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Applied and Environmental Microbiology, April 2005, p. 2095-2105, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2095-2105.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Gerald D. Baldridge,* Nicole Burkhardt, Michael J. Herron, Timothy J. Kurtti, and Ulrike G. Munderloh

Department of Entomology, University of Minnesota, St. Paul, Minnesota

Received 23 August 2004/ Accepted 23 October 2004

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 into Rickettsia monacensis, a rickettsial symbiont isolated from the tick Ixodes ricinus. Each transposon contained an expression cassette consisting of the rickettsial ompA promoter and a green fluorescent protein (GFP) reporter gene (GFPuv) or the ompB promoter and a red fluorescent protein reporter gene (DsRed2), followed by the ompA transcription terminator and a second ompA promoter 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 in R. monacensis and in Escherichia coli, but DsRed2 was expressed only in E. 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 in R. monacensis. We demonstrated that our vectors provide a means to study rickettsia-host cell interactions by visualizing GFPuv-fluorescent R. monacensis associated with actin tails in tick host cells.

* Corresponding author. Mailing address: Department of Entomology, University of Minnesota, 1980 Folwell Ave., St. Paul, MN 55108. Phone: (612) 624-3688. Fax: (612) 625-5299. E-mail: baldr001{at}umn.edu.

Applied and Environmental Microbiology, April 2005, p. 2095-2105, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2095-2105.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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