Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in
Rickettsia (
Rickettsiales;
Rickettsiaceae) species, including
Rickettsia akari, “
Candidatus Rickettsia amblyommii,”
R. bellii,
R. rhipicephali, and REIS, the rickettsial endosymbiont of
Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks.
R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all
Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two “
Ca. Rickettsia amblyommii” isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple
parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that
hsp2 genes were plasmid specific and that
hsp1 genes, found only on plasmids of “
Ca. Rickettsia amblyommii,”
R. felis,
R. monacensis, and
R. peacockii, were probably acquired independently of the
hsp2 genes. Plasmid copy numbers in seven
Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.The alphaproteobacteria of the genus
Rickettsia (
Rickettsiales;
Rickettsiaceae) have undergone the reductive genome evolution typical of obligate intracellular bacteria, resulting in A/T-rich genomes (1.1 × 10
6 to 1.5 × 10
6 bp) with a high content of pseudogenes undergoing elimination (
3,
10,
20,
26). Initial sequencing of rickettsial genomes focused on the important arthropod-borne pathogens
Rickettsia prowazekii,
Rickettsia conorii, and
Rickettsia typhi and appeared to confirm the prevailing belief that plasmids were absent and transposons were rare among
Rickettsia spp. (
2,
28,
39,
44). As mobile genetic elements in bacteria, plasmids and transposons drive horizontal gene transfer (HGT) and the acquisition of virulence determinants and environmental adaptive traits (
30,
43,
60,
70). Subsequent sequencing of the
Rickettsia felis genome revealed the surprising presence of abundant transposase paralogs and the 63-kbp pRF plasmid, with 68 open reading frames (ORFs) encoding predicted proteins, as well as a 39-kbp deletion form, pRFδ (
45). Although pRF was suggested to be conjugative, it was initially thought to be unique among the rickettsiae, a reasonable inference given that plasmids are uncommon among the reduced genomes of obligate intracellular bacteria and were previously unknown in the
Rickettsiales (
3,
4,
13). However, a phylogenetic analysis implied an origin for pRF in ancestral rickettsiae and the possible existence of other rickettsial plasmids (
28), which was soon confirmed by the cloning of the 23.5-kbp pRM plasmid from
Rickettsia monacensis (
6). Some of the 23 ORFs on pRM had close pRF homologs, and both plasmids carried transposon genes and the molecular footprints of transposition events associated with HGT from other bacterial taxa.The discoveries of pRF and pRM made obsolete the long-held dogma that plasmids were not present in members of the genus
Rickettsia and implied a source of unexpected genetic diversity in the reduced rickettsial genomes, particularly if potentially conjugative plasmids carrying transposon genes proved to be common among members of the genus. That hypothesis gained credence when pulsed-field gel electrophoresis (PFGE) and Southern blot surveys (
7) using plasmid gene-specific probes demonstrated plasmids in
Rickettsia helvetica, “
Candidatus Rickettsia hoogstraalii” (
38), and
Rickettsia massiliae and possible multiple plasmids in “
Candidatus Rickettsia amblyommii” (
71) isolates. The same study demonstrated the loss of a plasmid in the nonpathogenic species
Rickettsia peacockii during long-term serial passage in cultured cells and the absence of a plasmid in
Rickettsia montanensis M5/6, an isolate with a long laboratory passage history. Genome sequencing of
R. massiliae and
Rickettsia africae revealed the 15.3-kbp pRMA and 12.4-kbp pRAF sequences, with 12 and 11 ORFs, respectively, that were more similar to those of pRF than to those of pRM (
11,
24).The absence of plasmids in
R. montanensis and important
Rickettsia pathogens maintained as laboratory isolates has left unresolved the question of the true extent of plasmid distribution among
Rickettsia spp. Until recently, the genus was thought to consist of closely related species, known chiefly as typhus and spotted fever pathogens transmitted by lice, fleas, mites, and ticks (
31). It is now apparent that many, and possibly most,
Rickettsia spp. inhabit a diverse range of arthropods that do not feed on blood, as well as leeches, helminths, crustaceans, and protozoans, suggesting an ancient and complex evolutionary history (
54). A multigene phylogenetic analysis of the
Rickettsiales resulted in a “molecular clock” which indicated that the order arose from a presumably free-living ancestor and then adapted to intracellular growth during the appearance of metazoan phyla in the Cambrian explosion (
76). A transition to a primary association with arthropods followed during the Ordovician and Silurian periods. The genus
Rickettsia arose approximately 150 million years ago and evolved into several clades, including the early-diverging hydra and torix lineages associated with leeches and protozoans. A rapid radiation occurred about 50 million years ago in the arthropod-associated lineages (
76).Whole-genome sequencing has led to a revision of phylogenetic relationships among
Rickettsia spp. associated with blood-feeding arthropods (
10,
26,
28). A newly defined ancestral group (AG) contains the earliest-diverging species,
Rickettsia bellii and
Rickettsia canadensis, while
R. prowazekii and
R. typhi, transmitted by lice and fleas, respectively, constitute the typhus group (TG). A proposed transitional group (TRG), consisting of the mite-borne
Rickettsia akari, the flea-borne
R. felis, and the tick-borne
Rickettsia australis, bridges the genotypic and phenotypic differences between the TG and the much larger spotted fever group (SFG), consisting of tick-borne rickettsiae (
28). However, some presumptive SFG rickettsiae remain poorly characterized and are of uncertain phylogenetic status, while the accumulation of genomic data from rickettsiae found in a diverse range of invertebrate hosts may have profound impacts on the currently understood phylogeny of rickettsiae associated with blood-feeding arthropods. For example, it appears that the above AG and TRG species have many close relatives in insects (
76). Despite the recent phylogenomic advances, the genetic and host-adaptive mechanisms underlying the evolution of arthropod-transmitted pathogens of vertebrates from ancestral
Rickettsia spp., including any possible role of plasmids, remain poorly understood.In this report, we have taken advantage of recent isolations of rickettsiae from North and South America to conclusively demonstrate that low-copy-number plasmids are indeed common in low-passage isolates of AG, TRG, and SFG rickettsiae. The only exceptions were multiple isolates of
R. parkeri, obtained from ticks and human eschar biopsy specimens and newly recognized as a mildly pathogenic SFG rickettsia (
49,
50,
52,
79), and the previously characterized species
R. montanensis (
7). We confirmed that some
Rickettsia isolates harbor more than one plasmid by cloning and sequencing multiple plasmids from “
Ca. Rickettsia amblyommii” isolates AaR/SC and Ac/Pa, and we obtained PCR- and gel-based evidence that supported genome sequence evidence for the existence of multiple plasmids in REIS, the rickettsial endosymbiont of
Ixodes scapularis. Phylogenetic analysis provided strong evidence for multiple plasmid incompatibility groups and possible multiple origins of plasmid-carried
parA genes in the genus
Rickettsia. Other than genes encoding plasmid replication initiation and partitioning proteins, the newly sequenced “
Ca. Rickettsia amblyommii” plasmids resembled the previously sequenced rickettsial plasmids in sharing limited similarities in coding capacity (
6,
7,
22). However, we have previously drawn attention to the presence of
hsp genes, encoding α-crystalline small heat shock proteins, as a conserved feature of most rickettsial plasmids that may play a role in host adaptation (
7). Phylogenetic analysis indicated that the
hsp2 genes were plasmid specific, while the
hsp1 genes found on four rickettsial plasmids may have been acquired by a chromosome-to-plasmid transfer event in a TRG-like species.
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