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- Title
Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species.
- Authors
Tlapák, Hana; Köppen, Kristin; Rydzewski, Kerstin; Grunow, Roland; Heuner, Klaus
- Abstract
We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 (F. tularensis subsp. novicida-like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val (Francisella Integration Vector-tRNAVal-specific), using the attL/R-sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli. The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp. where FIV-Val stably integrated site specifically into the tRNAVal gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.
- Subjects
FRANCISELLA; BACTERIOPHAGES; GENOMICS; ESCHERICHIA coli; GENETIC mutation
- Publication
Frontiers in Cellular & Infection Microbiology, 2018, Vol 8, p1
- ISSN
2235-2988
- Publication type
Article
- DOI
10.3389/fcimb.2018.00075