TetR-Based Gene Regulation Systems for Francisella tularensis

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>There are a number of genetic tools available for studying<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Francisella tularensis</jats:named-content>, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems for<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">F. tularensis</jats:named-content>based on the Tet repressor, TetR. For the inducible system, a<jats:italic>tet</jats:italic>operator sequence was cloned into a modified<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">F. tularensis groESL</jats:named-content>promoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon,<jats:italic>luxCDABE</jats:italic>, was cloned under the hybrid<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Francisella</jats:named-content>tetracycline-regulated promoter (<jats:italic>FTRp</jats:italic>), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association with<jats:italic>tetO.</jats:italic>Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml<jats:sup>−1</jats:sup>. In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists of<jats:italic>FTRp</jats:italic>regulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with the<jats:italic>lux</jats:italic>reporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploring<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">F. tularensis</jats:named-content>protein function.</jats:p>

PanG, a New Ketopantoate Reductase Involved in Pantothenate Synthesis

“…The putative panG (FTT1388) allele plus 300 bp of DNA flanking each end was PCR amplified from F. tularensis Schu S4 genomic DNA. The amplified fragment was cut with BamHI and NotI and then ligated into the sacB suicide vector, pEDL50 (30). Using splice junction PCR, FTT1388 (panG) was eliminated from the plasmid, creating a product with AatII restriction sites on either end.…”

“…The PCR product was digested with AatII and ligated, forming pSKI01. Conjugation and allelic exchange were then performed to introduce the clean in-frame deletion of panG into F. tularensis Schu S4 as previously described (30,33).…”

Genetic engineering ofFrancisella tularensisLVS for use as a novel live vaccine platform againstPseudomonas aeruginosainfections

“…Such systems have been developed for Francisella species. 24,25 However, whether or not the tetracycline-regulation for Francisella is effective inside an animal host remains to be determined. Another potential pitfall of the current study is that, for ease of manipulation, the genes encoding the exogenous proteins were harbored on plasmids.…”

The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host

“…The F. tularensis Lon substrates identified in the E. coli system were expressed as C-terminally His-tagged recombinant proteins from a tetracycline-inducible promoter in shuttle plasmid pEDL17 (57). The coding sequences of FTL478, FTL578, FTL663, FTL1017, FTL1217, FTL1228, FTL1957, and clpX were amplified from LVS genomic DNA with the following primer pairs, respectively: Pr7409/Pr7410, Pr10050/ Pr10051, Pr7405/Pr7406, Pr7411/Pr7412, Pr7869/Pr7870, Pr10229/ Pr10230, Pr7407/Pr7408, and Pr7631/Pr7632.…”