Search for: All records

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 livePseudoalteromonasbacteria is the primary stimulant of coral metamorphosis. In this study, we create aPseudoalteromonassp. PS5 mutant lacking the TBP brominase gene,bmp2. Using this mutant, we confirm that thebmp2gene is critical for TBP biosynthesis inPseudoalteromonassp. PS5. Mutation of this gene ablates the bacterium’s ability in live cultures to stimulate the metamorphosis of the stony coralPorites astreoides. We further demonstrate that expression of TBP biosynthesis genes is strongest in stationary and biofilm modes of growth, wherePseudoalteromonassp. PS5 might exist within surface-attached biofilms on the sea floor. Finally, we create a modular transposon plasmid for genomic integration and fluorescent labeling ofPseudoalteromonassp. 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.

 

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 bacteriumPseudoalteromonas 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.

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 asEscherichia 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 marinePseudoalteromonasbacterium to study their association with its host animalHydroides 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.

 

Covering: Up to December 2020 Enzymatic halogenation reactions are essential for the production of thousands of halogenated natural products. However, in recent years, scientists discovered several halogenases that transiently incorporate halogen atoms in intermediate biosynthetic molecules to activate them for further chemical reactions such as cyclopropanation, terminal alkyne formation, C-/O-alkylation, biaryl coupling, and C–C rearrangements. In each case, the halogen atom is lost in the course of biosynthesis to the final product and is hence termed “cryptic”. In this review, we provide an overview of our current knowledge of cryptic halogenation reactions in natural product biosynthesis. 

Isonitrile‐containing natural products have garnered attention for their manifold bioactivities but are difficult to detect and isolate due to the chemical lability of the isonitrile functional group. Here, we used the isonitrile‐chlorooxime ligation (INC) in a reactivity‐based screening (RBS) protocol for the detection and isolation of alkaloid and terpene isonitriles in the cyanobacteriumFischerella ambiguaand a marine sponge of the order Bubarida, respectively. A trifunctional probe bearing a chlorooxime moiety, a UV active aromatic moiety, and a bromine label facilitated the chemoselective reaction with isonitriles, UV‐Vis spectroscopic detection, and mass spectrometric analysis. The INC‐based RBS allowed for the detection, isolation, and structural elucidation of isonitriles in microgram quantities.