Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

摘要: Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, pseudomallei. Using bioinformatic approaches other experimental analyses, identified P0253 P1 potent promoters that drive optimal expression of single copy B. anthracis spp. well their surrogate strains, respectively. In comparison, Y. pestis its strain need two chromosomal copies cysZK promoter (P2cysZK) for fluorescence. The P0253-, P2cysZK-, P1-driven GFP RFP fusions were first cloned into vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or derivatives. resultant constructs delivered respective surrogates subsequently agent strains. GFP- RFP-tagged strains exhibited bright fluorescence at an exposure time less than 200 msec displayed same virulence traits wild-type parental utility tagged was proven by macrophage infection assays lactate dehydrogenase release analysis. Such will be extremely useful high-throughput screens novel compounds could either kill these organisms, interfere with critical processes important bioweapon agents during alveolar macrophages.