Green Fluorescent Protein as a Marker in Rickettsia typhi Transformation

Abstract

Transformation of rickettsiae is a recent accomplishment, but utility of this technique is limited due to the paucity of selectable markers suitable for use in this intracellular pathogen. We chose a green fluorescent protein variant optimized for fluorescence under UV lights (GFPUV) as a fluorometric marker and transformed Rickettsia typhi with anrpoB-GFPUV fusion construct. The rickettsiae were subsequently grown in Vero cells, and cultures were screened by PCR and restriction fragment length polymorphism (RFLP) to confirm incorporation of the rpoB-GFPUV construct. Cultures were then analyzed by flow cytometry for detection of GFPUV expression, and transformed R. typhi were isolated in a fluorescence-activated cell sorter. This is the first report of transformation of rickettsiae with a nonrickettsial (GFPUV) gene.

Dissecting the Rickettsia prowazekii Genome: Genetic and Proteomic Approaches

“…This situation is rapidly changing as rickettsiologists take advantage of an increasing number of rickettsial genome sequence databases and the development of techniques, such as site-directed mutagenesis and transposon insertions, for generating and isolating rickettsial gene mutants. [7][8][9][10][11][12] However, the obligate intracellular nature of rickettsial growth continues to impose severe restrictions on the genetic manipulation and isolation of rickettsial mutants. For example, one of the most straightforward tasks in the genetic analysis of free-living bacteria such as Escherichia coli, the isolation of individual clones, is quite problematic for a bacterium that does not form colonies on the surface of a solid medium.…”

Identification and Molecular Analysis of the Gene Encoding Rickettsia typhi Hemolysin

“…While the TG rickettsiae adhere to and lyse sheep and human erythrocytes, the SFG rickettsiae cannot lyse erythrocytes. The hemolytic activity of R. prowazekii has been extensively studied for four decades (6,12,14), but the DNA sequences of two genes (tlyA and tlyC) encoding hemolysins of R. prowazekii have been identified recently (2). Rickettsial entry and exit from phagosomes have also been indirectly associated with two phospholipases, A 2 and C (14,15), but the molecular events that lead to rickettsial intracellular placement, cell-to-cell spread, and eventual lysis of the host cells have yet to be elucidated.…”

“…The infected monolayers were harvested, and rickettsiae were purified by discontinuous Renografin gradient ultracentrifugation. Genomic DNAs were isolated as previously described (12). Briefly, 200 l of Renografinpurified rickettsiae were suspended overnight at 37°C in 400 l of TE buffer (10 mM Tris-HCl, [pH 8.0], 1 mM EDTA) supplemented with 0.1 mg of proteinase K per ml and 1.0% sodium dodecyl sulfate, and an equal volume of phenolchloroform-isoamyl alcohol was added.…”

“…at 37°C for 1 h, and subjected to heat inactivation at 75°C for 10 min. Following phenol-chloroform-isoamyl extraction, the linearized GFPuv was electrophoresed on a 1% agarose gel, excised, and eluted as previously described (12). The vector (linearized GFPuv) and insert (tlyC) at a 1:3 ratio were ligated at 16°C overnight with 10 U of the T 4 DNA ligase and 1ϫ buffer (New England BioLabs).…”

Genetic Manipulation of Rickettsiae: a Preview

“…Silent mutations were introduced to distinguish transformants and spontaneous mutants. Troyer et al (21) demonstrated transformation of R. typhi using expression of the green fluorescent protein (GFP) to detect recombinants. For the R. typhi experiments, transformation was accomplished with a PCR product in which the GFP gene was translationally fused to the rpoB gene.…”