A and B revealed promiscuous binding of iron, copper, cobalt, and zinc, with greatest preference for copper. Iron depletion and copper toxicity experiments support the hypothesis that leptochelin metallophores may play key ecological roles in iron acquisition and in copper detoxification. In addition, the leptochelins possess significant cytotoxicity against several cancer cell lines.