Horizontal inheritance of plasmid genes in Rickettsia felis Gillespie, J'J' 1,2, Beier, M'S' 2, Rahm

1
Horizontal inheritance of plasmid genes in
Rickettsia felisGillespie, J.J. 1,2, Beier,
M.S. 2, Rahman, M.S. 2, Ammerman, N.C. 2,
Shallom, J.M. 1, Purkayastha, A. 1, Sobral, B.S.
1, and Azad, A.F. 21Virginia Bioinformatics
Institute at Virginia Tech, Blacksburg, VA 24061,
USA2Department of Microbiology and Immunology,
University of Maryland, School of Medicine,
Baltimore, MD 21201, USA
Synopsis The complete genome sequence of
Rickettsia felis (Rickettsiales Rickettsiaceae)
(1), a causative agent of murine typhus-like
rickettsiosis, revealed a number of genetic
anomalies that likely contribute not only to a
large genome size relative to most other
rickettsiae, but also to the phenotypic oddities
that have confounded the categorization of R.
felis as a member of either typhus group (TG) or
spotted fever group (SFG) rickettsiae. Most
intriguing was the first ever report from
rickettsiae of a supposed conjugative plasmid
(pRF) that contains 68 putative genes, several of
which encode proteins with high similarity to
conjugative machinery in other plasmid-containing
bacteria. Given few published reports of
plasmids occurring in obligate intracellular
parasites and the fact that, of 32 pRF genes not
found in any other rickettsiae, 30 (some inactive
derivatives) are known from other distant
bacteria and/or non-bacterial organisms, we set
out to determine the mode of inheritance of pRF
genes relative to the history of the conserved
chromosomal genes of rickettsiae. Our
phylogenetic analysis unambiguously supports the
horizontal inheritance of pRF genes in R. felis,
and further in silico characterization of pRF
supports its role as a functional plasmid.
pRF Gene Distribution Thirty pRF genes are also
found on the R. felis chromosome (Table 1), while
38 pRF genes are exclusively found on the plasmid
(Table 2). This characterization of the
distribution of pRF genes allowed for us to
compare the phylogenetic history of pRF genes
relative to their orthologous counterparts on the
R. felis chromosome as well as the chromosomes
of nine other rickettsiae. Ten such genes were
analyzed (Table 1, dashed boxes).
Phylogenetic Resolution Comparison of a second
robust rickettsiae phylogeny (Fig. 2A) with the
phylogeny estimated from 10 pRF genes present on
the chromosomes of the sampled Rickettsia spp.
suggests that pRF genes are not monophyletic with
their orthologs on the R. felis chromosome (Fig.
2B). Rather, the 10 pRF genes seem to be more
related to AG rickettsiae. Interestingly,
several exclusively pRF genes (or inactive
derivatives) occur in AG rickettsiae, with eight
of these genes characteristic of typical plasmids
(Table 2). This evidence prompts us to
hypothesize not only that pRF genes were likely
acquired horizontally by means of conjugation,
but that AG rickettsiae possess (or once
possessed) the same conjugation machinery found
in R. felis and may have contributed to the
architecture and composition of the pRF plasmid.
The future determination of plasmids in AG
rickettsiae, as well as the other TRG member R.
akari, will test our claim.
Further Evidence for a Functional
Plasmid Minimum cumulative skew points in DNA
strand compositional asymmetry and sharp changes
in coding strand in pRF identify the putative
oriV and region of replication termination (Fig.
3). Additionally, genes involved in DNA
replication are present around the oriV, namely
parA, DnaA and lon. Collectively, our in silico
predictions suggest that pRF is likely capable of
replication independent of the R. felis
chromosome. Hence, it is justified to determine
the role of pRF in R. felis survival and
pathogenicity.
A
B
Fig. 1. Estimated rickettsiae phylogeny from
parsimony analysis (exhaustive search) of 15
proteins present in nine genomes (plus two
Wolbachia outgroups). Tree 3834 steps, with
branch support from one million bootstrap
replicates.
Rickettsiae Phylogenomics A robust phylogeny
estimation (Fig. 1) allows us to propose a novel
rickettsiae classification, grouping nine genomes
into four categories ancestral (AG), typhus
group (TG), transitional group (TRG), and true
spotted fever group (SFG). We use this as a
benchmark to compare the phylogenetic history of
the plasmid genes encoded on pRF that are also
present on the chromosomal genomes of all
lineages of rickettsiae for which a genome
sequence is available at PATRIC (PathoSystems
Resource Integration Center, https//patric.vbi.vt
.edu) (2).
Fig. 3. (A) Schematic map of pRF with shaded
regions containing the putative oriV (right) and
replication termination region (left). (B) AT-
and (C) CG-skews computed with GenSkew
(http//mips.gsf.de/services/analysis/genskew).
Fig. 2. (A) Estimated phylogeny of 21 exclusively
chromosomal proteins from 10 rickettsial strains.
(B) Estimated phylogeny of 10 proteins present
on the chromosome and plasmids of R. felis and
the chromosomes of other Rickettsia spp.
Aquamarine (TG) and light blue (TRG) boxes depict
the major differences in tree topologies. The
pRF genes are boxed and shaded. Results from
both analyses of amino acids are from an
exhaustive search under parsimony with branch
support from one million bootstrap replications.

• 1. Ogata H, et al. (2005) The genome sequence
  of Rickettsia felis identifies the first
• putative conjugative plasmid in an obligate
  intracellular parasite. PLoS Biol 3 e248.
• 2. Snyder EE, et al. (2007) The VBI
  PathoSystems Resource Integration Center
  (PATRIC). Nucleic Acids Res. In press.

This work is funded through NIAID contract
HHSN266200400035C to BS and NIH grant R01 AI59118
to AA.