Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species

Abstract

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of <i>Francisella hispaniensis</i> AS02-814 (<i>F. tularensis</i> subsp. <i>novicida</i>-like 3523). In this study, we constructed two variants of a <i>Francisella</i> phage integration vector, called pFIV1-Val and pFIV2-Val (<i>Francisella</i> Integration Vector-tRNA^Val-specific), using the <i>attL/R-</i>sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a <i>sacB</i> 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 <i>Escherichia coli</i>. The constructs generated a circular episomal form in <i>E. coli</i> which could be used to transform <i>Francisella spp</i>. where FIV-Val stably integrated site specifically into the tRNA^Val 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 <i>Francisella</i> mutant strain. The vectors were stable <i>in vitro</i> and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in <i>Francisella</i> research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different <i>Francisella</i> species.