The genomic and metabolic diversity of Rickettsia

Research in Microbiology 158 (2007) 745e753 www.elsevier.com/locate/resmic

The genomic and metabolic diversity of Rickettsia Hans-Henrik Fuxelius a, Alistair Darby a,1, Chan-Ki Min b, Nam-Hyuk Cho b, Siv G.E. Andersson a,* a

Department of Molecular Evolution, Evolutionary Biology Center, Uppsala University, Norbyva¨gen 18C, S-752 36 Uppsala, Sweden b Department of Microbiology and Immunology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Republic of Korea Received 7 July 2007; accepted 19 September 2007 Available online 4 October 2007

Abstract Comparative genomics of Rickettsia and Orientia has revealed an exciting interplay between reductive evolutionary forces acting on metabolic genes in all species and proliferation of mobile genetic elements in some species. These contradictory evolutionary forces highlight the influence of chance, adaptation and host-cell exploitation during the evolution of intracellular bacteria. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Mobile elements; Orientia; Reductive evolution; Rickettsia; Type IV secretion systems

1. The typhus pathogens The term typhus was first used in the 1760s; currently the typhus disease complex includes a group of related infectious agents causing diseases such as epidemic typhus, murine typhus and scrub typhus. Disease symptoms include high fever, headache and different neurological signs. Before the era of antibiotics, treatment mortality rates could be as high as 40% in infected patients. Charles Nicolle, former director of the Pasteur Institute in Tunis, received the Nobel Prize in 1928 for his discovery in 1909 that epidemic typhus is transmitted among humans by the human body louse [20]. Shortly thereafter, von Prowazek and Rocha-Lima demonstrated that the disease is transmitted via louse faeces rather than through their bites [17]. Today, we know that vector-borne transmission pathway is a characteristic feature of all rickettsial diseases, with the ecological zone of the vector restricting the spread of the infections. * Corresponding author. Tel.: þ46 18 471 4379; fax: þ46 18 471 6404. E-mail addresses: [email protected] (H.-H. Fuxelius), [email protected] (A. Darby), [email protected] (C.-K. Min), [email protected] (N.-H. Cho), [email protected] (S.G.E. Andersson). 1 Present address: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. 0923-2508/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2007.09.008

Ever since the disease symptoms of typhus were first described, the disease has been associated with human warfare. Starting in the Mediterranean region disease outbreaks followed armies moving up through Europe during the 16th century, causing fatalities among soldiers and spreading into the civilian populations. During the first and second World Wars hygiene measures were implemented to limit the spread of the human body lice, but millions of soldiers in Europe were still infected with epidemic typhus [2]. In South-East Asia, the scrub typhus pathogen caused more fatalities among Allied forces during the 2nd World War than the fighting per se [24]. The evident potency of these agents has led to their investigation as biological weapons [9] and two species, Rickettsia prowazekii (the agent of epidemic typhus) and Rickettsia rickettsii (the agent of Rocky Mountain spotted fever) are currently on the bioterrorism watch list [8].

2. The rickettsial genomes For a schematic illustration of the phylogenetic relationships of the Rickettsia species, the diseases and the vertebrate and vector hosts involved, see Fig. 1. Two rickettsial groups have traditionally been recognized, the typhus group (TG),

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Fig. 1. Summary information of Rickettsia spp. and O. tsutsugamushi. The inferred phylogeny, which is based on fifteen proteins, is taken from Ref. [16], with O. tsutsugamushi included as outgroup. Shown to the right of the phylogeny are the hosts and vectors involved, the diseases in incidental human hosts and the sizes of the sequenced genomes. SF under the heading disease refers to Spotted Fever. The Rickettsia species have been sorted into five groups as in Ref. [16]: TG, Typhus Group (R. prowazekii [7] (NC_000963), R. typhi [19] (NC_006142)); SFG, Spotted Fever Group (R. conorii [21] (NC_003103), R. sibirica (NZ_AABW01000001), R. rickettsii (NZ_AADJ01000001)); TRG, Transition Group (R. felis [23] (NC_007109), R. akari (NZ_AAFE01000001)); AG, Ancestral Group (R. bellii [22] (NC_007940), R. canadensis (NZ_AAFF01000001)). Also included is O. tsutsugamushi [13] (AM494475).

which contains the agents of epidemic (R. prowazekii) and murine typhus (Rickettsia typhi); and the spotted fever group (SFG), which includes the agent of Rocky Mountain spotted fever (R. rickettsii). The earliest diverging species in the genus are Rickettsia bellii and Rickettsia canadensis; these have recently been placed in a separate group called the ancestral group (AG) Rickettsia. A few species, Rickettisa felis and Rickettsia akari have genotypic and phenotypic characteristics intermediate of the other two groups and were recently proposed to be placed in a separate group, called the transitional group (TRG) Rickettsia [16]. Orientia tsutsugamushi, the agent of scrub typhus, was formerly called Rickettsia tsutsugamushi, but received a novel genus name when it was discovered that the extent of sequence divergence in the rRNA genes was higher than previously anticipated. Genome sizes for the different groups range from 1.1 Mb in the TG [7,19], 1.2e1.3 Mb in the SFG [21], 1.3e1.5 Mb in the TRG [23] and 1.5 Mb in the AG [22], with O. tsutsugamushi representing an extreme outlier with its 2.1 Mb genome [13]. Genome sizes correlate roughly with gene numbers, which ranges from 800 to 2000 per genome. Gene annotations are notoriously difficult in Rickettsia because of the gene degradation processes, yielding numerous examples of a gene in one species that is disrupted into multiple short fragments in another species. Not surprisingly, many of the species-specific genes are short in size and encode hypothetical proteins of

unknown functions with no similarity to genes in other species; these may not represent functional genes [1]. Much has been said previously about the rates and patterns of gene loss [3e7,21]. In this paper, we will concentrate on the metabolic streamlining and contrast it with the proliferation of mobile genetic elements. 3. Genome degradation and exploitation of host cellular processes For the purpose of this discussion, we have compared the genomes of Rickettsia spp. and O. tsutsugamushi with those of two Wolbachia species isolated from Drosophila melanogaster [25] and Brugia malayii [15]. Wolbachia is transovarially transmitted among arthropods or nematodes, but unlike the Rickettsia species it is not associated with mammalian infections. To get an overview of the shared and variably present genes, we first performed a simple gene prediction analysis on all genomes with the aid of Glimmer using similar settings [14]. Orthologous protein clusters were identified as described previously [13] and sorted into seven taxonomic groups depending on their conservation profiles in Rickettsia (R), Orientia (O) and Wolbachia (W) (Fig. 2a). The identified protein clusters were further sorted into functional groups based on homology searches against the cluster of orthologous groups (COG) database (Fig. 2b,c).

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Fig. 2. Universally and variably present genes in Rickettsia, Orientia and Wolbachia. a) Venn diagram illustrating the conservation profiles in comparisons of Rickettisa, Orientia and Wolbachia species. b) Table showing the number of orthologous protein clusters and the relative fraction of clusters with a classification in the COG database. c) Relative fraction of protein clusters per functional category sorted according to the taxonomic groupings. Putative protein-coding regions were identified with aid of Glimmer [4]. Shared orthologs were identified and clustered as described in Ref. [13]. Included in the comparison are one genome from Orientia (O. tsutsugamushi), two genomes from Wolbachia (Wolbachia wMel and wBm), and eight genomes from Rickettsia (Rickettsia conorii, R. sibirica, R. rickettsii, R. felis, R. akari, R. prowazekii, R. typhi and R. bellii). Genes were sorted into groups based on gene presence in Rickettsia (R), Orientia (O) and/or Wolbachia (W). Genes uniquely attributed to one genus included single-species ORFs as well as orthologs present in two or more genomes in the genus. Abbreviations for functional categories are as follows: C, energy production; D, cell cycle control; E. amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, protein synthesis; K, transcription; L, replication; M, cell wall/membrane; N, cell motility; O, posttranslational modification; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis; R, general function prediction; S, poorly characterized; T, signal transduction; U, intracellular trafficking; V, defence mechanisms.

We identified a core set of 458 different protein clusters that are present in at least one species of each genus (group ROW in Fig. 2a). We assume that these genes were present in the last common ancestor of the three intracellular genera. As anticipated, a high fraction of these clusters, 96%, belong to wellcharacterised cellular processes with almost 50% accounted for by only three categories; protein synthesis (J: n ¼ 106), replication (L: n ¼ 45) and energy production (C: n ¼ 52) (Fig. 2c). Also present in ROW were a few genes potentially acquired by horizontal gene transfers such as the paralogous gene family coding for proteins with ankyrin repeat domains.

Variably present genes may reflect host-adaptation processes and give hints to the genetic sources underlying phenotypic differences in lifestyle, host preferences and vector transmission pathways. For protein clusters identified in two of the three genera, circa 60e70% have homologs in the COG database and could be assigned putative functions (Fig. 2b). We identified only 22 clusters in the group WO, of which 13 have homologs in the COG database; these few proteins are randomly distributed among the functional classes with 0e3 genes per category. Five times as many, 129 protein clusters, were present in RW, 94 of which could be COG-classified. Dominant functions in

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RW were replication (L: n ¼ 14), cell surface biogenesis (M: n ¼ 11) and energy production (C: n ¼ 9) (Fig. 2c). In these groups, the likelihood of a gene loss in one lineage has to be weighted against the probability of independent gains in the other two lineages. We favour the loss scenario, suggesting that most of the 22 and 129 proteins assigned to groups WO and RW have been lost in Rickettsia and Orientia, respectively. The number of genus-specific ORFs suggested by Glimmer to be present in the R, O and W classes is highly variable and 80e90% of these are hypothetical ORFs, false predictions or pseudogenised gene fragments with unknown functions. Because the focus of this analysis was on the comparison of genes with functional annotations, we made no particular effort to distinguish hypothetical genes from false predictions or pseudogenes. At first sight, the total number of protein clusters uniquely assigned to Rickettsia (1224) appears 10-fold higher than those identified in Orientia (148). However, a majority of the clusters unique to Rickettsia are present only in a single species and hence the gene counts are inflated by the inclusion of 8 species in R relative to a single species in O. Indeed, repeating the analysis for each of the individual Rickettsia species reduced the number of Rickettsia-specific protein clusters to 284 in R. typhi, 592 in R. conorii and 619 in R. felis (data not shown). Of interest for our discussion were the 18, 123 and 206 protein clusters in O, W and R associated with functional annotations. The functional profile in O is dominated by a single functional group; among the protein clusters uniquely assigned to O. tsutsugamushi, close to 60% belong to replication, recombination and repair processes (L: n ¼ 12). In contrast, protein clusters assigned to R represent a broad spectrum of functional categories, including carbohydrate and lipid transport and metabolism as well as cell wall and membrane biosynthesis. This indicates a more extensive loss of metabolic enzymes in Orientia than in Rickettsia. Below, we illustrate the metabolic dependency and diversity of the two genera with examples taken from pathways involved in the biosynthesis of ATP, amino acids, co-factors and nucleotides. 3.1. ATP biosynthesis The glycolytic pathway is complete in most members of the Rickettisales [18], with the exception of Rickettsia and Orientia (Fig. 3) that exploit host-derived ATP and other imported compounds such as pyruvate and amino acids to fuel the TCA cycle. No glycolytic genes are present in the Rickettsia genomes and only three such genes (tpiA, gap and pgk) were identified in O. tsutsugamushi. We hypothesize that the glycolytic pathway became non-essential in the ancestor of Rickettsia and Orientia following the acquisition of transporters for ATP and ADP. This may have alleviated the selective pressure for a rapid in-house production of ATP during an early phase of the intracellular growth cycle, allowing deletion mutations to accumulate in genes coding for components of the glycolytic pathway. The fraction of protein clusters with a functional classification indicates several unique losses in the energy category in

O. tsustugamushi (Fig. 2c). For example, O. tsutsguamushi has lost the pdh genes for subunits of the pyruvate dehydrogenase complex, which converts imported pyruvate to acetylCoA, i.e. the first substrate in the TCA cycle. These losses are remarkable since Rickettsia, Wolbachia and all other members of the Rickettsiales as well as mitochondria are dependent on this enzyme to process pyruvate imported from the host cell cytosol. However, not even the TCA cycle is complete in O. tsutsugamushi; the gltA gene for citrate synthase, which converts acetyl-CoA to citrate, and acnA gene for aconitate hydratase, which converts citrate to isocitrate, are truncated in the middle of the protein and may be under pseudogenisation. A possible function of the remaining enzymes of the TCA cycle in O. tsutsugamushi is to convert isocitrate to oxaloacetate. This ‘‘minimal TCA cycle’’ may be fuelled by cytosolic glutamate to yield oxaloacetate, which subsequently is converted to aspartate. Thus, we speculate that the leftover enzymes in the TCA cycle of O. tsutsugamushi may serve a residual function in amino acid metabolism. 3.2. Small molecule biosynthesis The pathways for amino acid biosynthesis are absent or imperfect in most intracellular lineages, implying the use of host-derived amino acids. Also absent in Rickettsia spp. and O. tsutsugamushi are genes for the biosynthesis of riboflavin, vitamin B6 and nicotinamide and all Rickettsia species except R. bellli have a reduced set of genes for folate biosynthesis. Additionally, O. tsutsugamushi has lost the entire pathways for the production of pantothenate and biotin and none of the Rickettsia species contain all five genes required for biotin biosynthesis (bioB, birA, bpl1, bioY1 and bioY2) in a fulllength form [12]. The gene for the acyl carrier protein (ACP), which binds intermediates in fatty acid synthesis, is also missing or truncated in O. tsutsugamushi. Finally, neither Rickettsia, nor O. tsutsugamushi is capable of producing nucleoside monophosphates. The dramatic alterations of the gene repertoires in these intracellular lineages can most likely be attributed to the evolution or acquisition of transporters for amino acids, cofactors, nucleoside mono-phosphates and ATP. This points towards a gradually increased usage of host nutrients in Rickettsia, a process that has progressed even further in O. tsutsugamushi. 3.3. LPS and cell wall components Among the 206 protein clusters uniquely assigned to Rickettsia, as many as 45 belong to the category ‘‘biogenesis of the cell wall and cell membrane structures’’, suggesting the presence of more elaborate surface structures in Rickettsia than in Orientia and Wolbachia. Indeed, there are notable differences in the biosynthetic pathways for the peptidoglycan and the lipopolysaccharide (LPS). Aminosugars for cell wall structures might be generated from glucosamine-1-phosphate or from imported N-acetylglucosamine-1-phosphate. However, aminosugars were only barely detected in purified Orientia by biochemical analysis and the loss of the glmU gene in Orientia,

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Fig. 3. Metabolic pathway maps. (a) The overview map is colour-coded to indicate pathways that are complete, partial or absent in the Rickettsiaceae. (b) Detailed information showing the presence of enzymes for glycolysis, the TCA cycle and the biosynthesis of purines/pyrimidines, phospholipids and peptidoglycans in six members of the Rickettsiales. The inferred presence of metabolites and enzymes in Rickettsia represents a consensus majority from the genomes listed in the legend to Fig. 2. The colour-coding system is displayed in boxes.

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which converts N-acetyl glucosamine-1-phosphate into UDPN-acetyl glucosamine, indicates an inefficient pathway for peptidoglycan biosynthesis. The absence of alanine racemace (alr) and glutamate racemase (murI/glr) in Orientia is also potentially problematic, implying the usage of the L-variants of the amino acids or supply with D-amino acids from the hosts for biosynthesis of the peptidoglycan. The LPS, which consists of a polysaccharide that is covalently linked to lipid A, is essential for virulence in many bacteria. Identified in the Rickettsia genomes was a limited set of genes for LPS biosynthesis, including genes for lipid-A and keto-deoxyoctulonate (Kdo) biosynthesis [7]. None of these genes were recovered in either Orientia or Wolbachia. The LPS contributes to the low permeability of the outer membrane, protecting it against antibiotics and other harmful molecules. It is also a strong inducer of the innate immune system through Toll-like receptors, which are conserved from insects to mammals. The unique lipid-A molecule in Rickettsia and the shedding of the LPS in Orientia may have been important to avoid induction of host defence mechanisms [10], as a sideeffect of which the permeability to host cytosolic compounds might have increased. The Rickettsia cells are also covered by a crystalline protein layer, called the S-layer, which accounts for as much as 10e15% of the total protein mass. Two of the surface protein antigens identified in this layer have been shown to be important antigenic determinants and a total of 17 different subfamilies were recognised upon mining the Rickettsia genomes [11]. These proteins are thought to have evolved under positive selection in Rickettsia and may be involved in a co-evolutionary arms race conflict with the host cell [11]. We identified a total of 6 homologs of the surface cell antigen family in the O. tsutsugamushi genome [13]. 4. Genome expansion, instability and host-cell adaptation processes The impact of the overall reductive evolutionary forces on these genomes is contrasted with a scattered distribution of repeated sequences and mobile genetic elements. Both the reductive and the proliferative processes are particularly evident in O. tsutsugamushi; this is the metabolically most compact as well as the most highly repeated rickettsial genome due to the proliferation of conjugation-like type four secretion systems (TFSS). How have these opposing and variable characteristics affected the stability of the rickettsial genomes? 4.1. Rearrangements in Rickettsia At first, gene order structures of the Rickettsia genomes seemed to be highly conserved, as inferred from comparisons both within and across the TG and the SFG Rickettsia. For example, a comparison of the genomic structures of R. prowazekii and R. conorii revealed almost perfect gene synteny (Fig. 4), despite a nucleotide sequence divergence for this species pair that approaches saturation at synonymous sites. However, comparisons of the TG or SFG with members of

the TRG Rickettsia revealed multiple inversions, as here exemplified with a gene order plot of R. prowazekii and R. felis (Fig. 4). Even more radically disrupted is the gene order structure in comparisons with the AG Rickettsia, here exemplified with comparisons of R. prowazekii and R. bellii (Fig. 4). Extending these comparisons even further to include O. tsutsugamushi results in a complete breakdown of the synteny. Since the gene order structure is highly conserved in most pairwise genome comparisons of species belonging to the TG and the SFG, a higher rearrangement frequency has to be inferred in R. felis and R. bellii. It is probably no coincidence that these genomes, unlike the others, encode both transposases and conjugative type IV secretion systems. The proliferation of these elements in O. tsutsugamushi along with a more distal divergence node explains the complete lack of conserved gene order structures for this species and the Rickettsia species. 4.2. The Rickettsia plasmid R. felis has puzzled scientists for a long time due to its genotypic and phenotypic characteristics, which are intermediate of the TG and the SFG Rickettsia. Like the TG Rickettsia it is associated with fleas rather than ticks, but like the SFG Rickettsia it is transovarially maintained in the vector. Additionally, it was the first Rickettsia species reported to contain a plasmid with genes for conjugative pili, transposases, integrases, recombinases, SpoT and proteins with ankyrin (ANK) and tetratricopeptide (TPR) repeats [23]. Also located on the plasmid are genes for a patatin homolog with a phospholipase activity and hyaluronidase, an enzyme that increases host permeability [23]. Many of the 68 genes identified on the R. felis plasmid are homologous to chromosomal genes identified in the AG Rickettsia and O. tsutsugamushi, 30 of which also have chromosomal homologs in R. felis. Whereas phylogenetic inferences based on 21 conserved chromosomal genes suggested that R. felis and R. akari cluster with the SFG Rickettsia, a combined set of 10 genes with homologs on both the chromosome as well as the plasmid in R. felis indicated a basal position for the plasmid genes in the Rickettsia tree [16]. Furthermore, none of the individual plasmid gene trees was in concordance with the chromosomal gene tree, suggesting different evolutionary trajectories [16]. However, the tree topologies were often not well resolved and the chromosomal homologs of R. felis, whenever present, showed no consistent clustering with either group [16]. At present, we do not know when the plasmid was acquired; some suggest that the plasmid genes were acquired in a single event that either occurred in R. felis or in an ancestor of the TRG [23], whereas others speculate that the rickettsial ancestor contained plasmids and that the plasmid genes were acquired at different time points from different hosts [16]. It is argued that the importance of genes coding for host penetration functions may have selected for plasmid maintenance in R. felis [16]. The location of 16 toxin and 14 antitoxin genes on the chromosome may also have prevented plasmid-less strains of R. felis from emerging. Future experimental studies

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Fig. 4. Conserved and scrambled gene order structures in Rickettsia. The dot-plots show the order of sequences in comparisons of R. prowazekii to four Rickettsia species that represent each of the four rickettsial groups and O. tsutsugamushi. Abbreviations of species names are as follows; RC, R. conorii; RF, R. felis, RT, R. typhi; RP, R. prowazekii; RB, R. bellii; OT, O. tsutsugamushi.

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and plasmid-curing experiments are needed to address questions concerning fitness effects of the plasmid [16]. 4.3. Chromosomal conjugative type IV secretion systems The identification of a complete set of conjugal DNA transfer (tra) genes in Rickettsia bellii was another key finding [22]. This is consistent with the observation of sex pili-like cell surface structures in this species as well as in R. canadensis. Yet, in comparison to the identification of conjugative transfer operons at more than 20 sites in the genome in the O. tsutsugamushi genome [13], even this finding seems bleak. The many copies of the tra operon in O. tsutsugamushi are often rearranged, shortened and there is evidence of gene conversion among duplicates present in two or more clusters [13]. The presence of conjugative elements in these genomes is correlated with an increased number of transposons, breakpoints and a general breakdown in genome synteny between taxa that are closely related. It is unclear whether these clusters originated by intragenomic duplication events or by multiple, independent integration events, and whether they have facilitated the spread of other mobile elements. Nevertheless, all data accumulated to date indicate that genes for conjugation systems, possibly introduced by plasmids, have flourished in early diverging species of Rickettsia and Orientia. 4.4. Acquisition and spread of eukaryotic-like genes Located in the vicinity of operons for conjugation machineries are genes for proteins that contain eukaryotic proteinprotein interaction motifs, such as tetratricopeptide (TPR) and ankyrin (ANK) repeats [13,22]. These are short repetitive motives of 33e34 amino acids that form scaffolds to mediate interaction among proteins. Such protein motives are extremely common in eukaryotes, where they are involved in diverse cellular functions, such as cell cycle control, transcriptional regulation and protein transport. In O. tsutsugamushi, each of the TPR and ANK protein families can be sorted into a few groups that typically contain one long master gene and multiple shorter duplication remnants. Likewise, numerous duplication remnants of genes coding for proteins involved in global regulation, such as histidine kinases (the sensor protein of a two-component system regulon) and SpoT (involved in the synthesis of the global regulator ppGpp), were identified in the O. tsutsugamushi genome. These genes are physically linked to the mobile clusters and may have been amplified together with the tra-clusters, transposons and reverse transcriptases. Although much less abundant, a few have also been identified in other rickettsial genomes. There are currently no published data to support a detailed functional description of the TPR and ANK-repeat proteins in Rickettsia and Orientia and whether these have contributed to host-parasite interactions and microbial pathogenesis. Rather, several copies for the tra-associated genes are truncated and these may have been silenced. Thus, their function, if any, remains to be elucidated.

Phylogenetic inferences indicate a distant relationship with tra genes in environmental chlamydiae inhabiting amoeba [22]. Sequence similarity to other bacterial species that normally inhabit amoeba was also detected, like for example Legionella and Parachlamydiae. Since 20% of Acantamoebae spp. was found to contain bacterial endosymbionts of various different taxonomic affiliations, it was speculated that the ancestor of the rickettsiae could have used amoeba as the natural host and that this might have been the site for elaborate gene exchange [22]. Supporting this scenario is that R. bellii efficiently multiplies in the nucleus of eukaryotic cells and survives in phagocytic amoeba, Acanthamoeba polyphaga [22]. The immediate ancestral host of Rickettsia and Orientia was probably an arthropod since all species of the Rickettsiales inhabit arthropods. However, this does not exclude that some of the modern species may also inhabit amoeba; if so, arthropods could repeatedly be re-infected with these bacteria from a second reservoir, such for example as amoeba.

5. Conclusions The first important take-home message from the past ten years of research on rickettsial genomes is that the underlying erosion process is ongoing in all species of Rickettsia and Orientia. Many of the enzymes already discarded belong to pathways that have been supplemented with substrates and metabolites from the host cell cytoplasm, in accordance with the theory that the deterioration process in intracellular bacteria is associated with an increased reliance on host cellular functions [6]. The other main finding is that these reductive tendencies have not prevented mobile genetic elements from invading and proliferating in these genomes. The first isolation of a plasmid in R. felis [23] followed by the identification of chromosomally encoded operons for conjugation systems in R. bellii [22] have demonstrated that mechanisms exist for the influx of novel DNA sequences into these genomes. Last but not least, the recent sequencing of the O. tsutsugamushi genome has shown that these elements can not only exist in rickettsial genomes, but also proliferate extensively [13]. Strikingly, the two processes have operated on different gene sets; genome reduction has acted on genes involved in housekeeping functions and metabolic processes, whereas gene gains have mostly involved selfishly propagating elements and genes putatively involved in host-cell interaction processes. Interestingly, both processes have driven the O. tsutsugamushi genome to its extreme; it has not only the metabolically most dependent rickettsial genome of those studied until date, but also the highest fraction of repeated sequences and mobile elements. Thus, the two evolutionary processes, loss and gain, may be two sides of the same coin; on the one hand, there is selection for host-cell exploitation and interaction and on the other hand, chance evolution may be more dominant in small bacterial populations that undergo repeated bottlenecks. In combination, these processes could drive bacterial genomes towards a minimal set of genes with a maximal number of repetitions. It will be interesting to see

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if even more extreme variants of rickettsial genomes will be identified in the near future.

[14]

Acknowledgements [15]

This work was supported by grants from the Ministry of Health and Welfare, Republic of Korea (Grant A010379) to N.-H. Cho, the Helge Ax:son Johnson Foundation to H.-H. Fuexlius and the Swedish Research Council, the Swedish Foundation for Strategic Research, The Knut and Alice Wallenberg Foundation and the Goran Gustafsson Foundation to S.G.E. Andersson.

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