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)

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.

Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei

“…So far we have utilized different vectors based on such system to make the fluorescent labeling in gram‐negative bacteria F. tularensis Schu S4 and LVS (Su et al. ), Y. pestis , Y. pseudotuberculosis , B. thailandensis , B. pseudomallei , and B. mallei . Despite the Tn7 transposition has not yet been documented in gram‐positive bacteria, the markerless allelic exchange system developed by Janes and Stibitz () also worked efficiently in tagging both Sterne and Ames strains of B. anthracis , a gram‐positive bacteria.…”

“…The wild-type gfp and rfp have been mutated or optimized based on codon usage preferred by bacteria to improve detection and expression of the fluorescent protein in prokaryotes (Heim et al 1995;Cormack et al 1996;Miller and Lindow 1997;Norris et al 2010). We chose the GFP Superfolder and RFP Tur-boRed (Su et al 2013) as fluorescent proteins for B. anthracis, Y. pestis, and Y. pseudotuberculosis, and the eGFP and optimized RFP (Norris et al 2010) for B. mallei and B. pseudomallei, which have been utilized successfully in fluorescent tagging of Francisella spp. (Su et al 2013) and Burkholderia spp.…”

“…We chose the GFP Superfolder and RFP Tur-boRed (Su et al 2013) as fluorescent proteins for B. anthracis, Y. pestis, and Y. pseudotuberculosis, and the eGFP and optimized RFP (Norris et al 2010) for B. mallei and B. pseudomallei, which have been utilized successfully in fluorescent tagging of Francisella spp. (Su et al 2013) and Burkholderia spp. (Norris et al 2010).…”

“…When applicable, the mini-Tn7 transposon system is a convenient and most efficient delivery tool for site-specific genomic tagging of bacteria in which the tagging DNA is stably inserted at a unique and neutral chromosomal site. So far we have utilized different vectors based on such system to make the fluorescent labeling in gramnegative bacteria F. tularensis Schu S4 and LVS (Su et al 2013), Y. pestis, Y. pseudotuberculosis, B. thailandensis, B. pseudomallei, and B. mallei. Despite the Tn7 transposition has not yet been documented in gram-positive bacteria, the markerless allelic exchange system developed by Janes and Stibitz (2006) also worked efficiently in tagging both Sterne and Ames strains of B. anthracis, a gram-positive bacteria.…”

DNA Transposition at Work

“…Not only is glmS highly conserved across many bacterial species, but it appears to be present in essentially all bacterial genomes sequenced to date and most bacteria have only one attTn7 site. One use of Tn 7 transposition has been to generate bacterial strains that express reporter genes such as GFP or luciferase for in vivo studies. − By introducing additional attTn7 sites into a genome, it is also possible to increase and modulate the expression level of a transgene . Genes encoding antibiotic resistance can be stably integrated into organisms that are already multidrug-resistant (for example, the hygromycin resistance gene, hph , in Acinetobacter baumannii ), or metabolic pathways modulated by adding heterolous genes …”