Mariner-based transposon mutagenesis of Rickettsia prowazekii

Abstract

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP(UV)) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP(UV) were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP(UV) fluorescence.

FIG. 1.

Physical map of pMW1650. The transposase, Himar1, is under the control of the borrelial promoter, flgBp (16). The region including rpsLp-arr-2_Rp (Rif^r), ompAp-GFP_UV, and ColE1 represents the transposable element and is bounded by inverted repeats (IR), denoted by triangles. The small arrows indicate oligonucleotides (DW317 and DW1100) used for subsequent sequencing after rescue in E. coli. ColE1, an E. coli origin of replication; Kan^r, the kanamycin resistance gene.

FIG. 2.

Southern blot analysis of transformed rickettsiae. (A) Lane 1, a mixture of chromosomal DNA isolated from R. prowazekii Madrid E (1 μg) and from L929 host cells (1.5 μg); lane 2, DNA (2 μg) isolated from a population of rifampin-resistant rickettsiae following transformation. (B) Lanes 1, 2, and 4 to 7, rickettsial chromosomal DNA (2 μg) from putative clones isolated by limiting dilution; lane 3, a mixture of chromosomal DNA isolated from R. prowazekii Madrid E (2 μg) and from L929 host cells (0.5 μg). In all analyses, chromosomal DNA was digested with HindIII and membranes were probed with a ³²P-labeled GFP_UV ORF. Lanes M, molecular size markers.

FIG. 3.

Schematic of the R. prowazekii chromosome showing the locations of Himar1 insertions. Insertions into coding regions are indicated with red arrowheads and the respective ORF number. Intergenic insertions are indicated with blue lines. Specific insertion sites are listed in Table S1 in the supplementary material.

FIG. 4.

PCR analysis of a limiting-dilution clone. DNA, isolated from rifampin-resistant rickettsiae at selected time points, was used as a template in a PCR designed to detect the RP401 gene. The presence of wild-type sequence results in the amplification of a 420-bp fragment. A transposon insertion within the RP401 gene results in a 3,011-bp fragment. Lane 1, negative control; lane 2, wild-type Madrid E chromosomal DNA; lane 3, DNA isolated from a population of rifampin-resistant rickettsiae harvested at day 18; lane 4, DNA isolated from rickettsiae harvested on day 49 following an initial round of limiting dilution; lane 5, DNA isolated on day 83 from a cloned population following a second round of limiting dilution. Lane M, molecular size markers.

FIG. 5.

Fluorescence microscopy of rickettsial transformants. (A) The image is an overlay of intracellular rickettsiae expressing the GFP_UV protein (green) visualized with an FITC filter set and the nuclear counterstain DAPI visualized with a DAPI filter set (blue) on acetone-fixed cells. As a negative control, the inset in panel A shows an L929 cell infected with wild-type R. prowazekii viewed with the same FITC and DAPI filter sets. An increase in gain was used to visualize the nonfluorescent rickettsiae counterstained with DAPI. (B) Live mouse L929 cell infected with viable rickettsiae expressing GFP_UV protein visualized with CytoViva 150 Exfo X-Cite 120 fluorescent light source. (C) The same cell visualized in panel B with fluorescent rickettsiae (green) viewed with the CytoViva and a universal fluorescent filter set.