Applied Microbiology and Biotechnology

, Volume 97, Issue 20, pp 9029–9041

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)

  • Shengchang Su
  • Roland Saldanha
  • Adin Pemberton
  • Hansraj Bangar
  • Steven A. Kawamoto
  • Bruce Aronow
  • Daniel J. Hassett
  • Thomas J. Lamkin
Applied genetics and molecular biotechnology

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.

Keywords

Stable Constitutive Chromosomal GFP/RFP reporters Francisella tularensis 

Supplementary material

253_2013_5081_MOESM1_ESM.xlsx (293 kb)
ESM 1(XLSX 293 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Shengchang Su
    • 1
  • Roland Saldanha
    • 2
  • Adin Pemberton
    • 2
  • Hansraj Bangar
    • 1
  • Steven A. Kawamoto
    • 2
  • Bruce Aronow
    • 3
  • Daniel J. Hassett
    • 1
  • Thomas J. Lamkin
    • 1
    • 4
  1. 1.Department of Molecular Genetics, Biochemistry and MicrobiologyUniversity of Cincinnati College of MedicineCincinnatiUSA
  2. 2.UES, Inc.DaytonUSA
  3. 3.Division of Biomedical InformaticsCincinnati Children’s Hospital Medical CenterCincinnatiUSA
  4. 4.Air Force Research Laboratory711th HPW/RHPC, Wright Patterson Air Force BaseDaytonUSA

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