3a) and ripA’-lacZ fusion alleles (Fig. 3b) on the chromosome (Fig. 3c). The insertions did not impact intracellular replication of
the reporter strains and thus were unlikely to significantly impact expression of the wild type ripA gene. Figure 3 Reporter plasmids and co-integrates. Cabozantinib cell line Cartoon representations of the F. tularensis LVS genomic organizations of the ripA locus (a), pBSK ripA’-lacZ2 transcriptional reporter plasmid (b), and the ripA::pBSK ripA’lacZ cointegrate (c). The ripA locus is present in only one copy in ripA::pBSK ripA’-lacZ2 however the promoter is duplicated by the insertion resulting maintenance of the entire wild type ripA locus as well as the ripA’-lacZ reporter. The predicted ripA promoter is represented by a black arrow (a-c). pBSK ripA’-lacZ2 is shown in gray while the alleles of the native locus are white. We examined the effects of specific mutations in the predicted ripA promoter, ribosome binding site, and translation frame on the expression of β-galactosidase. Mutations in the predicted -10 sequence, RBS, and the introduction
of a frameshift mutation (Fig. 2a) in the translational fusion construct each resulted in decreased β-galactosidase activity as compared to the wild type reporter (Fig. 2c). The β-galactosidase activity expressed by the chromosomal click here reporters was less than 25% of that produced by the plasmid reporters (Fig. 2b). The ripA’-lacZ1 translational fusion produced significantly less activity than the ripA’-lacZ2 transcriptional fusion in both the chromosomal and plasmid version of the reporter (Fig. 2b). These differences might reflect post transcriptional regulation of expression or simply a difference in the efficiency of translation initiation between the two constructs. Quantification of RipA protein We were unable to quantify native RipA protein concentrations in Francisella cultures since our polyclonal anti-RipA antisera produced high background in Western blots and ELISA . We therefore generated a construct that expressed a RipA – tetracysteine (TC) fusion protein DOK2 to facilitate the use of FlAsH™ (Invitrogen) reagents to directly measure RipA protein concentrations.
Both plasmid and chromosomal integrant strains (Fig. 4a) expressing RipA-TC (Fig. 4b) were constructed in a ΔripA background. Intracellular replication was restored in each of these strains demonstrating that the RipA-TC fusion protein was functional and did not confer a detectable mutant phenotype (data not shown). Figure 4 Tetracysteine tag construction and expression. (a) Graphical depiction of F. tularensis LVS ripA locus showing the location of SOE PCR primers used to insert the C terminal TC tag (marked in gray). (b) Nucleotide and amino acid sequence of the C terminal TCtag showing the overlapping sequence of the SOE PCR primers. (c) In gel fluorescence of RipA-TC (black arrow) from dilution series of F. tularensis LVS (plasmid) pKK ripA’-TC and F.