Transformation of Rickettsia prowazekii to rifampin resistance

Abstract

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 beta 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.

FIG. 1

(A) Schematic map of the R. prowazekii rpoB gene region. The orientation of transcription is indicated by arrows. Plasmids containing the rickettsial fragments are indicated by horizontal lines. The lengths of the lines indicate the corresponding segment of the gene map contained within the recombinant plasmid. DW89, DW71, DW83, and DW92 mark the positions of the specific oligonucleotides used to amplify the inserts of the indicated plasmids. E, EcoRI; H, HindIII; P, PstI; X, XbaI. (B) Nucleotide sequence of the 5′ half of the R. prowazekii rpoB gene. The DNA sequence of the noncoding strand is shown. The predicted amino acid sequence is given in the single-letter code. The putative Shine-Dalgarno-like sequence is doubly underlined. Relevant oligonucleotide sequences discussed in the text are indicated, and the direction of DNA synthesis is indicated by arrows. Mutations are indicated at the appropriate positions above and below the sequence.

FIG. 2

Detection of plasmid DNA in electroporated rickettsiae. The sdhA gene and pBluescript II SK+ (pSK) and pMOB plasmids are indicated. Numbers are sizes in kilobases. (A) Autoradiograph of DNA extracted from DNase-treated R. prowazekii cells immediately following electroporation, digested with HindIII, and probed with pMW489, a plasmid containing the R. prowazekii sdhA gene on a 6.0-kb HindIII fragment. Lanes 1 and 3, R. prowazekii cells electroporated in the presence of 1 μg of pBluescript II SK+ plasmid; lanes 2 and 4, R. prowazekii cells mixed with 1 μg of pBluescript II SK+ plasmid but not electroporated. (B) Detection of electroporated DNA after infection of host cells. R. prowazekii cells were electroporated with 10 μg of pMOB and the electroporated rickettsiae were allowed to infect L929 cells. At 1 h (lane 1) and 48 h (lane 2) after infection, the rickettsiae were harvested and treated with DNase. After inactivating the DNase, rickettsial DNA was extracted, digested with HindIII, and probed with pMW489.

FIG. 3

(A) Sequencing reactions (only C and T lanes are shown) comparing different ratios of templates that differ in sequence at one position. The template present at the indicated percentages contains two C residues. (B) DNA sequence of PCR products from Rif^s and Rif^r rickettsiae. The mutations described in the text are indicated on the right. Asterisks identify the sequence residues of the minor background Rif^r population.