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J Bacteriol, April 1998, p. 2118-2124, Vol. 180, No. 8
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Transformation of Rickettsia prowazekii
to Rifampin Resistance
Lyudmila I.
Rachek,
Aimee M.
Tucker,
Herbert H.
Winkler, and
David O.
Wood*
Department of Microbiology and Immunology,
Laboratory of Molecular Biology, University of South Alabama
College of Medicine, Mobile, Alabama 36688
Received 4 December 1997/Accepted 16 February 1998
Rickettsia prowazekii, the causative agent of epidemic
typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence
of techniques for genetic manipulation hampers the study of this organism's unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we
identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic
exchange in R. prowazekii. Comparison
of the rpoB sequences from the rifampin-sensitive
(Rifs) Madrid E strain and a rifampin-resistant
(Rifr) mutant identified a single point mutation
that results in an arginine-to-lysine change at position 546 of the
R. prowazekii RNA polymerase
subunit.
A plasmid containing this mutation and two additional silent
mutations created in codons flanking the Lys-546 codon was introduced
into the Rifs Madrid E strain of R. prowazekii by electroporation, and in the presence of
rifampin, resistant rickettsiae were selected. Transformation, via
homologous recombination, was demonstrated by DNA sequencing of PCR
products containing the three mutations in the Rifr
region of rickettsial rpoB. This is the first successful
demonstration of genetic transformation of Rickettsia
prowazekii and represents the initial step in the
establishment of a genetic system in this obligate intracellular
pathogen.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Laboratory of Molecular Biology,
University of South Alabama College of Medicine, Mobile, AL 36688. Phone: (334) 460-6324. Fax: (334) 460-7269. E-mail:
wood{at}sungcg.usouthal.edu.
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