Designer diatom episomes delivered by bacterial conjugation.

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research

Domoic acid biosynthesis in the red alga Chondria armata suggests a complex evolutionary history for toxin production.

Domoic acid (DA), the causative agent of amnesic shellfish poisoning, is produced by select organisms within two distantly related algal clades: planktonic diatoms and red macroalgae. The biosynthetic pathway to isodomoic acid A was recently solved in the harmful algal bloom-forming diatom Pseudonitzschia multiseries, establishing the genetic basis for the global production of this potent neurotoxin. Herein, we sequenced the 507-Mb genome of Chondria armata, the red macroalgal seaweed from which DA was first isolated in the 1950s, identifying several copies of the red algal DA (rad) biosynthetic gene cluster. The rad genes are organized similarly to the diatom DA biosynthesis cluster in terms of gene synteny, including a cytochrome P450 (CYP450) enzyme critical to DA production that is notably absent in red algae that produce the simpler kainoid neurochemical, kainic acid. The biochemical characterization of the N-prenyltransferase (RadA) and kainoid synthase (RadC) enzymes support a slightly altered DA biosynthetic model in C. armata via the congener isodomoic acid B, with RadC behaving more like the homologous diatom enzyme despite higher amino acid similarity to red algal kainic acid synthesis enzymes. A phylogenetic analysis of the rad genes suggests unique origins for the red macroalgal and diatom genes in their respective hosts, with native eukaryotic CYP450 neofunctionalization combining with the horizontal gene transfer of N-prenyltransferases and kainoid synthases to establish DA production within the algal lineages

Biosynthesis of the neurotoxin domoic acid in a bloom-forming diatom

Oceanic harmful algal blooms of Pseudo-nitzschia diatoms produce the potent mammalian neurotoxin domoic acid (DA). Despite decades of research, the molecular basis for its biosynthesis is not known. By using growth conditions known to induce DA production in Pseudo-nitzschia multiseries, we implemented transcriptome sequencing in order to identify DA biosynthesis genes that colocalize in a genomic four-gene cluster. We biochemically investigated the recombinant DA biosynthetic enzymes and linked their mechanisms to the construction of DA's diagnostic pyrrolidine skeleton, establishing a model for DA biosynthesis. Knowledge of the genetic basis for toxin production provides an orthogonal approach to bloom monitoring and enables study of environmental factors that drive oceanic DA production

Illuminating the Dark Metabolome of Pseudo-nitzschia-microbiome Associations

The exchange of metabolites mediates algal and bacterial interactions that maintain ecosystem function. Yet, while 1000s of metabolites are produced, only a few molecules have been identifiedin these associations. Using the ubiquitous microalgae Pseudo-nitzschia sp., as a model, we employed an untargeted metabolomics strategy to assign structural characteristics to themetabolites that distinguished specific diatom-microbiome associations. We cultured five species of Pseudo-nitzschia, including two species that produced the toxin domoic acid, and examinedtheir microbiomes and metabolomes. A total of 4826 molecular features were detected by tandem mass spectrometry. Only 229 of these could be annotated using available mass spectral libraries,but by applying new in-silico annotation tools, characterization was expanded to 2710 features. The metabolomes of the Pseudo-nitzschia-microbiome associations were distinct and distinguished by structurally diverse nitrogen compounds, ranging from simple amines andamides to cyclic compounds such as imidazoles, pyrrolidines, and lactams. By illuminating the dark metabolomes, this study expands our capacity to discover new chemical targets that facilitatemicrobial partnerships and uncovers the chemical diversity that underpins algae-bacteria interactions

Resilience Is Not Enough: Toward a More Meaningful Rangeland Adaptation Science

Rangeland ecosystems, and their managers, face the growing urgency of climate change impacts. Researchers are therefore seeking integrative social-ecological frameworks that can enhance adaptation by managers to these climate change dynamics through tighter linkages among multiple scientific disciplines and manager contexts. Social-ecological framings, including resilience and vulnerability, are popular in such efforts, but their potential to inform meaningful rangeland adaptation science is limited by traditional disciplinary silos. Here, we provide reflective lessons learned from a multidisciplinary Rangelands, Ranching, and Resilience (R3) project on U.S. western rangelands that addressed 1) biophysical science projections of forage production under future climate scenarios, 2) ranchers’ views of resilience using social science methods, and 3) outreach efforts coordinated through extension professionals. Despite the project's initial intentions, human dimensions and ecological researchers largely worked in parallel sub-teams during the project, rather than weaving their expertise together with managers. The R3 project was multidisciplinary, but it provides a case study on lessons learned to suggest how social and ecological researchers can move towards approaches that transcend individual disciplines. Transdisciplinary science and management in rangelands requires more than just conceptual social-ecological frameworks. Additional methodological concepts need to include: 1) relationship building; 2) shared meaning making; and 3) a commitment to continual conversations and learning, or staying with the trouble, following Haraway (2016). If the goal is to address meaningful rangeland adaptation science rather than just produce academic products, researchers, outreach professionals, and rangeland-based communities should address a series of critical troubling questions. In the process of addressing these, deeper engagement among and beyond disciplines will occur as relationship building, shared meaning, and continual conversations and learning facilitate staying with the trouble.USDA-ARS24 month embargo; first published 20 May 2024This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Designer diatom episomes delivered by bacterial conjugation.

Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research