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Updated: Jun 7, 2026
Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi
Published on: September 28, 2022
New vector tools with a hygromycin resistance marker for use with opportunistic pathogens
Eric J Kalivoda1, Joseph Horzempa, Nicholas A Stella
1The Charles T. Campbell Laboratory of Ophthalmic Microbiology, Department of Ophthalmology, University of Pittsburgh Eye Center, Pittsburgh, PA 15213, USA.
Abstract:
The ability of many bacterial strains to tolerate antibiotics can limit the number of molecular tools available for research of these organisms. To help address this problem, we have modified a diverse set of vectors to include a broadly expressed hygromycin resistance (HmR) marker. Hygromycin B is an aminoglycoside antibiotic not used to treat infections in humans and has antimicrobial activity against a wide range of microorganisms. Vectors with four replication origins are represented, with potential applications including general cloning, allelic replacement, and transcriptional analysis. We show that vectors with the broad host range pBBR1-replicon conferred HmR to Achromobacter xylosoxidans, Acinetobacter baumannii, Pseudomonas aeruginosa, and Serratia marcescens, and a pC194-based vector was able to confer HmR to Francisella tularensis. We also used a subset of these plasmids to manipulate the genome of S. marcescens. Each vector has an origin of transfer for conjugation, and is also able to replicate in Saccharomyces cerevisiae to take advantage of the powerful yeast recombineering system.
Insights
Researchers developed new molecular tools for studying antibiotic-resistant bacteria by incorporating a hygromycin resistance (HmR) marker into versatile vectors. These tools enhance genetic manipulation in challenging bacterial species.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Antibiotic resistance in bacteria limits the availability of molecular tools for research.
- Developing novel genetic manipulation methods is crucial for studying diverse bacterial pathogens.
Purpose of the Study:
- To engineer a set of versatile vectors conferring hygromycin resistance (HmR) for broad-spectrum bacterial research.
- To expand the toolkit for genetic manipulation in antibiotic-resistant and clinically relevant bacterial strains.
Main Methods:
- Modification of diverse vectors to include a hygromycin resistance (HmR) marker.
- Testing vector efficacy in conferring HmR across multiple bacterial species, including *Achromobacter xylosoxidans*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Serratia marcescens*, and *Francisella tularensis*.
- Utilizing plasmids for genomic manipulation in *S. marcescens* and leveraging yeast recombineering capabilities.
Main Results:
- Successfully conferred hygromycin resistance (HmR) in *A. xylosoxidans*, *A. baumannii*, *P. aeruginosa*, *S. marcescens*, and *F. tularensis* using modified vectors.
- Demonstrated the utility of these vectors for genomic manipulation in *S. marcescens*.
- Engineered vectors capable of replication in both bacteria and *Saccharomyces cerevisiae*, enabling cross-kingdom genetic engineering.
Conclusions:
- The developed hygromycin resistance (HmR) vectors provide a broadly applicable tool for genetic manipulation in a wide range of bacterial species.
- These vectors overcome limitations posed by antibiotic resistance, facilitating research on microbial pathogens.
- The dual-replicating nature of the vectors enhances their versatility for molecular biology applications.
