Identification and Characterization of Novel and Potent Transcription Promoters of Francisella tularensis

Abstract

Two alternative promoter trap libraries, based on the green fluorescence protein (gfp) reporter and on the chloramphenicol acetyltransferase (cat) cassette, were constructed for isolation of potent Francisella tularensis promoters. Of the 26,000 F. tularensis strain LVS gfp library clones, only 3 exhibited visible fluorescence following UV illumination and all appeared to carry the bacterioferritin promoter (Pbfr). Out of a total of 2,000 chloramphenicol-resistant LVS clones isolated from the cat promoter library, we arbitrarily selected 40 for further analysis. Over 80% of these clones carry unique F. tularensis DNA sequences which appear to drive a wide range of protein expression, as determined by specific chloramphenicol acetyltransferase (CAT) Western dot blot and enzymatic assays. The DNA sequence information for the 33 unique and novel F. tularensis promoters reported here, along with the results of in silico and primer extension analyses, suggest that F. tularensis possesses classical Escherichia coli 70 -related promoter motifs. These motifs include the ؊10 (TATAAT) and ؊35 [TTGA(C/T)A] domains and an AT-rich region upstream from ؊35, reminiscent of but distinct from the E. coli upstream region that is termed the UP element. The most efficient promoter identified (Pbfr) appears to be about 10 times more potent than the F. tularensis groEL promoter and is probably among the strongest promoters in F. tularensis. The battery of promoters identified in this work will be useful, among other things, for genetic manipulation in the background of F. tularensis intended to gain better understanding of the mechanisms involved in pathogenesis and virulence, as well as for vaccine development studies.

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

“…We hypothesized that perhaps expression of FliC of P. aeruginosa (FliC Pa ) in LVS is detrimental to this bacterium. Since groE is an especially robust promoter, 9 we reasoned using a weaker promoter to drive expression of fliC may reduce the apparent unfavorable effect that overexpression of this heterologous gene was having on LVS. Therefore, we cloned fliC into pGRP so that this P. aeruginosa gene was under the control of the FTL_0580 (FGRp) promoter 17 which produces substantially fewer transcripts than the groE promoter 18 (Fig.…”

“…However, a plasmid-based expression system is more practical to provide proof of concept. Therefore, we modified a stable Francisella plasmid, pFNLTP8 8 to encode the robust groE promoter of F. tularensis 9 (Fig. 1A).…”

“…9 Zaide et al showed these 2 promoters, along with groE, are the most potent of F. tularensis. 9 An alternative strategy to achieve greater protein expression could be to utilize tandem promoters to maximize transcript levels. 23 However, the Figure 3.…”

“…This strategy employed the use of a stable Francisella shuttle vector in which the exogenous genes were under the control of either the groE or FGRp promoter. 9,17 These recombinant F. tularensis LVS strains were used to immunize mice to determine if the heterologously-expressed proteins could generate a robust adaptive immune response against P. aeruginosa. Mice that were immunized with recombinant LVS expressing FliC of P. aeruginosa produced a significant level of antibodies specific for P. aeruginosa relative to mock-immunized mice.…”

“…Primers groE1 and groE2 were used to PCR-amplify the F. tularensis LVS groE promoter. 9 This amplicon was digested with KpnI and EcoRI, gel-purified, and ligated with pFNLTP8 that had been digested with these same enzymes yielding pABST.…”