A single diiron enzyme catalyses the oxidative rearrangement of tryptophan to indole nitrile.

Nitriles are uncommon in nature and are typically constructed from oximes through the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third nitrile biosynthesis strategy featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the eagle-killing neurotoxin, aetokthonotoxin, AetD transforms the 2-aminopropionate portion of 5,7-dibromo-L-tryptophan to a nitrile. Employing a combination of structural, biochemical and biophysical techniques, we characterized AetD as a non-haem diiron enzyme that belongs to the emerging haem-oxygenase-like dimetal oxidase superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation, which we propose proceeds via an aziridine intermediate. Our work presents a unique template for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other haem-oxygenase-like dimetal oxidase enzymes.

Molecular forecasting of domoic acid during a pervasive toxic diatom bloom.

In 2015, the largest recorded harmful algal bloom (HAB) occurred in the Northeast Pacific, causing nearly 100 million dollars in damages to fisheries and killing many protected marine mammals. Dominated by the toxic diatom Pseudo-nitzschia australis, this bloom produced high levels of the neurotoxin domoic acid (DA). Through molecular and transcriptional characterization of 52 near-weekly phytoplankton net-tow samples collected at a bloom hotspot in Monterey Bay, California, we identified active transcription of known DA biosynthesis (dab) genes from the three identified toxigenic species, including P. australis as the primary origin of toxicity. Elevated expression of silicon transporters (sit1) during the bloom supports the previously hypothesized role of dissolved silica (Si) exhaustion in contributing to bloom physiology and toxicity. We find that coexpression of the dabA and sit1 genes serves as a robust predictor of DA one week in advance, potentially enabling the forecasting of DA-producing HABs. We additionally present evidence that low levels of iron could have colimited the diatom population along with low Si. Iron limitation represents an overlooked driver of both toxin production and ecological success of the low-iron-adapted Pseudo-nitzschia genus during the 2015 bloom, and increasing pervasiveness of iron limitation may fuel the escalating magnitude and frequency of toxic Pseudo-nitzschia blooms globally. Our results advance understanding of bloom physiology underlying toxin production, bloom prediction, and the impact of global change on toxic blooms.

Giant polyketide synthase enzymes in the biosynthesis of giant marine polyether toxins.

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.

Methods for the discovery and characterization of octocoral terpene cyclases.

Octocorals are the most prolific source of terpenoids in the marine environment, with more than 4000 different compounds known from the phylum to date. However, the biochemical and genetic origin of their production remained elusive until recent studies showed that octocorals encode genes responsible for the biosynthesis of terpenoids in their own chromosomal DNA rather than from microbial symbionts as originally proposed. The identified coral genes include those encoding a new group of class I terpene cyclases (TCs) clustered among other candidate classes of tailoring enzymes. Phylogenetic analyses established octocoral TCs as a monophyletic clade, distinct from TCs of plants, bacteria, and other organisms. The newly discovered group of TCs appears to be ubiquitous in octocorals and is evolutionarily ancient. Given the recent discovery of octocoral terpenoid biochemistry and only limited genomic data presently available, there is substantial potential for discovering new biosynthetic pathways from octocorals for terpene production. The following chapter outlines practical experimental procedures for octocoral DNA and RNA extraction, genome and transcriptome assembly and mining, TC cloning and gene expression, protein purification, and in vitro analyses.

Discovery of Latent Cannabichromene Cyclase Activity in Marine Bacterial Flavoenzymes.

Production of phytocannabinoids remains an area of active scientific interest due to the growing use of cannabis by the public and the underexplored therapeutic potential of the over 100 minor cannabinoids. While phytocannabinoids are biosynthesized by Cannabis sativa and other select plants and fungi, structural analogs and stereoisomers can only be accessed synthetically or through heterologous expression. To date, the bioproduction of cannabinoids has required eukaryotic hosts like yeast since key, native oxidative cyclization enzymes do not express well in bacterial hosts. Here, we report that two marine bacterial flavoenzymes, Clz9 and Tcz9, perform oxidative cyclization reactions on phytocannabinoid precursors to efficiently generate cannabichromene scaffolds. Furthermore, Clz9 and Tcz9 express robustly in bacteria and display significant tolerance to organic solvent and high substrate loading, thereby enabling fermentative production of cannabichromenic acid in Escherichia coli and indicating their potential for biocatalyst development.

Giant polyketide synthase enzymes biosynthesize a giant marine polyether biotoxin.

Prymnesium parvum are harmful haptophyte algae that cause massive environmental fish-kills. Their polyketide polyether toxins, the prymnesins, are amongst the largest nonpolymeric compounds in nature, alongside structurally-related health-impacting "red-tide" polyether toxins whose biosynthetic origins have been an enigma for over 40 years. Here we report the 'PKZILLAs', massive P. parvum polyketide synthase (PKS) genes, whose existence and challenging genomic structure evaded prior detection. PKZILLA-1 and -2 encode giant protein products of 4.7 and 3.2 MDa with 140 and 99 enzyme domains, exceeding the largest known protein titin and all other known PKS systems. Their predicted polyene product matches the proposed pre-prymnesin precursor of the 90-carbon-backbone A-type prymnesins. This discovery establishes a model system for microalgal polyether biosynthesis and expands expectations of genetic and enzymatic size limits in biology.

The shikimate pathway: gateway to metabolic diversity.

Covering: 1997 to 2023The shikimate pathway is the metabolic process responsible for the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Seven metabolic steps convert phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) into shikimate and ultimately chorismate, which serves as the branch point for dedicated aromatic amino acid biosynthesis. Bacteria, fungi, algae, and plants (yet not animals) biosynthesize chorismate and exploit its intermediates in their specialized metabolism. This review highlights the metabolic diversity derived from intermediates of the shikimate pathway along the seven steps from PEP and E4P to chorismate, as well as additional sections on compounds derived from prephenate, anthranilate and the synonymous aminoshikimate pathway. We discuss the genomic basis and biochemical support leading to shikimate-derived antibiotics, lipids, pigments, cofactors, and other metabolites across the tree of life.

Unveiling a CAAX Protease-Like Protein Involved in Didemnin Drug Maturation and Secretion.

The assembly line biosynthesis of the powerful anticancer-antiviral didemnin cyclic peptides is proposed to follow a prodrug release mechanism in Tristella bacteria. This strategy commences with the formation of N-terminal prodrug scaffolds and culminates in their cleavage during the cellular export of the mature products. In this study, a comprehensive exploration of the genetic and biochemical aspects of the enzymes responsible for both the assembly and cleavage of the acylated peptide prodrug scaffolds is provided. This process involves the assembly of N-acyl-polyglutamine moieties orchestrated by the nonribosomal peptide synthetase DidA and the cleavage of these components at the post-assembly stage by DidK, a transmembrane CAAX hydrolase homolog. The findings not only shed light on the complex prodrug mechanism that underlies the synthesis and secretion of didemnin compounds but also offer novel insights into the expanded role of CAAX hydrolases in microbes. Furthermore, this knowledge can be leveraged for the strategic design of genome mining approaches aimed at discovering new bioactive natural products that employ similar prodrug biochemical strategies.

Biosynthesis of Haloterpenoids in Red Algae via Microbial-like Type I Terpene Synthases.

Red algae or seaweeds produce highly distinctive halogenated terpenoid compounds, including the pentabromochlorinated monoterpene halomon that was once heralded as a promising anticancer agent. The first dedicated step in the biosynthesis of these natural product molecules is expected to be catalyzed by terpene synthase (TS) enzymes. Recent work has demonstrated an emerging class of type I TSs in red algal terpene biosynthesis. However, only one such enzyme from a notoriously haloterpenoid-producing red alga (Laurencia pacifica) has been functionally characterized and the product structure is not related to halogenated terpenoids. Herein, we report 10 new type I TSs from the red algae Portieria hornemannii, Plocamium pacificum, L. pacifica, and Laurencia subopposita that produce a diversity of halogenated mono- and sesquiterpenes. We used a combination of genome sequencing, terpenoid metabolomics, in vitro biochemistry, and bioinformatics to establish red algal TSs in all four species, including those associated with the selective production of key halogenated terpene precursors myrcene, trans-ß-ocimene, and germacrene D-4-ol. These results expand on a small but growing number of characterized red algal TSs and offer insight into the biosynthesis of iconic halogenated algal compounds that are not without precedence elsewhere in biology.

Molecular Forecasting of Domoic Acid during a Pervasive Toxic Diatom Bloom.

In 2015, the largest recorded harmful algal bloom (HAB) occurred in the Northeast Pacific, causing nearly 100 million dollars in damages to fisheries and killing many protected marine mammals. Dominated by the toxic diatom Pseudo-nitzschia australis , this bloom produced high levels of the neurotoxin domoic acid (DA). Through molecular and transcriptional characterization of 52 near-weekly phytoplankton net-tow samples collected at a bloom hotspot in Monterey Bay, California, we identified active transcription of known DA biosynthesis ( dab ) genes from the three identified toxigenic species, including P. australis as the primary origin of toxicity. Elevated expression of silicon transporters ( sit1 ) during the bloom supports the previously hypothesized role of dissolved silica (Si) exhaustion in contributing to bloom physiology and toxicity. We find that co-expression of the dabA and sit1 genes serves as a robust predictor of DA one week in advance, potentially enabling the forecasting of DA-producing HABs. We additionally present evidence that low levels of iron could have co-limited the diatom population along with low Si. Iron limitation represents a previously unrecognized driver of both toxin production and ecological success of the low iron adapted Pseudo-nitzschia genus during the 2015 bloom, and increasing pervasiveness of iron limitation may fuel the escalating magnitude and frequency of toxic Pseudo-nitzschia blooms globally. Our results advance understanding of bloom physiology underlying toxin production, bloom prediction, and the impact of global change on toxic blooms. Significance: Pseudo-nitzschia diatoms form oceanic harmful algal blooms that threaten human health through production of the neurotoxin domoic acid (DA). DA biosynthetic gene expression is hypothesized to control DA production in the environment, yet what regulates expression of these genes is yet to be discovered. In this study, we uncovered expression of DA biosynthesis genes by multiple toxigenic Pseudo-nitzschia species during an economically impactful bloom along the North American West Coast, and identified genes that predict DA in advance of its production. We discovered that iron and silica co-limitation restrained the bloom and likely promoted toxin production. This work suggests that increasing iron limitation due to global change may play a previously unrecognized role in driving bloom frequency and toxicity.

Linking bacterial tetrabromopyrrole biosynthesis to coral metamorphosis.

An important factor dictating coral fitness is the quality of bacteria associated with corals and coral reefs. One way that bacteria benefit corals is by stimulating the larval to juvenile life cycle transition of settlement and metamorphosis. Tetrabromopyrrole (TBP) is a small molecule produced by bacteria that stimulates metamorphosis with and without attachment in a range of coral species. A standing debate remains, however, about whether TBP biosynthesis from live Pseudoalteromonas bacteria is the primary stimulant of coral metamorphosis. In this study, we create a Pseudoalteromonas sp. PS5 mutant lacking the TBP brominase gene, bmp2. Using this mutant, we confirm that the bmp2 gene is critical for TBP biosynthesis in Pseudoalteromonas sp. PS5. Mutation of this gene ablates the bacterium's ability in live cultures to stimulate the metamorphosis of the stony coral Porites astreoides. We further demonstrate that expression of TBP biosynthesis genes is strongest in stationary and biofilm modes of growth, where Pseudoalteromonas sp. PS5 might exist within surface-attached biofilms on the sea floor. Finally, we create a modular transposon plasmid for genomic integration and fluorescent labeling of Pseudoalteromonas sp. PS5 cells. Our results functionally link a TBP biosynthesis gene from live bacteria to a morphogenic effect in corals. The genetic techniques established here provide new tools to explore coral-bacteria interactions and could help to inform future decisions about utilizing marine bacteria or their products for coral restoration.

Oxidative rearrangement of tryptophan to indole nitrile by a single diiron enzyme.

Nitriles are uncommon in nature and are typically constructed from oximes via the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third strategy of nitrile biosynthesis featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the 'eagle-killing' neurotoxin, aetokthonotoxin, AetD converts the alanyl side chain of 5,7-dibromo-L-tryptophan to a nitrile. Employing a combination of structural, biochemical, and biophysical techniques, we characterized AetD as a non-heme diiron enzyme that belongs to the emerging Heme Oxygenase-like Diiron Oxidase and Oxygenase (HDO) superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation that we propose proceeds via an aziridine intermediate. Our work presents a new paradigm for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other HDO enzymes.

Enzymatic Halogenation of Terminal Alkynes.

The biosynthetic installation of halogen atoms is largely performed by oxidative halogenases that target a wide array of electron-rich substrates, including aromatic compounds and conjugated systems. Halogenated alkyne-containing molecules are known to occur in Nature; however, halogen atom installation on the terminus of an alkyne has not been demonstrated in enzyme catalysis. Herein, we report the discovery and characterization of an alkynyl halogenase in natural product biosynthesis. We show that the flavin-dependent halogenase from the jamaicamide biosynthetic pathway, JamD, is not only capable of terminal alkyne halogenation on a late-stage intermediate en route to the final natural product but also has broad substrate tolerance for simple to complex alkynes. Furthermore, JamD is specific for terminal alkynes over other electron-rich aromatic substrates and belongs to a newly identified family of halogenases from marine cyanobacteria, indicating its potential as a chemoselective biocatalyst for the formation of haloalkynes.

A modular plasmid toolkit applied in marine bacteria reveals functional insights during bacteria-stimulated metamorphosis.

A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacterium Pseudoalteromonas luteoviolacea, which stimulates the metamorphosis of the model tubeworm, Hydroides elegans. Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for testing the genetic tractability of marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts resulted in the successful conjugation of 12 marine strains from the Alphaproteobacteria and Gammaproteobacteria classes. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with potential implications for environmental restoration and biotechnology. IMPORTANCE Marine Proteobacteria are attractive targets for genetic engineering due to their ability to produce a diversity of bioactive metabolites and their involvement in host-microbe symbioses. Modular cloning toolkits have become a standard for engineering model microbes, such as Escherichia coli, because they enable innumerable mix-and-match DNA assembly and engineering options. However, such modular tools have not yet been applied to most marine bacterial species. In this work, we adapt a modular plasmid toolkit for use in a set of 12 marine bacteria from the Gammaproteobacteria and Alphaproteobacteria classes. We demonstrate the utility of this genetic toolkit by engineering a marine Pseudoalteromonas bacterium to study their association with its host animal Hydroides elegans. This work provides a proof of concept that modular genetic tools can be applied to diverse marine bacteria to address basic science questions and for biotechnology innovations.

Mechanistic and Structural Insights into a Divergent PLP-Dependent l-Enduracididine Cyclase from a Toxic Cyanobacterium.

Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cyanobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure. The ncAA l-enduracididine is an early intermediate in guanitoxin biosynthesis and is produced by GntC, a unique pyridoxal-5'-phosphate (PLP)-dependent enzyme. GntC catalyzes a cyclodehydration from a stereoselectively γ-hydroxylated l-arginine precursor via a reaction that functionally and mechanistically diverges from previously established actinobacterial cyclic arginine ncAA pathways. Herein, we interrogate l-enduracididine biosynthesis from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using spectroscopy, stable isotope labeling techniques, and X-ray crystallography structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the α- and ß-positions of its substrate before catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison of holo- and substrate-bound GntC structures and activity assays on site-specific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enable an improved understanding of how nature divergently produces cyclic arginine ncAAs and generate additional tools for their biocatalytic production and downstream biological applications.

Linking bacterial tetrabromopyrrole biosynthesis to coral metamorphosis.

An important factor dictating coral fitness is the quality of bacteria associated with corals and coral reefs. One way that bacteria benefit corals is by stimulating the larval to juvenile life cycle transition of settlement and metamorphosis. Tetrabromopyrrole (TBP) is a small molecule produced by bacteria that stimulates metamorphosis in a range of coral species. A standing debate remains, however, about whether TBP biosynthesis from live Pseudoalteromonas bacteria is the primary stimulant of coral metamorphosis. In this study, we create a Pseudoalteromonas sp. PS5 mutant lacking the TBP brominase gene, bmp2 . Using this mutant, we confirm that the bmp2 gene is critical for TBP biosynthesis in Pseudoalteromonas sp. PS5. Mutation of this gene ablates the bacterium's ability in live cultures to stimulate the metamorphosis of the stony coral Porites astreoides . We further demonstrate that expression of TBP biosynthesis genes is strongest in stationary and biofilm modes of growth, where Pseudoalteromonas sp. PS5 might exist within surface-attached biofilms on the sea floor. Finally, we create a modular transposon plasmid for genomic integration and fluorescent labeling of Pseudoalteromonas sp. PS5 cells. Our results functionally link a TBP biosynthesis gene from live bacteria to a morphogenic effect in corals. The genetic techniques established here provide new tools to explore coral-bacteria interactions and could help to inform future decisions about utilizing marine bacteria or their products for restoring degraded coral reefs.

Extreme genome diversity and cryptic speciation in a harmful algal-bloom-forming eukaryote.

Harmful algal blooms of the toxic haptophyte Prymnesium parvum are a recurrent problem in many inland and estuarine waters around the world. Strains of P. parvum vary in the toxins they produce and in other physiological traits associated with harmful algal blooms, but the genetic basis for this variation is unknown. To investigate genome diversity in this morphospecies, we generated genome assemblies for 15 phylogenetically and geographically diverse strains of P. parvum, including Hi-C guided, near-chromosome-level assemblies for two strains. Comparative analysis revealed considerable DNA content variation between strains, ranging from 115 to 845 Mbp. Strains included haploids, diploids, and polyploids, but not all differences in DNA content were due to variation in genome copy number. Haploid genome size between strains of different chemotypes differed by as much as 243 Mbp. Syntenic and phylogenetic analyses indicate that UTEX 2797, a common laboratory strain from Texas, is a hybrid that retains two phylogenetically distinct haplotypes. Investigation of gene families variably present across the strains identified several functional categories associated with metabolic and genome size variation in P. parvum, including genes for the biosynthesis of toxic metabolites and proliferation of transposable elements. Together, our results indicate that P. parvum comprises multiple cryptic species. These genomes provide a robust phylogenetic and genomic framework for investigations into the eco-physiological consequences of the intra- and inter-specific genetic variation present in P. parvum and demonstrate the need for similar resources for other harmful algal-bloom-forming morphospecies.

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium.

Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cya-nobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure. The ncAA L-enduracididine is an early intermediate in guanitoxin biosynthesis and is produced by GntC, a unique pyridoxal-5'-phosphate (PLP)-dependent enzyme. GntC catalyzes a cyclodehydration from a stereoselectively γ-hydroxylated L-arginine precursor via a reaction that functionally and mechanistically diverges from previously established actinobacterial cyclic arginine ncAA pathways. Herein, we interrogate L-enduracididine biosynthesis from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using spectroscopic, stable isotope labeling techniques, and X-ray crystal structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the α- and ß-positions of its substrate prior to catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison of holo- and substrate bound GntC structures and activity assays on sitespecific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enables an improved understanding of how Nature divergently produces cyclic arginine ncAAs and generates additional tools for their biocatalytic production and downstream biological applications.

Terpene biosynthesis in marine sponge animals.

Sea sponges are the largest marine source of small-molecule natural products described to date. Sponge-derived molecules, such as the chemotherapeutic eribulin, the calcium-channel blocker manoalide, and antimalarial compound kalihinol A, are renowned for their impressive medicinal, chemical, and biological properties. Sponges contain microbiomes that control the production of many natural products isolated from these marine invertebrates. In fact, all genomic studies to date investigating the metabolic origins of sponge-derived small molecules concluded that microbes-not the sponge animal host-are the biosynthetic producers. However, early cell-sorting studies suggested the sponge animal host may play a role particularly in the production of terpenoid molecules. To investigate the genetic underpinnings of sponge terpenoid biosynthesis, we sequenced the metagenome and transcriptome of an isonitrile sesquiterpenoid-containing sponge of the order Bubarida. Using bioinformatic searches and biochemical validation, we identified a group of type I terpene synthases (TSs) from this sponge and multiple other species, the first of this enzyme class characterized from the sponge holobiome. The Bubarida TS-associated contigs consist of intron-containing genes homologous to sponge genes and feature GC percentage and coverage consistent with other eukaryotic sequences. We identified and characterized TS homologs from five different sponge species isolated from geographically distant locations, thereby suggesting a broad distribution amongst sponges. This work sheds light on the role of sponges in secondary metabolite production and speaks to the possibility that other sponge-specific molecules originate from the animal host.

A modular plasmid toolkit applied in marine Proteobacteria reveals functional insights during bacteria-stimulated metamorphosis.

A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacterium Pseudoalteromonas luteoviolacea , which stimulates the metamorphosis of the model tubeworm, Hydroides elegans . Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for rapidly creating and iteratively testing genetic tractability by modifying marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts enabled the successful transformation of twelve marine strains across two Proteobacteria classes, four orders and ten genera. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with broader implications for environmental restoration and biotechnology.