BACKGROUND
rickettsiae are obligate intracellular gram-negative bacteria mostly associated to arthropods, some of which causing mild to severe diseases in humans.
pathogenic species are classified into two groups based on phylogenetic analyses  <cit> .
the typhus group  includes two rickettsia prowazekii  and r. typhi, and the spotted fever group  includes  <dig> pathogenic species and numerous species of unknown pathogenicity  <cit> .
two additional validated species, r. bellii and r. canadensis, and a variety of unvalidated species from insects or leeches are organized into the most outer outgroups of the genus rickettsia  <cit> .
the relatively low rate of lateral gene transfer, the continuous gene loss and the colinearity of most of their genomes make rickettsia species an outstanding model for comparative genomics  <cit> .
indeed, genome reduction  <cit>  paradoxically results in higher virulence in r. prowazekii.
the pathogenic mechanisms of rickettsiae are unclear.
within ticks, rickettsiae remain quiescent during the starvation of their vector but undergo a reversion to the virulent state, termed reactivation, following incubation at 37°c or blood meal  <cit> .
this phenomenon is marked in r. rickettsii by morphological changes in the microcapsular and slime layers  <cit> .
the precise molecular mechanisms of this change, however, are only poorly understood.
during human infection, attachment to and invasion of host cells were suggested to involve the outer membrane proteins rompa and rompb and the adhesins adr <dig> and adr <dig>  <cit> .
a phospholipase d activity was proposed to play a role in escape from phagosomes  <cit> , and intracellular motility was demonstrated to rely on actin polymerization  <cit> .
none of these factors nor the presence of a type iv secretion system  <cit> , however, explain the virulence differences observed among rickettsia species  <cit> .
over the last ten years, r. africae has emerged as the causative agent of african tick-bite fever  <cit> , the most common sfg rickettsiosis both in terms of seroprevalence  <cit>  and incidence  <cit> .
such an epidemiologic success is due to various factors, including the increase of tourism to wildlife parks in sub-saharan africa, the attack host-seeking behavior of its vector ticks,amblyomma sp., and the elevated prevalence of r. africae in these ticks, with infection rates of up to 100%  <cit> .
in addition, the bacterium has been identified in other areas with warm climates, such as the west indies, where it was found in guadeloupe, martinique, st kitts and nevis, and antigua islands  <cit> .
such a distribution, as well as the presence of r. africae in reunion island, is likely to result from the transfer from africa of cattle bearing infected ticks  <cit> .
tick-associated rickettsiae may infect ticks feeding on infected hosts or may be passed from one generation to the next transovarially.
r. africae is transmitted transovarially and appears to be the most successful rickettsia in its adaptation to its vector tick, as the prevalence of tick infection is higher than that of any other rickettsia  <cit> .
in addition, infection does not appear to alter tick fitness .
these data highlight the fact that r. africae is an extremely successful and fit bacterium.
by comparison with r. conorii, the second most prevalent sfg rickettsia in africa, whose genome has previously been sequenced  <cit> , r. africae exhibits a higher prevalence in ticks  <cit> , a lower virulence in humans  <cit> , and a greater genetic homogeneity  <cit> .
the genetic factors underlying these characteristics are, however, unknown.
we assumed that the r. africae genome sequence might help understand the characteristics of this species and the genetic mechanisms associated with the difference in virulence.
here, we present the sequence of the r. africae genome and additional data that suggest that this species has emerged recently.
in support of this hypothesis, we show that r. africae is a clonal population.
we also present data that support the assumption that rickettsial virulence increases following gene inactivation.
RESULTS
general features of the genome
the genome of r. africae consists of two replicons: a circular chromosome of  <dig> , <dig> base pairs   and a  <dig>  bp circular plasmid .
we acknowledge the fact that the esf- <dig> strain, first isolated in  <dig>  <cit> , may have undergone loss or rearrangement of plasmid or chromosomal genes during multiple passages in cell culture.
sequences were deposited in genbank under accession number .
the chromosome has a g + c content of  <dig> %, in the range of other sfg rickettsial genomes , whereas the plasmid has a g + c content of  <dig> %, similar to those of r. felis   <cit>  but higher than that of r. massiliae plasmids .
the predicted total complement of  <dig>  open reading frames ,  <dig>  chromosomal , and  <dig> plasmidic  orfs , is in the range of genomes from sfg rickettsiae with the exception of r. felis, which exhibits a larger genome .
of these,  <dig>   exhibited homologs in the non-redundant database, and  <dig>   were assigned putative functions .
overall, the  <dig>  chromosomal orfs encoded  <dig>  protein-coding genes, with  <dig> of these being split into  <dig> to  <dig> orfs by the presence of one to several stop codons.
by comparison with other sfg genomes, r. africae had fewer split genes than any other species with the exception of r. felis .
in addition, r. africae exhibited a single rrna operon, with non-contiguous 16s and 23s rrna genes as in other rickettsial genomes,  <dig> trnas and another three rnas.
the r. africae chromosome exhibited an almost perfect colinearity with the r. conorii genome  <cit> , with the exception of a  <dig> -bp inversion .
at both extremities of the inversion, there were repeats of the rickettsia palindromic element –  <dig>  familly.
in this inverted fragment, r. africae exhibited  <dig> orfs and  <dig> rpes that were absent from r. conorii.
among these  <dig> orfs, a cluster of  <dig> consecutive orfs had orthologs in the 3'-extremity of the tra cluster previously identified in the r. massiliae genome  <cit> .
these  <dig> orfs included tradf , a transposase , spot <dig> , a split spot <dig> , a split spot <dig> , a split signal transduction histidine kinase , dam <dig>  a site-specific dna adenine methylase , and orf <dig> of unknown function .
in addition to the orthologs in r. massiliae, these genes had orthologs in similar clusters in r. felis, r. bellii, r. canadensis and o. tsutsugamushi but were absent from all other species.
as in r. massiliae, r. bellii and r. canadensis, the r. africae cluster was bounded at its 3'-end by a trna-val, but, in contrast with these three species, neither an integrase with its atti site nor a trna-val fragment marker of integration was present at the 5' end .
the presence of a similar gene cluster inserted at the same position in several rickettsia species, with a gc content different from that of the genome  suggests that it was acquired horizontally from a common ancestor and then transmitted vertically.
in r. africae, an attc site, specific to integron-inserted gene cassettes, located at the 3'-end  of the spot <dig> gene , supports the role of integration in the insertion of this gene cluster.
attc sites were also identified in r. massiliae , r. felis , and r. bellii .
nevertheless, the presence of transposases in all species and the fact that, in r. felis, nine of these genes are located in the prf plasmid support the role of several genetic mechanisms at the origin of this cluster, possibly involving plasmids, integrons and transposons.
in comparison with other species containing this gene cluster, r. africae had the smallest number of genes.
in particular, it lacked most of the tra cluster, with the exception of tradf, but retained three spot genes, including two degraded to pseudogenes.
in r. bellii and r. massiliae, tra genes were described as encoding components of a type iv secretion system  for conjugal dna transfer  <cit> .
in terms of gene content, the r. africae cluster was more similar to those of r. felis and r. canadensis, with the loss of the tra cluster, the conservation of spot genes and the presence of pseudogenes, than to those of r. massiliae and r. bellii, in which the tra cluster was intact but spot genes were partially degraded.
such findings suggest that species-specific evolution of this gene cluster occurred, which likely resulted from gene excisions in r. africae, r. felis and r. canadensis, or gene expansion by transposase duplication in r. massiliae.
* unpublished genomes available in genbank: r. akari  ,r. rickettsii  , r. sibirica  , r. bellii  , and r. canadensis  .
in addition to the tradf gene described above, the r. africae chromosome retained many of the components of the type iv secretion system  involved in both dna transfer and effector translocation in other bacteria  <cit> , including virb <dig>  virb <dig> , virb <dig> , virb <dig> , virb <dig> , virb <dig> , virb <dig> , virb <dig> , virb <dig>  and vird <dig> .
in addition, r. africae possessed a trax  and a split fimd  gene but lacked other tra cluster genes found in r. massiliae, r. felis, r. bellii and o. tsutsugamushi, such as trac and tragf <cit> .
therefore, the tra cluster was mostly eliminated from the r. africae, and, following a "use it or lose it" scheme, this species probably did not need a tra gene-linked conjugation system.
in addition, the pra plasmid did not contain genes encoding proteins involved in conjugation.
six transposase-encoding genes were identified in the chromosome, including one split into two orfs  and one present as a remnant and two in the pra plasmid, including one present as a fragment.
this contrasts with the large expansion of transposases caused by gene duplications previously detected in r. felis and r. bellii  <cit> .
common rickettsial gene set and phylogeny
when compared to eight other available rickettsial genomes, a total of  <dig> genes and  <dig> rna-encoding genes of r. africae had orthologs in all genomes.
in addition, another  <dig> r. africae genes had orthologs only in sfg rickettsiae and were either absent or remnant in tg rickettsiae.
consequently, we identified  <dig> genes as constituting the core gene set of all available rickettsial genomes and  <dig> orfs as the core gene set of sfg rickettsiae.
following concatenation of the  <dig> core genes, a reliable phylogenetic organization  was obtained using three analysis methods that was consistent with previous phylogenetic studies of rickettsia species  <cit> .
in comparison with other rickettsia genomes, r. africae had  <dig>   <dig> and  <dig> fewer genes than r. bellii, r. felis and r massiliae, respectively, but  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> more genes than r. typhi, r. prowazekii, r. akari, r. rickettsii, r. sibirica, and r. conorii, respectively.
when comparing the numbers of degraded genes , r. africae, with  <dig> degraded genes, had a significantly less degraded genome  than that of other spotted fever group rickettsiae including r. akari , r. conorii , r. massiliae , r. rickettsii  and r. sibirica  .
it had, however, significantly more degraded genes than r. felis .
transcription of genes conserved in r. africae but absent from highly pathogenic species
r. africae had  <dig> intact genes that were either absent or degraded in all three virulent species r. conorii, r. rickettsii and r. prowazekii.
of these,  <dig> encoded proteins of unknown functions   .
the remaining six genes encoded a plasmid maintenance system antidote protein , the spot <dig> gene , a site-specific dna adenine methylase  , an ankyrin repeat , a putative integral membrane protein , and a protein  exhibiting a high degree of amino acid sequence identity  with proteins of γ-proteobacteria classified within the cog <dig> as putative virulence proteins.
when investigating the transcription of these  <dig> genes in r. africae grown at  <dig>   <dig> and 37°c, we observed a significantly higher transcription level at 37°c than at lower temperatures for two genes, raf_orf <dig> and raf_orf <dig>  the former gene contained a putative protease domain site, but the latter had no known function.
the r. africae plasmid
the r. africae plasmid  is a new example of a plasmid in rickettsia species, following those in r. felis  <cit> , r. massiliae  <cit> , r. monacensis  <cit> , r. helvetica, r. peacockii, r. amblyommii and r. hoogstraalii  <cit> .
this plasmid, named pra, is smaller  than those of r. felis , r. monacensis , and r. massiliae .
the pra plasmid is predicted to contain  <dig> genes,  <dig> of which  have homologs in public databases and are associated with functional attributes.
these six genes encode for a chromosomal replication initiator dnaa-like protein , a site-specific recombinase , two contiguous transposases exhibiting 100% sequence similarity  but with one  shorter than the other, the auto-transporter protein sca <dig> , and a para-like plasmid stability protein .
five genes  have orthologs in the r. massiliae plasmid, six have orthologs in the r. felis plasmids , and three have orthologs in the r. monacensis plasmid .
the presence of two genes  conserved in plasmids from four species suggests that these plasmids have a common origin.
the presence of two almost identical successive transposases in r. africae matching a single gene in r. massiliae and r. felis suggests a duplication event in the former species.
the pra plasmid lacks heat shock protein-encoding genes found in other rickettsial plasmids.
in contrast, orf <dig>  a site-specific recombinase, is absent from other species.
its closest phylogenetic neighbour is a site-specific recombinase from magnetospirillum magnetotacticum, a high g-c content α-proteobacterium living in aquatic environments  <cit> .
the sca <dig> gene  found intact in r. africae pra was absent from the r. massiliae and r. monacensis plasmids and present but fragmented within r. felis prf, but it was absent from prfδ as well all other rickettsia species.
as outlined by baldridge et al.
<cit> , the plasmid content of a rickettsia species may vary according to the passage history of rickettsial strains.
when estimating the prevalence of the plasmid among r. africae strains, we detected it in the  <dig> tested isolates from south africa and in the  <dig> eschar biopsies from patients with atbf contracted in the same country and in 20/ <dig> r. africae-positive amblyomma ticks .
therefore, it appears from these results that, depending on the geographic location, the plasmid of r. africae may be unstable.
whether the plasmid has been lost by pcr-negative strains or cannot be amplified with the primers we used is as yet unknown.
such inter-strain differences in plasmid content were also observed in r. felis .
stress response
rickettsiae live intracellularly in both arthropod and mammal hosts.
this implies that periods of tick starvation and feeding cause bacterial dormancy and multiplication following reactivation  <cit> .
as a consequence, and despite their obligate intracellular location, rickettsiae may face, and thus have to adapt to, highly variable and extreme environmental conditions.
known as the stringent response, this bacterial adaptation to nutritional stress has been described to be mediated by the accumulation of guanosine nucleotides pppgpp  and ppgpp   <cit> .
accordingly, the transcriptional analysis of r. conorii exposed to a nutrient deprivation was characterized by the up-regulation of gmk and of genes from the spot family, suggesting a role for these nucleotides as effectors of the stringent response  <cit> .
the r. africae genome exhibited eight spot genes phylogenetically classified within two major clades .
the largest clade included spot genes with hydrolase activity , while the second included those with a synthetase domain.
with eight genes, r. africae had more spot genes than r. rickettsii , r. conorii , r. sibirica , r. akari , r. canadensis , r. typhi  and r. prowazekii  but fewer genes than r. felis  and r. bellii  .
altogether, our data suggest that r. africae is more regulated than more pathogenic species.
infection of mammal hosts
the r. africae genome encoded rompa  and rompb , two surface-exposed and immunodominant proteins belonging to the paralogous "surface cell antigen"  family and known in rickettsia species to be responsible for antigenic differences between species  <cit>  and to elicit an immune response in patients  <cit> .
experimental studies suggested that these two auto-transporter proteins could function as adhesins  <cit> .
in addition, another eight sca-encoding genes were found in the genome.
these  <dig> genes were represented by  <dig> orfs due to partial degradation of some of the paralogs .
among the  <dig> sca-encoding genes detected in rickettsia species  <cit> , r. africae had similar sets of conserved , degraded  and absent  sca genes as r. conorii and r. rickettsii.
in addition to these  <dig> sca-encoding genes, r. africae exhibited a degraded sca <dig> gene and a complete sca <dig> gene carried by the pra plasmid, only shared with r. felis, where it was also found partially degraded on the prf plasmid.
the sca <dig> genes from both species were grouped into a distinct cluster close to the sca <dig>   <dig> and  <dig> genes .
this result further supports a common origin of the pra and prf plasmids.
a proteomic approach recently allowed the identification of two paralogous proteins encoded by the genes rc1281-rc <dig> and rp827-rp <dig>  as putative adhesins adr <dig> and adr <dig>  these proteins may be key actors for entry and infection in both r. conorii and r. prowazekii  <cit> .
both proteins are ubiquitously present within the rickettsia genus  <cit> .
their presence within the r. africae genome   reinforces their suspected key role in rickettsial life.
both pld and tlyc, encoding phospholipase d  <cit>  and hemolysin c  <cit> , respectively, which play a role in phagosomal escape  <cit> , were conserved in the r. africae genome .
this bacterium also exhibited genes encoding other proteins with membranolytic activity, including tlya  and pat <dig>   <cit> .
as expected, the genome of r. africae has a ricka gene  orthologous to all rickettsial ricka genes and coding a protein activating the arp2/ <dig> complex, whose nucleation triggers actin polymerisation  <cit>  .
the rick a protein in r. africae is slightly different from those of other species, with a phenylalanine instead of a serine within the g-actin-binding site, an ennip  motif repeated twice instead of four times in the central proline-rich region of the protein , and an aspartate and an isoleucine instead of an asparagine and an alanine or valine, respectively, in the carboxy-terminal region.
despite these differences, the ricka protein of r. africae appeared to be functional as demonstrated by its ability to polymerize actin and multiply intranuclearly .
sixteen vir gene paralogs were found in the r. africae genome.
virulence genes of the vir family belong to the type iv secretion machinery, a system that allows the delivery of virulence factors from bacterial and eukaryotic host membranes to the cytoplasm of the host cell  <cit> .
all  <dig> genes were found to be intact and common to all rickettsia genomes with the exception of virb6- <dig> in r. africae and virb6- <dig> in r. massiliae .
in both species, these genes were split into two orfs.
phylogenetic analysis of the virb6- <dig> gene distinguished clearly the sfg and tg and showed that the r. africae virb6- <dig> protein is phylogenetically closer to that of r. sibirica .
clonality of r. africae
of the  <dig> amblyomma ticks tested,  <dig>  were pcr-positive for r. africae .
therefore, infection rates of amblyomma ticks with r. africae may be higher than previously described  <cit> , which suggests an extreme fitness of this rickettsia for its vector.
in addition, such infection rates are the highest among rickettsia species .
using mst, pcr products of the expected sizes were obtained from the dksa-xerc, mppa-purc and rpme-trnafmet intergenic spacers from all tested specimens.
sequences obtained from these amplicons were in all cases identical to those previously obtained for r. africae , , and , for the dksa-xerc, mppa-purc and rpme-trnafmet spacers, respectively).
this is the first rickettsia demonstrated to be clonal.
other tested rickettsia species, including r. conorii , r. massiliae , r. sibirica , and r. felis , were significantly more genetically variable than r. africae .
discussion
using a comparative study of rickettsial genomes, we found that virulence in rickettsia species is not correlated with acquisition of foreign dna but may rather result from a reduction in regulation due to genome decay  <cit> .
comparative genomics sheds light on a much wider spectrum of virulence acquisition mechanisms in bacteria than initially thought  <cit> .
based on the examples of enterobacteria and staphylococci, gain in pathogenicity in bacteria was mainly thought to result from horizontal gene transfer, either directly or through mobile genetic elements  <cit> .
however, a recent study of rickettsia species associated with arthropods, insects, leeches and protists clearly demonstrated that horizontal gene transfer was a rare event within this genus  <cit> .
in addition, genomic studies demonstrated that rickettsiae are undergoing genome decay, affecting in priority horizontally-acquired genes  <cit> , and that there is no association between pathogenicity and acquisition of virulence markers  <cit> .
in fact, the genome of the most virulent species, r. prowazekii  <cit> , is a subset of the less pathogenic species r. conorii  <cit> , thus highlighting a paradoxical relationship between smaller genome size and higher pathogenicity.
careful comparison of the r. prowazekii and r. typhi genomes also demonstrated that the former species, more pathogenic than the latter, had a more decayed genome despite a 12-kb insertion that likely resulted from a single genetic event  <cit> .
when investigating the genomic characteristics associated with the milder virulence of r. africae, we first ruled out a potential role of the plasmid by the fact that it is unstable in this species.
then, we compared the gene contents of r. africae with r. conorii, r. rickettsii, and r. prowazekii, which exhibit a higher pathogenicity in humans and their arthropod hosts.
we observed that r. africae showed no gene loss but had  <dig> genes fully conserved that were either absent or degraded in the other species .
we speculated that, because r. africae had more intact genes than more virulent species, some of these genes may be involved in maintaining a low virulence level.
such a behavior may not be unique to rickettsiae.
it was found that gene knockout resulted in increased virulence in mycoplasma, streptococcus pyogenes, and vibrio cholerae  <cit> .
in m. ulcerans, genome reduction was also linked to gain in virulence  <cit> .
it emerges as a concept that virulence may be increased by gene loss  <cit> .
we assume that a similar phenomenon may happen in rickettsiae, and that inactivation of some genes may deregulate the control of bacterial multiplication, in particular during the reactivation phenomenon following warming, thus enhancing pathogenesis.
among the  <dig> putative candidate genes unique to r. africae, we identified only two genes  that were significantly more transcribed at 37°c than at lower temperatures.
of these, one  encoded a protein that had a putative protease domain.
a protease was previously shown in vibrio cholerae to be a virulence repressor  <cit> .
however, whether this differentially-transcribed protease plays a role in virulence repression in r. africae is as yet unknown.
in contrast, the spot <dig> gene  unique to r. africae was not upregulated, and this species retained another two spot pseudogenes  that were completely lost by other species.
spot genes, effectors of the stringent response, were shown to play a major role in adaptation to stress in r. conorii, in particular when subjected to abrupt temperature variations similar to those occurring during a tick blood meal  <cit> .
r. africae, however, has more spot genes than r. conorii or r. rickettsii and does not show any modification of expression of its specific spot <dig> gene during temperature variations.
we speculate that higher regulation ability in r. africae is linked to lower pathogenicity.
in addition, when compared to other tick-borne rickettsia species, r. africae exhibited several unique characteristics.
first, this species is extremely successful and fit: it is highly adapted and harmless to its tick host, being efficiently transmitted both transtadially and transovarially in amblyomma sp.
ticks, which consequently act as efficient reservoirs  <cit> .
in contrast, r. rickettsii  <cit>  and r. conorii  <cit>  have a negative effect on their tick vectors in experimental models.
as a result, the prevalence of r. africae in its host ticks is higher than that of most other rickettsiae.
similarly, r. africae is less pathogenic for humans than other sfg species such as r. conorii and r. rickettsii, in particular because the infection is never lethal  <cit> .
this observation was later supported by the demonstration that inoculation eschars in atbf were histologically different from those in msf  <cit> .
in particular, in contrast with other sfg rickettsioses where eschars are characterized by perivascular infiltration of t cells and macrophages, with some b lymphocytes and few polymorphonuclears, the vasculitis in atbf is made of a large infiltrate of neutrophils causing an extensive cutaneous inflammation and necrosis   <cit> .
such a local reaction, in addition to the few r. africae cells detected in eschars  <cit> , suggests that the bacterium replicated poorly in human tissues.
second, r. africae has significantly fewer degraded genes than other sfg species , except r. felis.
specifically, this characteristic suggests that r. africae is undergoing a slower degradation process than other rickettsiae.
third, the identification of a single mst genotype among  <dig> strains suggested that r. africae was clonal  <cit> .
this contrasted with the variable plasmid content of this species.
originally thought to be absent in rickettsia species, plasmids have been detected in eight species to date  <cit> , and their plasmid content may exhibit intraspecies variability.
in r. felis, two plasmid forms have been sequenced  <cit> , and baldridge et al.
found two plasmids in both r. peacockii and r. amblyommii  <cit> .
in addition, these authors showed that r. peacockii lost its plasmids during long-term serial passages in cell culture  <cit> .
in r. africae, the pra plasmid may also be unstable, as shown by the absence of plasmid detection in 12/ <dig> amblyomma ticks tested.
this plasmid encodes  <dig> orfs, two of which are common to r. felis, r. massiliae and r. monacensis plasmids , which strongly suggests a common source for these mobile elements.
we suspect that rickettsial plasmids and tra clusters are vertically inherited but are apparently unstable and are currently degrading.
CONCLUSIONS
based on its genome and lifestyle, we suspect that the clonal r. africae is more regulated and more specifically adapted to its host and warm environment than other tick-associated rickettsiae.
we speculate that losing this regulation, as observed in several intracellular pathogens, is a critical cause of virulence  <cit> .
further transcriptomic analysis of r. africae and other rickettsia species grown at various temperatures is currently ongoing to identify putative other candidate genes involved in stress response.

 rickettsiae are obligate intracellular gram-negative bacteria mostly associated to arthropods, some of which causing mild to severe diseases in humans. pathogenic species are classified into two groups based on phylogenetic analyses. the typhus group includes two rickettsia prowazekii and r. typhi, and the spotted fever group includes pathogenic species and numerous species of unknown pathogenicity. two additional validated species, r. bellii and r. canadensis, and a variety of unvalidated species from insects or leeches are organized into the most outer outgroups of the genus rickettsia. the relatively low rate of lateral gene transfer, the continuous gene loss and the colinearity of most of their genomes make rickettsia species an outstanding model for comparative genomics. indeed, genome reduction paradoxically results in higher virulence in r. prowazekii. the pathogenic mechanisms of rickettsiae are unclear. within ticks, rickettsiae remain quiescent during the starvation of their vector but undergo a reversion to the virulent state, termed reactivation, following incubation at 37°c or blood meal. this phenomenon is marked in r. rickettsii by morphological changes in the microcapsular and slime layers. the precise molecular mechanisms of this change, however, are only poorly understood. during human infection, attachment to and invasion of host cells were suggested to involve the outer membrane proteins rompa and rompb and the adhesins adr and adr. a phospholipase d activity was proposed to play a role in escape from phagosomes, and intracellular motility was demonstrated to rely on actin polymerization. none of these factors nor the presence of a type iv secretion system, however, explain the virulence differences observed among rickettsia species. over the last ten years, r. africae has emerged as the causative agent of african tick-bite fever, the most common sfg rickettsiosis both in terms of seroprevalence and incidence. such an epidemiologic success is due to various factors, including the increase of tourism to wildlife parks in sub-saharan africa, the attack host-seeking behavior of its vector ticks,amblyomma sp., and the elevated prevalence of r. africae in these ticks, with infection rates of up to 100%. in addition, the bacterium has been identified in other areas with warm climates, such as the west indies, where it was found in guadeloupe, martinique, st kitts and nevis, and antigua islands. such a distribution, as well as the presence of r. africae in reunion island, is likely to result from the transfer from africa of cattle bearing infected ticks. tick-associated rickettsiae may infect ticks feeding on infected hosts or may be passed from one generation to the next transovarially. r. africae is transmitted transovarially and appears to be the most successful rickettsia in its adaptation to its vector tick, as the prevalence of tick infection is higher than that of any other rickettsia. in addition, infection does not appear to alter tick fitness. these data highlight the fact that r. africae is an extremely successful and fit bacterium. by comparison with r. conorii, the second most prevalent sfg rickettsia in africa, whose genome has previously been sequenced, r. africae exhibits a higher prevalence in ticks, a lower virulence in humans, and a greater genetic homogeneity. the genetic factors underlying these characteristics are, however, unknown. we assumed that the r. africae genome sequence might help understand the characteristics of this species and the genetic mechanisms associated with the difference in virulence. here, we present the sequence of the r. africae genome and additional data that suggest that this species has emerged recently. in support of this hypothesis, we show that r. africae is a clonal population. we also present data that support the assumption that rickettsial virulence increases following gene inactivation.  general features of the genome the genome of r. africae consists of two replicons: a circular chromosome of, base pairs and a bp circular plasmid. we acknowledge the fact that the esf- strain, first isolated in, may have undergone loss or rearrangement of plasmid or chromosomal genes during multiple passages in cell culture. sequences were deposited in genbank under accession number. the chromosome has a g + c content of %, in the range of other sfg rickettsial genomes, whereas the plasmid has a g + c content of %, similar to those of r. felis but higher than that of r. massiliae plasmids. the predicted total complement of open reading frames, chromosomal, and plasmidic orfs, is in the range of genomes from sfg rickettsiae with the exception of r. felis, which exhibits a larger genome. of these, exhibited homologs in the non-redundant database, and were assigned putative functions. overall, the chromosomal orfs encoded protein-coding genes, with of these being split into to orfs by the presence of one to several stop codons. by comparison with other sfg genomes, r. africae had fewer split genes than any other species with the exception of r. felis. in addition, r. africae exhibited a single rrna operon, with non-contiguous 16s and 23s rrna genes as in other rickettsial genomes, trnas and another three rnas. the r. africae chromosome exhibited an almost perfect colinearity with the r. conorii genome, with the exception of a -bp inversion. at both extremities of the inversion, there were repeats of the rickettsia palindromic element – familly. in this inverted fragment, r. africae exhibited orfs and rpes that were absent from r. conorii. among these orfs, a cluster of consecutive orfs had orthologs in the 3'-extremity of the tra cluster previously identified in the r. massiliae genome. these orfs included tradf, a transposase, spot, a split spot, a split spot, a split signal transduction histidine kinase, dam a site-specific dna adenine methylase, and orf of unknown function. in addition to the orthologs in r. massiliae, these genes had orthologs in similar clusters in r. felis, r. bellii, r. canadensis and o. tsutsugamushi but were absent from all other species. as in r. massiliae, r. bellii and r. canadensis, the r. africae cluster was bounded at its 3'-end by a trna-val, but, in contrast with these three species, neither an integrase with its atti site nor a trna-val fragment marker of integration was present at the 5' end. the presence of a similar gene cluster inserted at the same position in several rickettsia species, with a gc content different from that of the genome suggests that it was acquired horizontally from a common ancestor and then transmitted vertically. in r. africae, an attc site, specific to integron-inserted gene cassettes, located at the 3'-end of the spot gene, supports the role of integration in the insertion of this gene cluster. attc sites were also identified in r. massiliae, r. felis, and r. bellii. nevertheless, the presence of transposases in all species and the fact that, in r. felis, nine of these genes are located in the prf plasmid support the role of several genetic mechanisms at the origin of this cluster, possibly involving plasmids, integrons and transposons. in comparison with other species containing this gene cluster, r. africae had the smallest number of genes. in particular, it lacked most of the tra cluster, with the exception of tradf, but retained three spot genes, including two degraded to pseudogenes. in r. bellii and r. massiliae, tra genes were described as encoding components of a type iv secretion system for conjugal dna transfer. in terms of gene content, the r. africae cluster was more similar to those of r. felis and r. canadensis, with the loss of the tra cluster, the conservation of spot genes and the presence of pseudogenes, than to those of r. massiliae and r. bellii, in which the tra cluster was intact but spot genes were partially degraded. such findings suggest that species-specific evolution of this gene cluster occurred, which likely resulted from gene excisions in r. africae, r. felis and r. canadensis, or gene expansion by transposase duplication in r. massiliae. * unpublished genomes available in genbank: r. akari,r. rickettsii, r. sibirica, r. bellii, and r. canadensis. in addition to the tradf gene described above, the r. africae chromosome retained many of the components of the type iv secretion system involved in both dna transfer and effector translocation in other bacteria, including virb virb, virb, virb, virb, virb, virb, virb, virb and vird. in addition, r. africae possessed a trax and a split fimd gene but lacked other tra cluster genes found in r. massiliae, r. felis, r. bellii and o. tsutsugamushi, such as trac and tragf. therefore, the tra cluster was mostly eliminated from the r. africae, and, following a "use it or lose it" scheme, this species probably did not need a tra gene-linked conjugation system. in addition, the pra plasmid did not contain genes encoding proteins involved in conjugation. six transposase-encoding genes were identified in the chromosome, including one split into two orfs and one present as a remnant and two in the pra plasmid, including one present as a fragment. this contrasts with the large expansion of transposases caused by gene duplications previously detected in r. felis and r. bellii. common rickettsial gene set and phylogeny when compared to eight other available rickettsial genomes, a total of genes and rna-encoding genes of r. africae had orthologs in all genomes. in addition, another r. africae genes had orthologs only in sfg rickettsiae and were either absent or remnant in tg rickettsiae. consequently, we identified genes as constituting the core gene set of all available rickettsial genomes and orfs as the core gene set of sfg rickettsiae. following concatenation of the core genes, a reliable phylogenetic organization was obtained using three analysis methods that was consistent with previous phylogenetic studies of rickettsia species. in comparison with other rickettsia genomes, r. africae had and fewer genes than r. bellii, r. felis and r massiliae, respectively, but and more genes than r. typhi, r. prowazekii, r. akari, r. rickettsii, r. sibirica, and r. conorii, respectively. when comparing the numbers of degraded genes, r. africae, with degraded genes, had a significantly less degraded genome than that of other spotted fever group rickettsiae including r. akari, r. conorii, r. massiliae, r. rickettsii and r. sibirica. it had, however, significantly more degraded genes than r. felis. transcription of genes conserved in r. africae but absent from highly pathogenic species r. africae had intact genes that were either absent or degraded in all three virulent species r. conorii, r. rickettsii and r. prowazekii. of these, encoded proteins of unknown functions. the remaining six genes encoded a plasmid maintenance system antidote protein, the spot gene, a site-specific dna adenine methylase, an ankyrin repeat, a putative integral membrane protein, and a protein exhibiting a high degree of amino acid sequence identity with proteins of γ-proteobacteria classified within the cog as putative virulence proteins. when investigating the transcription of these genes in r. africae grown at and 37°c, we observed a significantly higher transcription level at 37°c than at lower temperatures for two genes, raf_orf and raf_orf the former gene contained a putative protease domain site, but the latter had no known function. the r. africae plasmid the r. africae plasmid is a new example of a plasmid in rickettsia species, following those in r. felis, r. massiliae, r. monacensis, r. helvetica, r. peacockii, r. amblyommii and r. hoogstraalii. this plasmid, named pra, is smaller than those of r. felis, r. monacensis, and r. massiliae. the pra plasmid is predicted to contain genes, of which have homologs in public databases and are associated with functional attributes. these six genes encode for a chromosomal replication initiator dnaa-like protein, a site-specific recombinase, two contiguous transposases exhibiting 100% sequence similarity but with one shorter than the other, the auto-transporter protein sca, and a para-like plasmid stability protein. five genes have orthologs in the r. massiliae plasmid, six have orthologs in the r. felis plasmids, and three have orthologs in the r. monacensis plasmid. the presence of two genes conserved in plasmids from four species suggests that these plasmids have a common origin. the presence of two almost identical successive transposases in r. africae matching a single gene in r. massiliae and r. felis suggests a duplication event in the former species. the pra plasmid lacks heat shock protein-encoding genes found in other rickettsial plasmids. in contrast, orf a site-specific recombinase, is absent from other species. its closest phylogenetic neighbour is a site-specific recombinase from magnetospirillum magnetotacticum, a high g-c content α-proteobacterium living in aquatic environments. the sca gene found intact in r. africae pra was absent from the r. massiliae and r. monacensis plasmids and present but fragmented within r. felis prf, but it was absent from prfδ as well all other rickettsia species. as outlined by baldridge et al., the plasmid content of a rickettsia species may vary according to the passage history of rickettsial strains. when estimating the prevalence of the plasmid among r. africae strains, we detected it in the tested isolates from south africa and in the eschar biopsies from patients with atbf contracted in the same country and in 20/ r. africae-positive amblyomma ticks. therefore, it appears from these results that, depending on the geographic location, the plasmid of r. africae may be unstable. whether the plasmid has been lost by pcr-negative strains or cannot be amplified with the primers we used is as yet unknown. such inter-strain differences in plasmid content were also observed in r. felis. stress response rickettsiae live intracellularly in both arthropod and mammal hosts. this implies that periods of tick starvation and feeding cause bacterial dormancy and multiplication following reactivation. as a consequence, and despite their obligate intracellular location, rickettsiae may face, and thus have to adapt to, highly variable and extreme environmental conditions. known as the stringent response, this bacterial adaptation to nutritional stress has been described to be mediated by the accumulation of guanosine nucleotides pppgpp and ppgpp. accordingly, the transcriptional analysis of r. conorii exposed to a nutrient deprivation was characterized by the up-regulation of gmk and of genes from the spot family, suggesting a role for these nucleotides as effectors of the stringent response. the r. africae genome exhibited eight spot genes phylogenetically classified within two major clades. the largest clade included spot genes with hydrolase activity, while the second included those with a synthetase domain. with eight genes, r. africae had more spot genes than r. rickettsii, r. conorii, r. sibirica, r. akari, r. canadensis, r. typhi and r. prowazekii but fewer genes than r. felis and r. bellii. altogether, our data suggest that r. africae is more regulated than more pathogenic species. infection of mammal hosts the r. africae genome encoded rompa and rompb, two surface-exposed and immunodominant proteins belonging to the paralogous "surface cell antigen" family and known in rickettsia species to be responsible for antigenic differences between species and to elicit an immune response in patients. experimental studies suggested that these two auto-transporter proteins could function as adhesins. in addition, another eight sca-encoding genes were found in the genome. these genes were represented by orfs due to partial degradation of some of the paralogs. among the sca-encoding genes detected in rickettsia species, r. africae had similar sets of conserved, degraded and absent sca genes as r. conorii and r. rickettsii. in addition to these sca-encoding genes, r. africae exhibited a degraded sca gene and a complete sca gene carried by the pra plasmid, only shared with r. felis, where it was also found partially degraded on the prf plasmid. the sca genes from both species were grouped into a distinct cluster close to the sca and genes. this result further supports a common origin of the pra and prf plasmids. a proteomic approach recently allowed the identification of two paralogous proteins encoded by the genes rc1281-rc and rp827-rp as putative adhesins adr and adr these proteins may be key actors for entry and infection in both r. conorii and r. prowazekii. both proteins are ubiquitously present within the rickettsia genus. their presence within the r. africae genome reinforces their suspected key role in rickettsial life. both pld and tlyc, encoding phospholipase d and hemolysin c, respectively, which play a role in phagosomal escape, were conserved in the r. africae genome. this bacterium also exhibited genes encoding other proteins with membranolytic activity, including tlya and pat. as expected, the genome of r. africae has a ricka gene orthologous to all rickettsial ricka genes and coding a protein activating the arp2/ complex, whose nucleation triggers actin polymerisation. the rick a protein in r. africae is slightly different from those of other species, with a phenylalanine instead of a serine within the g-actin-binding site, an ennip motif repeated twice instead of four times in the central proline-rich region of the protein, and an aspartate and an isoleucine instead of an asparagine and an alanine or valine, respectively, in the carboxy-terminal region. despite these differences, the ricka protein of r. africae appeared to be functional as demonstrated by its ability to polymerize actin and multiply intranuclearly. sixteen vir gene paralogs were found in the r. africae genome. virulence genes of the vir family belong to the type iv secretion machinery, a system that allows the delivery of virulence factors from bacterial and eukaryotic host membranes to the cytoplasm of the host cell. all genes were found to be intact and common to all rickettsia genomes with the exception of virb6- in r. africae and virb6- in r. massiliae. in both species, these genes were split into two orfs. phylogenetic analysis of the virb6- gene distinguished clearly the sfg and tg and showed that the r. africae virb6- protein is phylogenetically closer to that of r. sibirica. clonality of r. africae of the amblyomma ticks tested, were pcr-positive for r. africae. therefore, infection rates of amblyomma ticks with r. africae may be higher than previously described, which suggests an extreme fitness of this rickettsia for its vector. in addition, such infection rates are the highest among rickettsia species. using mst, pcr products of the expected sizes were obtained from the dksa-xerc, mppa-purc and rpme-trnafmet intergenic spacers from all tested specimens. sequences obtained from these amplicons were in all cases identical to those previously obtained for r. africae,, and, for the dksa-xerc, mppa-purc and rpme-trnafmet spacers, respectively). this is the first rickettsia demonstrated to be clonal. other tested rickettsia species, including r. conorii, r. massiliae, r. sibirica, and r. felis, were significantly more genetically variable than r. africae. discussion using a comparative study of rickettsial genomes, we found that virulence in rickettsia species is not correlated with acquisition of foreign dna but may rather result from a reduction in regulation due to genome decay. comparative genomics sheds light on a much wider spectrum of virulence acquisition mechanisms in bacteria than initially thought. based on the examples of enterobacteria and staphylococci, gain in pathogenicity in bacteria was mainly thought to result from horizontal gene transfer, either directly or through mobile genetic elements. however, a recent study of rickettsia species associated with arthropods, insects, leeches and protists clearly demonstrated that horizontal gene transfer was a rare event within this genus. in addition, genomic studies demonstrated that rickettsiae are undergoing genome decay, affecting in priority horizontally-acquired genes, and that there is no association between pathogenicity and acquisition of virulence markers. in fact, the genome of the most virulent species, r. prowazekii, is a subset of the less pathogenic species r. conorii, thus highlighting a paradoxical relationship between smaller genome size and higher pathogenicity. careful comparison of the r. prowazekii and r. typhi genomes also demonstrated that the former species, more pathogenic than the latter, had a more decayed genome despite a 12-kb insertion that likely resulted from a single genetic event. when investigating the genomic characteristics associated with the milder virulence of r. africae, we first ruled out a potential role of the plasmid by the fact that it is unstable in this species. then, we compared the gene contents of r. africae with r. conorii, r. rickettsii, and r. prowazekii, which exhibit a higher pathogenicity in humans and their arthropod hosts. we observed that r. africae showed no gene loss but had genes fully conserved that were either absent or degraded in the other species. we speculated that, because r. africae had more intact genes than more virulent species, some of these genes may be involved in maintaining a low virulence level. such a behavior may not be unique to rickettsiae. it was found that gene knockout resulted in increased virulence in mycoplasma, streptococcus pyogenes, and vibrio cholerae. in m. ulcerans, genome reduction was also linked to gain in virulence. it emerges as a concept that virulence may be increased by gene loss. we assume that a similar phenomenon may happen in rickettsiae, and that inactivation of some genes may deregulate the control of bacterial multiplication, in particular during the reactivation phenomenon following warming, thus enhancing pathogenesis. among the putative candidate genes unique to r. africae, we identified only two genes that were significantly more transcribed at 37°c than at lower temperatures. of these, one encoded a protein that had a putative protease domain. a protease was previously shown in vibrio cholerae to be a virulence repressor. however, whether this differentially-transcribed protease plays a role in virulence repression in r. africae is as yet unknown. in contrast, the spot gene unique to r. africae was not upregulated, and this species retained another two spot pseudogenes that were completely lost by other species. spot genes, effectors of the stringent response, were shown to play a major role in adaptation to stress in r. conorii, in particular when subjected to abrupt temperature variations similar to those occurring during a tick blood meal. r. africae, however, has more spot genes than r. conorii or r. rickettsii and does not show any modification of expression of its specific spot gene during temperature variations. we speculate that higher regulation ability in r. africae is linked to lower pathogenicity. in addition, when compared to other tick-borne rickettsia species, r. africae exhibited several unique characteristics. first, this species is extremely successful and fit: it is highly adapted and harmless to its tick host, being efficiently transmitted both transtadially and transovarially in amblyomma sp. ticks, which consequently act as efficient reservoirs. in contrast, r. rickettsii and r. conorii have a negative effect on their tick vectors in experimental models. as a result, the prevalence of r. africae in its host ticks is higher than that of most other rickettsiae. similarly, r. africae is less pathogenic for humans than other sfg species such as r. conorii and r. rickettsii, in particular because the infection is never lethal. this observation was later supported by the demonstration that inoculation eschars in atbf were histologically different from those in msf. in particular, in contrast with other sfg rickettsioses where eschars are characterized by perivascular infiltration of t cells and macrophages, with some b lymphocytes and few polymorphonuclears, the vasculitis in atbf is made of a large infiltrate of neutrophils causing an extensive cutaneous inflammation and necrosis. such a local reaction, in addition to the few r. africae cells detected in eschars, suggests that the bacterium replicated poorly in human tissues. second, r. africae has significantly fewer degraded genes than other sfg species, except r. felis. specifically, this characteristic suggests that r. africae is undergoing a slower degradation process than other rickettsiae. third, the identification of a single mst genotype among strains suggested that r. africae was clonal. this contrasted with the variable plasmid content of this species. originally thought to be absent in rickettsia species, plasmids have been detected in eight species to date, and their plasmid content may exhibit intraspecies variability. in r. felis, two plasmid forms have been sequenced, and baldridge et al. found two plasmids in both r. peacockii and r. amblyommii. in addition, these authors showed that r. peacockii lost its plasmids during long-term serial passages in cell culture. in r. africae, the pra plasmid may also be unstable, as shown by the absence of plasmid detection in 12/ amblyomma ticks tested. this plasmid encodes orfs, two of which are common to r. felis, r. massiliae and r. monacensis plasmids, which strongly suggests a common source for these mobile elements. we suspect that rickettsial plasmids and tra clusters are vertically inherited but are apparently unstable and are currently degrading.  based on its genome and lifestyle, we suspect that the clonal r. africae is more regulated and more specifically adapted to its host and warm environment than other tick-associated rickettsiae. we speculate that losing this regulation, as observed in several intracellular pathogens, is a critical cause of virulence. further transcriptomic analysis of r. africae and other rickettsia species grown at various temperatures is currently ongoing to identify putative other candidate genes involved in stress response. 
we also speculate that in rickettsia species virulence is mostly associated with gene loss. to study the genetic characteristics associated with virulence, we compared this species to r. prowazekii, r. rickettsii and r. conorii. the genome sequence was deposited in genbank under accession number. among these, the pathogenicity varies greatly, from the highly virulent r. prowazekii, which causes epidemic typhus and kills its arthropod host, to the mild pathogen r. africae, the agent of african tick-bite fever, which does not affect the fitness of its tick vector. we evaluated the clonality of r. africae in patients and ticks, and determined its genome sequence, which comprises a circular chromosome of, bp including a tra operon and an unstable -bp plasmid. r. africae and r. prowazekii have, respectively, the less and most decayed genomes. eighteen genes are present only in r. africae including one with a putative protease domain upregulated at 37°c.
 the rickettsia genus includes validated species, of which are proven human pathogens. among these, the pathogenicity varies greatly, from the highly virulent r. prowazekii, which causes epidemic typhus and kills its arthropod host, to the mild pathogen r. africae, the agent of african tick-bite fever, which does not affect the fitness of its tick vector.  we evaluated the clonality of r. africae in patients and ticks, and determined its genome sequence, which comprises a circular chromosome of, bp including a tra operon and an unstable -bp plasmid. to study the genetic characteristics associated with virulence, we compared this species to r. prowazekii, r. rickettsii and r. conorii. r. africae and r. prowazekii have, respectively, the less and most decayed genomes. eighteen genes are present only in r. africae including one with a putative protease domain upregulated at 37°c.  based on these data, we speculate that a loss of regulatory genes causes an increase of virulence of rickettsial species in ticks and mammals. we also speculate that in rickettsia species virulence is mostly associated with gene loss. the genome sequence was deposited in genbank under accession number. 