This content will become publicly available on August 2, 2024
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
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
- Award ID(s):
- 1942251
- NSF-PAR ID:
- 10481025
- Editor(s):
- Ruby, Edward G.
- Publisher / Repository:
- American Society for Microbiology
- Date Published:
- Journal Name:
- mBio
- ISSN:
- 2150-7511
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Summary Pseudoalteromonas luteoviolacea is a globally distributed marine bacterium that stimulates the metamorphosis of marine animal larvae, an important bacteria–animal interaction that can promote the recruitment of animals to benthic ecosystems. Recently, differentP .luteoviolacea isolates have been shown to produce two stimulatory factors that can induce tubeworm and coral metamorphosis; Metamorphosis‐Associated Contractile structures (MACs) and tetrabromopyrrole (TBP) respectively. However, it remains unclear what proportion ofP .luteoviolacea isolates possess the genes encoding MACs, and what phenotypic effect MACs and TBP have on other larval species. Here, we show that 9 of 19 sequencedP .luteoviolacea genomes genetically encode both MACs and TBP. WhileP .luteoviolacea biofilms producing MACs stimulate the metamorphosis of the tubewormHydroides elegans , TBP biosynthesis genes had no effect under the conditions tested. Although MACs are lethal to larvae of the cnidarianHydractinia symbiologicarpus ,P .luteoviolacea mutants unable to produce MACs are capable of stimulating metamorphosis. Our findings reveal a hidden complexity of interactions between a single bacterial species, the factors it produces and two species of larvae belonging to different phyla. -
more » « less
Abstract 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 aPseudoalteromonas sp. PS5 mutant lacking the TBP brominase gene,bmp2 . Using this mutant, we confirm that thebmp2 gene is critical for TBP biosynthesis inPseudoalteromonas sp. 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, wherePseudoalteromonas 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 ofPseudoalteromonas 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. -
Harnessing the unique biochemical capabilities of non-model microorganisms would expand the array of biomanufacturing substrates, process conditions, and products. There are non-model microorganisms that fix nitrogen and carbon dioxide, derive energy from light, catabolize methane and lignin-derived aromatics, are tolerant to physiochemical stresses and harsh environmental conditions, store lipids in large quantities, and produce hydrogen. Model microorganisms often only break down simple sugars and require low stress conditions, but they have been engineered for the sustainable manufacture of numerous products, such as fragrances, pharmaceuticals, cosmetics, surfactants, and specialty chemicals, often by using tools from synthetic biology. Transferring complex pathways has proven to be exceedingly difficult, as the cofactors, cellular conditions, and energy sources necessary for this pathway to function may not be present in the host organism. Utilization of unique biochemical capabilities could also be achieved by engineering the host; although, synthetic biology tools developed for model microbes often do not perform as designed in other microorganisms. The metabolically versatile Rhodopseudomonas palustris CGA009, a purple non-sulfur bacterium, catabolizes aromatic compounds derived from lignin in both aerobic and anaerobic conditions and can use light, inorganic, and organic compounds for its source of energy. R. palustris utilizes three nitrogenase isozymes to fulfill its nitrogen requirements while also generating hydrogen. Furthermore, the bacterium produces two forms of RuBisCo in response to carbon dioxide/bicarbonate availability. While this potential chassis harbors many beneficial traits, stable heterologous gene expression has been problematic due to its intrinsic resistance to many antibiotics and the lack of synthetic biology parts investigated in this microbe. To address these problems, we have characterized gene expression and plasmid maintenance for different selection markers, started a synthetic biology toolbox specifically for the photosynthetic R. palustris , including origins of replication, fluorescent reporters, terminators, and 5′ untranslated regions, and employed the microbe’s endogenous plasmid for exogenous protein production. This work provides essential synthetic biology tools for engineering R. palustris ’ many unique biochemical processes and has helped define the principles for expressing heterologous genes in this promising microbe through a methodology that could be applied to other non-model microorganisms.more » « less
-
more » « less
Abstract Unlike biologically available nitrogen and phosphorus, which are often at limiting concentrations in surface seawater, sulfur in the form of sulfate is plentiful and not considered to constrain marine microbial activity. Nonetheless, in a model system in which a marine bacterium obtains all of its carbon from co-cultured phytoplankton, bacterial gene expression suggests that at least seven dissolved organic sulfur (DOS) metabolites support bacterial heterotrophy. These labile exometabolites of marine dinoflagellates and diatoms include taurine, N-acetyltaurine, isethionate, choline-O-sulfate, cysteate, 2,3-dihydroxypropane-1-sulfonate (DHPS), and dimethylsulfoniopropionate (DMSP). Leveraging from the compounds identified in this model system, we assessed the role of sulfur metabolites in the ocean carbon cycle by mining the Tara Oceans dataset for diagnostic genes. In the 1.4 million bacterial genome equivalents surveyed, estimates of the frequency of genomes harboring the capability for DOS metabolite utilization ranged broadly, from only 1 out of every 190 genomes (for the C2 sulfonate isethionate) to 1 out of every 5 (for the sulfonium compound DMSP). Bacteria able to participate in DOS transformations are dominated by Alphaproteobacteria in the surface ocean, but by SAR324, Acidimicrobiia, and Gammaproteobacteria at mesopelagic depths, where the capability for utilization occurs in higher frequency than in surface bacteria for more than half the sulfur metabolites. The discovery of an abundant and diverse suite of marine bacteria with the genetic capacity for DOS transformation argues for an important role for sulfur metabolites in the pelagic ocean carbon cycle.
-
more » « less
Abstract Insects have repeatedly forged symbioses with heritable microbes, gaining novel traits. For the microbe, the transition to symbioses can lead to the degeneration of the symbiont's genome through transmission bottlenecks, isolation, and the loss of DNA repair enzymes. However, some insect-microbial symbioses have persisted for millions of years, suggesting that natural selection slows genetic drift and maintains functional consistency between symbiont populations. By sampling in multiple countries, we examine genomic diversity within a symbiont species, a heritable symbiotic bacterium found only in human head lice. We find that human head louse symbionts contain genetic diversity that appears to have arisen contemporaneously with the appearance of anatomically modern humans within Africa and/or during the colonization of Eurasia by humans. We predict that the observed genetic diversity underlies functional differences in extant symbiont lineages, through the inactivation of genes involved in symbiont membrane construction. Furthermore, we find evidence of additional gene losses prior to the appearance of modern humans, also impacting the symbiont membrane. From this, we conclude that symbiont genome degeneration is proceeding, via gene inactivation and subsequent loss, in human head louse symbionts, while genomic diversity is maintained. Collectively, our results provide a look into the genomic diversity within a single symbiont species and highlight the shared evolutionary history of humans, lice, and bacteria.
