Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens

Abstract

The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair-long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic "melting pot" where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.

Figure 1. Phylogenetic Relationships of R. bellii with Other Rickettsiae

The tree was built using the comparison of concatenated nucleotide sequences of the 16S rDNA, gltA, pheS, pheT, nusA, valS, and smpB genes using the neighbor-joining method with Kimura-2 parameter. Bootstrap values are indicated at the nodes. The tree was rooted using the orthologous sequences of Anaplasma marginale as an outgroup (not shown). The scale bar corresponds to 5% divergence in nucleotide sequences.

Figure 2. Comparison of R. bellii and R. conorii Genomes, and the Cumulative Nucleotide-Excess Curve for the R. bellii Genome

In the top panel, dots represent homologous genomic segments detected by BLASTN (E-value < 0.001). Bottom panel shows the GT-excess plot.

Figure 3. Alignment of the tra Gene Clusters Found in the Chromosomes of R. bellii and P. amoebophila

Homologous genes are indicated by the same color. For R. bellii, regions similar to tra genes in E. coli F plasmid, A. tumefacience Ti plasmid, and R. felis pRF plasmid are indicated by red, blue, and green bars, respectively.

Figure 4. Sex Pili-Like Surface Appendage of R. bellii

(A) Electron micrograph of pili observed after negative staining. R. bellii shows long and flexible pilus between 2 bacteria observed from supernatant of XTC-2 cells infected for 48 h at 28 °C. Bacteria were fixed by addition (1:1) of 2% formaldehyde-saline and samples were applied to copper Formvar grids (Electron Microscopy Sciences) for 2 min before negative staining with 1% phosphotungstic acid (pH 6.5) for 2 to 4 min. Samples were examined with a Hitachi 7000 transmission electron microscope. (B) Immunofluorescence performed on the same bacterial culture. R. bellii were probed with a rabbit polyclonal anti-R. belli antibody and with a FITC-conjugated goat anti rabbit Ig as secondary antibody. Slides were examined with an Olympus BX 51 microscope (magnification 100×).

Figure 5. Phylogenetic Tree of the Proteins Encoded in the F-Like tra Gene Clusters

The tree was built using a maximum likelihood method with JTT substitution model and midpoint rooting based on the concatenated sequence alignment of TraD_F and TraG_F. Plasmid encoded sequences are indicated by asterisks. Other sequences are encoded in the chromosomes.

Figure 6. Proportions of BLAST Best Hits to Different Organism Groups

The percentages are given relative to the total number of ORFs exhibiting database hits (E-value < 10⁻⁵).

Figure 7. R. bellii Grown in XTC-2 Cell Line

(A) Gimenez-stained bacteria observed by optic microscopy. (B) Electronic microscopy of infected cells evidences multiplication of R. bellii within the nucleus. (A) and (B) show the intranuclear accumulation of R. bellii. (C) Higher-magnification image showing the intrusion of R. bellii into the nucleus.