Analysis of Fluorescent Protein Expression in Transformants ofRickettsia monacensis, an Obligate Intracellular Tick Symbiont

Abstract

We developed and applied transposon-based transformation vectors for molecular manipulation and analysis of spotted fever group rickettsiae, which are obligate intracellular bacteria that infect ticks and, in some cases, mammals. Using the Epicentre EZ::TN transposon system, we designed transposons for simultaneous expression of a reporter gene and a chloramphenicol acetyltransferase (CAT) resistance marker. Transposomes (transposon-transposase complexes) were electroporated intoRickettsia monacensis, a rickettsial symbiont isolated from the tickIxodes ricinus. Each transposon contained an expression cassette consisting of the rickettsialompApromoter and a green fluorescent protein (GFP) reporter gene (GFPuv) or theompBpromoter and a red fluorescent protein reporter gene (DsRed2), followed by theompAtranscription terminator and a secondompApromoter CAT gene cassette. Selection with chloramphenicol gave rise to rickettsial populations with chromosomally integrated single-copy transposons as determined by PCR, Southern blotting, and sequence analysis. Reverse transcription-PCR and Northern blots demonstrated transcription of all three genes. GFPuv transformant rickettsiae exhibited strong fluorescence in individual cells, but DsRed2 transformants did not. Western blots confirmed expression of GFPuv inR. monacensisand inEscherichia coli, but DsRed2 was expressed only inE. coli. The DsRed2 gene, but not the GFPuv gene, contains many GC-rich amino acid codons that are rare in the preferred codon suite of rickettsiae, possibly explaining the failure to express DsRed2 protein inR. monacensis. We demonstrated that our vectors provide a means to study rickettsia-host cell interactions by visualizing GFPuv-fluorescentR. monacensisassociated with actin tails in tick host cells.