The Orientia tsutsugamushi genome reveals massive proliferation of conjugative type IV secretion system and host-cell interaction genes

Abstract

Scrub typhus is caused by the obligate intracellular rickettsia Orientia tsutsugamushi (previously called Rickettsia tsutsugamushi). The bacterium is maternally inherited in trombicuid mites and transmitted to humans by feeding larvae. We report here the 2,127,051-bp genome of the Boryong strain, which represents the most highly repeated bacterial genome sequenced to date. The repeat density of the scrub typhus pathogen is 200-fold higher than that of its close relative Rickettsia prowazekii, the agent of epidemic typhus. A total of 359 tra genes for components of conjugative type IV secretion systems were identified at 79 sites in the genome. Associated with these are >200 genes for signaling and host-cell interaction proteins, such as histidine kinases, ankyrin-repeat proteins, and tetratrico peptide-repeat proteins. Additionally, the O. tsutsugamushi genome contains >400 transposases, 60 phage integrases, and 70 reverse transcriptases. Deletions and rearrangements have yielded unique gene combinations as well as frequent pseudogenization in the tra clusters. A comparative analysis of the tra clusters within the genome and across strains indicates sequence homogenization by gene conversion, whereas complexity, diversity, and pseudogenization are acquired by duplications, deletions, and transposon integrations into the amplified segments. The results suggest intragenomic duplications or multiple integrations of a massively proliferating conjugative transfer system. Diversifying selection on host-cell interaction genes along with repeated population bottlenecks may drive rare genome variants to fixation, thereby short-circuiting selection for low complexity in bacterial genomes.

Fig. 1.

Circular map of the O. tsutsugamushi genome. The outer circle shows plus-strand genes and the second circle, minus-strand genes; in red, fragmented genes, and in orange, full-length genes. The third circle shows Rickettsial core genes in yellow; the fourth circle, identical repeats >200 bp in green; the fifth circle, tra genes for components of conjugative TFSSs in blue; the sixth circle, transposases in pink; in the seventh circle, numbered color blocks represent the tra gene clusters in Fig. 3; in the eighth circle, colored letters represent tra-associated gene clusters in Fig. 3; and the innermost circle shows the GC skew plot (G−C)/(G+C).

Fig. 2.

Repeat density in obligate intracellular bacteria. The frequency of repeats in the genomes of (a) O. tsutsugamushi and (b) R. prowazekii, with each dot representing a repeat >95% identical and >200 bp in size; blue, direct repeats; red, reverse repeats. (c) Scrambled gene order structures in O. tsutsugamushi and R. bellii. The yellow box indicates the location of the single tra cluster for TFSSs in the R. bellii genome and the homologous amplified tra clusters in the O. tsutsugamushi genomes. (d) Repeat density is plotted against genome size (% identical repeats >200 bp). Bacterial groupings are indicated by circles; red, obligate intracellular members of the Rickettsiales including Orientia, O. tsutsugamushi; Ana, Anaplasma marginale; Ehl, Ehrlichia ruminantium; Rb, R. bellii; Rc, Rickettsia conorii; Rf, R. felis; Rp, R. prowazekii; Rt, Rickettsia typhi; Trs, Wolbachia endosymbiont; TRS, wMel, Wolbachia wMel; green, Hel, Helicobacter pylori; Nei.c58, Neisseria meningitidis C58; Nei.z2491, N. meningitidis z2491; blue, the most highly repeated bacterial genomes until the time of this study are those of Myco, Mycoplasma mycoides; and Phyt, Phytoplasma strain OY M.

Fig. 3.

Gene order structure of (a) 24 long repeated tra gene clusters and (b) the flanking tra-associated gene clusters encoding putative effector proteins. The position of the clusters in the genome is indicated in Fig. 1.

Fig. 4.

A sequence comparison of selected O. tsutsugamushi repeated regions from different clinical isolates. (a) Expansion of region due to transposon insertion. (b) Reduction of the region due to deletion. The top line shows the gene order structure in the Boryong strain (B), and the bars underneath represent homologs regions found in strains Karp (K), Kato (KT), and Gilliam (G). Blue indicates inferred transposon insertions and red color sequence deletions. Green indicates the insertion of a transposon in strain G.