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Characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agent bacterium, Francisella tularensis Schu S4 and the surrogate type B live vaccine strain (LVS)

  • Applied genetics and molecular biotechnology
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Abstract

Here, we constructed stable, constitutively expressed, chromosomal green (GFP) and red fluorescent (RFP) reporters in the genome of the surrogate strain, Francisella tularensis spp. holarctica LVS (herein LVS), and the select agent, F. tularensis Schu S4. A bioinformatic approach was used to identify constitutively expressed genes. Two promoter regions upstream of the FTT1794 and rpsF(FTT1062) genes were selected and fused with GFP and RFP reporter genes in pMP815, respectively. While the LVS strains with chromosomally integrated reporter fusions exhibited fluorescence, we were unable to deliver the same fusions into Schu S4. Neither a temperature-sensitive Francisella replicon nor a pBBR replicon in the modified pMP815 derivatives facilitated integration. However, a mini-Tn7 integration system was successful at integrating the reporter fusions into the Schu S4 genome. Finally, fluorescent F. tularensis LVS and a mutant lacking MglA were assessed for growth in monocyte-derived macrophages (MDMs). As expected, when compared to wild-type bacteria, replication of an mglA mutant was significantly diminished, and the overall level of fluorescence dramatically decreased with infection time. The utility of the fluorescent Schu S4 strain was also examined within infected MDMs treated with clarithromycin and enrofloxacin. Taken together, this study describes the development of an important reagent for F. tularensis research, especially since the likelihood of engineered antibiotic resistant strains will emerge with time. Such strains will be extremely useful in high-throughput screens for novel compounds that could interfere with critical virulence processes in this important bioweapons agent and during infection of alveolar macrophages.

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Acknowledgments

We acknowledge support from the United States Defense Threats Reduction Agency INSIGHTS Program. We also wish to thank Drs. M. Pavelka (University of Rochester) and T. Zahrt (Medical College of Wisconsin) for provision of plasmid vectors.

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Authors and Affiliations

  1. Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH, 45267-0524, USA

    Shengchang Su, Hansraj Bangar, Daniel J. Hassett & Thomas J. Lamkin

  2. UES, Inc., Dayton, OH, 45432, USA

    Roland Saldanha, Adin Pemberton & Steven A. Kawamoto

  3. Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229-3039, USA

    Bruce Aronow

  4. Air Force Research Laboratory, 711th HPW/RHPC, Wright Patterson Air Force Base, Dayton, OH, 45433, USA

    Thomas J. Lamkin

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  1. Shengchang Su
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  2. Roland Saldanha
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  3. Adin Pemberton
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  4. Hansraj Bangar
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  5. Steven A. Kawamoto
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  6. Bruce Aronow
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  7. Daniel J. Hassett
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  8. Thomas J. Lamkin
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Correspondence to Daniel J. Hassett or Thomas J. Lamkin.

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Su, S., Saldanha, R., Pemberton, A. et al. Characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agent bacterium, Francisella tularensis Schu S4 and the surrogate type B live vaccine strain (LVS). Appl Microbiol Biotechnol 97, 9029–9041 (2013). https://doi.org/10.1007/s00253-013-5081-9

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  • DOI: https://doi.org/10.1007/s00253-013-5081-9

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