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1. Expression of endogenous Anopheles gambiae microRNAs using an Anopheles gambiae densovirus (AgDNV) intronic expression system

   https://doi.org/10.1186/s13071-025-06994-7  

   Johnson, Rebecca_M; Metz, Hillery_C; Suzuki, Yasutsugu; McLean, Kyle_J; Rasgon, Jason_L (August 2025, Parasites & Vectors)

   Abstract BackgroundAnopheles gambiaedensovirus (AgDNV) is a highly species-specific parvovirus that reaches high titers in adultAnopheles gambiaemosquitoes with few transcriptomic effects and minimal significant fitness effects. Given these characteristics, AgDNV has been proposed as a viral vector for basic research and mosquito control. Previous work created an AgDNV co-expression system with a wild-type AgDNV helper plasmid and a transducing plasmid expressing enhanced green fluorescent protein (EGFP) that can be used to co-transfect cells to generate infectious recombinant transducing AgDNV virions. Generated virions infect theAn. gambiaemidgut, fat body, and ovaries, yet this viral vector system is limited in the size of transgenes that can be expressed due to capsid packaging limitations. MethodsConsidering these size constraints, we created an artificial intron within the EGFP gene of the transducing construct that can express small pieces of genetic material such as microRNAs (miRNAs), microRNA sponges, or other small sequences. Placement of this intron in EGFP created a fluorescent reporter such that incorrect splicing produces a frameshift mutation in EGFP and an early stop codon, whereas correct splicing results in normal EGFP expression and co-transcription of the intronic genetic cargo. A selection of miRNAs with predicted or demonstrated importance in mosquito immunity and reproduction with expression localized to the fat body or ovaries were chosen as intronic cargo. Construct expression and splicing was evaluated, and the impact of miRNA expression on putative miRNA targets was measuredin vitroandin vivo. ResultsThe created intron was correctly spliced in cells and mosquitoes; however, miRNA delivery resulted in inconsistent changes to miRNA and predicted target gene transcript levels—possibly due to organ-specific miRNA expression or inaccurate putative target predictions leading to miRNA–target gene sequence mismatch. ConclusionsAlthough our results on target gene expression were inconsistent, with optimization this viral vector and developed intron have potential as an expression tool withinAn. gambiaemosquitoes or cell lines. Graphical Abstract 

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2. Plasmid Characteristics Modulate the Propensity of Gene Exchange in Bacterial Vesicles

   https://doi.org/10.1128/JB.00430-18  

   Tran, Frances; Boedicker, James Q.; Becker, Anke (April 2019, Journal of Bacteriology)

   ABSTRACT Horizontal gene transfer is responsible for the exchange of many types of genetic elements, including plasmids. Properties of the exchanged genetic element are known to influence the efficiency of transfer via the mechanisms of conjugation, transduction, and transformation. Recently, an alternative general pathway of horizontal gene transfer has been identified, namely, gene exchange by extracellular vesicles. Although extracellular vesicles have been shown to facilitate the exchange of several types of plasmids, the influence of plasmid characteristics on genetic exchange within vesicles is unclear. Here, a set of different plasmids was constructed to systematically test the impact of plasmid properties, specifically, plasmid copy number, size, and origin of replication, on gene transfer in vesicles. The influence of each property on the production, packaging, and uptake of vesicles containing bacterial plasmids was quantified, revealing how plasmid properties modulate vesicle-mediated horizontal gene transfer. The loading of plasmids into vesicles correlates with the plasmid copy number and is influenced by characteristics that help set the number of plasmids within a cell, including size and origin of replication. Plasmid origin also has a separate impact on both vesicle loading and uptake, demonstrating that the origin of replication is a major determinant of the propensity of specific plasmids to transfer within extracellular vesicles. IMPORTANCE Extracellular vesicle formation and exchange are common within bacterial populations. Vesicles package multiple types of biomolecules, including genetic material. The exchange of extracellular vesicles containing genetic material facilitates interspecies DNA transfer and may be a promiscuous mechanism of horizontal gene transfer. Unlike other mechanisms of horizontal gene transfer, it is unclear whether characteristics of the exchanged DNA impact the likelihood of transfer in vesicles. Here, we systematically examine the influence of plasmid copy number, size, and origin of replication on the loading of DNA into vesicles and the uptake of DNA containing vesicles by recipient cells. These results reveal how each plasmid characteristic impacts gene transfer in vesicles and contribute to a greater understanding of the importance of vesicle-mediated gene exchange in the landscape of horizontal gene transfer. 

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3. Expanding the capabilities of MuGENT for large-scale genetic engineering of the fastest-replicating species, Vibrio natriegens

   https://doi.org/10.1128/spectrum.03964-23  

   Glasgo, Liz D; Lukasiak, Katie L; Zinser, Erik R (June 2024, Microbiology Spectrum)

   Cardona, Silvia T (Ed.)

   ABSTRACT The fastest replicating bacteriumVibrio natriegensis a rising workhorse for molecular and biotechnological research with established tools for efficient genetic manipulation. Here, we expand on the capabilities of multiplex genome editing by natural transformation (MuGENT) by identifying a neutral insertion site and showing how two selectable markers can be swapped at this site for sequential rounds of natural transformation. Second, we demonstrated that MuGENT can be used for complementation by gene insertion at an ectopic chromosomal locus. Additionally, we developed a robust method to cure the competence plasmid required to induce natural transformation. Finally, we demonstrated the ability of MuGENT to create massive deletions; the 280 kb deletion created in this study is one of the largest artificial deletions constructed in a single round of targeted mutagenesis of a bacterium. These methods each advance the genetic potential ofV. natriegensand collectively expand upon its utility as an emerging model organism for synthetic biology. IMPORTANCEVibrio natriegensis an emerging model organism for molecular and biotechnological applications. Its fast growth, metabolic versatility, and ease of genetic manipulation provide an ideal platform for synthetic biology. Here, we develop and apply novel methods that expand the genetic capabilities of theV. natriegensmodel system. Prior studies developed a method to manipulate multiple regions of the chromosome in a single step. Here, we provide new resources that diversify the utility of this method. We also provide a technique to remove the required genetic tools from the cell once the manipulation is performed, thus establishing “clean” derivative cells. Finally, we show the full extent of this technique’s capability by generating one of the largest chromosomal deletions reported in the literature. Collectively, these new tools will be beneficial broadly to theVibriocommunity and specifically to the advancement ofV. natriegensas a model system. 

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4. Diverse and abundant phages exploit conjugative plasmids

   https://doi.org/10.1038/s41467-024-47416-z  

   Quinones-Olvera, Natalia; Owen, Siân V.; McCully, Lucy M.; Marin, Maximillian G.; Rand, Eleanor A.; Fan, Alice C.; Martins Dosumu, Oluremi J.; Paul, Kay; Sanchez Castaño, Cleotilde E.; Petherbridge, Rachel; et al (April 2024, Nature Communications)

   Abstract Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 65 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the evolutionarily promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids. 

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

   https://doi.org/10.1128/mbio.01502-23  

   Alker, Amanda T.; Farrell, Morgan V.; Aspiras, Alpher E.; Dunbar, Tiffany L.; Fedoriouk, Andriy; Jones, Jeffrey E.; Mikhail, Sama R.; Salcedo, Gabriella Y.; Moore, Bradley S.; Shikuma, Nicholas J. (August 2023, mBio)

   Ruby, Edward G. (Ed.)

   ABSTRACT 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. IMPORTANCEMarine 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. 

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