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

摘要

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 σ~(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.