Transposon mutagenesis of the obligate intracellular pathogen Rickettsia prowazekii

Abstract

Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsL(P)), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsL(P)-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes.

FIG. 1.

Schematic outlining the synthesis of Rparr-2 and the construction of the rpsL^P-Rparr-2 transposon. Arrows at the top of the figure represent oligonucleotides, while the solid black bar represents the synthesized Rparr-2 gene.

FIG. 2.

Hybridization of the R. prowazekii rpsL^P-Rparr-2 gene cassette to R. prowazekii chromosomal DNA digested with HindIII. Lane 1, DNA isolated from the Madrid E strain; lane 2, DNA isolated from rifampin-resistant rickettsiae of transformation T-12; lane 3, DNA isolated from rifampin-resistant rickettsiae of transformation T-15. Molecular size markers are indicated, as is the 5.9-kb rpsL HindIII fragment.

FIG. 3.

(A) Schematic of the R. prowazekii chromosome showing the locations of Rparr-2 transposon insertions. (B) Gene map showing the insertion of the Rparr-2 transposon into the R. prowazekii RP689 ORF. Dotted arrows indicate the positions and directions of synthesis of primer sites. Numbers indicate size in base pairs. ME, mosaic ends; rpsL^P, rpsL promoter.

FIG. 4.

(A) PCR analysis of the RP689 insertion. Chromosomal primers that amplify a portion of the RP689 gene (DW615 and DW616) (Fig. 3B) were used in PCRs with template DNAs from R. prowazekii. Lane 1, T-12B1 RP689 insertion clone DNA; lane 2, Madrid E DNA; lane 3, L-929 cell DNA; lane 4, water. Size markers (M) are indicated in base pairs. (B) Southern blot analysis. Lane 1, T-12 DNA; lane 2, T-12B1 DNA; lane 3, Madrid E DNA; lane 4, L-929 cell DNA.