Sequence and expression analysis of the ompA gene of Rickettsia peacockii, an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni.

Abstract

The transmission dynamics of Rocky Mountain spotted fever in Montana appears to be regulated by Rickettsia peacockii, a tick symbiotic rickettsia that interferes with transmission of virulent Rickettsia rickettsii. To elucidate the molecular relationships between the two rickettsiae and glean information on how to possibly exploit this interference phenomenon, we studied a major rickettsial outer membrane protein gene, ompA, presumed to be involved in infection and pathogenesis of spotted fever group rickettsiae (SFGR) but which is not expressed in the symbiont. Based on PCR amplification and DNA sequence analysis of the SFGR ompA gene, we demonstrate that R. peacockii is the most closely related of all known SFGR to R. rickettsii. We show that R. peacockii, originally described as East Side agent in Dermacentor andersoni ticks from the east side of the Bitterroot Valley in Montana, is still present in that tick population as well as in D. andersoni ticks collected at two widely separated locations in Colorado. The ompA genes of R. peacockii from these locations share three identical premature stop codons and a weakened ribosome binding site consensus sequence relative to ompA of R. rickettsii. The R. peacockii ompA promoter closely resembles that of R. rickettsii and is functional based on reverse transcription-PCR results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that OmpA translation products were not detected in cultured tick cells infected with R. peacockii. Double immunolabeling studies revealed actin tail structures in tick cells infected with R. rickettsii strain Hlp#2 but not in cells infected with R. peacockii.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2004, p. 6628–6636
0099-2240/04/$08.00?0DOI: 10.1128/AEM.70.11.6628–6636.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 11
Sequence and Expression Analysis of the ompA Gene of
Rickettsia peacockii, an Endosymbiont of the Rocky
Mountain Wood Tick, Dermacentor andersoni
Gerald D. Baldridge,1Nicole Y. Burkhardt,1Jason A. Simser,2Timothy J. Kurtti,1* and
Ulrike G. Munderloh1
Department of Entomology, University of Minnesota, St. Paul, Minnesota,1and Department of Microbiology and
Immunology, University of Maryland, Baltimore, Maryland2
Received 8 April 2004/Accepted 29 June 2004
The transmission dynamics of Rocky Mountain spotted fever in Montana appears to be regulated by
Rickettsia peacockii, a tick symbiotic rickettsia that interferes with transmission of virulent Rickettsia rickettsii.
To elucidate the molecular relationships between the two rickettsiae and glean information on how to possibly
exploit this interference phenomenon, we studied a major rickettsial outer membrane protein gene, ompA,
presumed to be involved in infection and pathogenesis of spotted fever group rickettsiae (SFGR) but which is
not expressed in the symbiont. Based on PCR amplification and DNA sequence analysis of the SFGR ompA
gene, we demonstrate that R. peacockii is the most closely related of all known SFGR to R. rickettsii. We show
that R. peacockii, originally described as East Side agent in Dermacentor andersoni ticks from the east side of
the Bitterroot Valley in Montana, is still present in that tick population as well as in D. andersoni ticks collected
at two widely separated locations in Colorado. The ompA genes of R. peacockii from these locations share three
identical premature stop codons and a weakened ribosome binding site consensus sequence relative to ompA
of R. rickettsii. The R. peacockii ompA promoter closely resembles that of R. rickettsii and is functional based on
reverse transcription-PCR results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western
blotting showed that OmpA translation products were not detected in cultured tick cells infected with R.
peacockii. Double immunolabeling studies revealed actin tail structures in tick cells infected with R. rickettsii
strain Hlp#2 but not in cells infected with R. peacockii.
Ticks are major vectors of emerging and reemerging disease
agents in North America, Europe, and elsewhere in temperate
zones. Because their complex life cycle alternates between
arthropods and vertebrates and commonly involves a wildlife
reservoir host, vector-borne pathogens are difficult to eradi-
cate. In addition, growing concerns over toxicity of acaricides
have renewed interest in environmentally compatible strate-
gies for control of ticks and tick-borne pathogens. In addition
to bacterial pathogens, obligate intracellular proteobacteria
are sometimes found in ticks as apparently nonpathogenic
endosymbionts that are transmitted transovarially from gener-
ation to generation (29). Closely related microorganisms are
more likely to interfere with each other in arthropod vectors
than are distant relatives (4, 10, 23), and endosymbionts could
possibly be exploited to ultimately render ticks incapable of
transmitting closely related pathogens.
Rickettsia peacockii, an endosymbiont of Dermacentor ander-
soni, the Rocky Mountain wood tick (28), is of particular in-
terest because it may have played a role in the declining prev-
alence in the North American Rockies of Rickettsia rickettsii,
the etiologic agent of Rocky Mountain spotted fever (RMSF)
(9). It has long been known that in the Bitterroot Valley of
Montana the absence of RMSF outbreaks correlates with the
presence of nonpathogenic spotted fever group rickettsiae
(SFGR) (6, 7, 18). R. peacockii, also known as the East Side
agent, was first detected in ticks collected from the east side of
Montana’s Bitterroot Valley with a prevalence of up to 80% (7,
28). In contrast, R. rickettsii is nearly absent in ticks on the east
side of the valley (34), and most cases of RMSF occur on the
west side of the valley, where prevalence of R. rickettsii in ticks
is considerably lower (less than 1% [16]) than that of R. pea-
cockii on the valley’s east side. While a low prevalence of R.
rickettsii may be due to its pathogenic effects on ticks (27), a
high prevalence of R. peacockii in tick ovaries might further
interfere with maintenance and transmission of R. rickettsii (7).
Although the genomes of R. rickettsii, Rickettsia sibirica, and
Rickettsia conorii (31) have been sequenced, very few DNA
sequence data are available for nonpathogenic SFGR that
could help define their symbiotic nature on a molecular basis.
Several genes have been used to establish rickettsial relation-
ships, principally the 16S rRNA gene, citrate synthase gene
(gltA), rickettsial outer membrane protein genes A and B
(ompA and ompB, respectively), and gene D (15, 40–42, 44).
The latter four genes encode proteins and undergo stabilizing
(neutral) selection (14), indicating that they are suitable for
taxonomic purposes. The molecular and biological traits that
differentiate R. peacockii from R. rickettsii (28, 47) include an
inability of the former to make a functional OmpA, which
could underlie its endosymbiotic nature and apparent confine-
ment to D. andersoni. Pathogenic R. rickettsii expresses OmpA
and polymerizes host cell actin, facilitating its adhesion to host
cells, cell-to-cell spread, and replication within mammalian
and tick hosts (19, 22, 24). Unlike surface protein genes that
* Corresponding author. Mailing address: Department of Entomol-
ogy, University of Minnesota, 1980 Folwell Ave., St. Paul, MN 55108.
Phone: (612) 624-4740. Fax: (612) 625-5299. E-mail: kurtt001@umn
.edu.
6628

Page 2

rapidly evolve in response to host antibodies (52), Fournier et
al. (14, 15) found by calculating the ratio of synonymous and
nonsynonymous amino acid substitution rates that the ompA
and -B genes undergo neutral selection. Because SFGR are
predominantly maintained in nature by transovarial transmis-
sion through successive tick generations, their evolution is
probably more influenced by tick factors than by those of the
mammalian host. In order to gain a better understanding of the
biological and evolutionary relationship between R. rickettsii
and R. peacockii, we expanded analysis of the R. peacockii
ompA gene to include a 3.5-kb region downstream of its tan-
dem repeats (Fig. 1). We also sequenced the promoter region
and examined ompA transcription using reverse transcription-
PCR (RT-PCR). Our analysis included PCR-amplified ompA
sequences from genomic DNA of R. peacockii-infected ticks
collected in Colorado and from ticks on the east and west sides
of the Bitterroot Valley. We anticipate that the new data pre-
sented here will aid in developing methods for the genetic
manipulation and transformation of tick symbiotic rickettsiae
with the aim of improving their potential for the control of
tick-borne pathogens.
MATERIALS AND METHODS
Rickettsiae and DNA extraction. R. peacockii strain DAE100R (isolate from
Rustic, Colo.) was grown in the chronically infected D. andersoni cell line
DAE100 (46). In addition, for routine maintenance R. peacockii was grown in
Ixodes scapularis, the black-legged tick, cell line ISE6 (25). Initially, rickettsiae
were released from infected DAE100 cells by forcing cell suspensions through a
27-gauge needle and removing large debris by low-speed centrifugation (275 ?
g for 10 min). The supernatant was filtered through 0.8-?m syringe filters, and
the host cell-free rickettsial suspension was added to a culture of ISE6 cells.
Subsequently, R. peacockii was transferred every 2 weeks by introducing an
aliquot (0.1 to 0.5 ml) of an infected ISE6 culture to an uninfected ISE6 culture
(5 ml of ISE6 cells at 5 ? 106cells/ml in a 25-cm2flask). All cultures were
maintained in antibiotic-free L15B300 medium supplemented with fetal bovine
serum (5%), tryptose phosphate broth (5%), and bovine lipoprotein concentrate
(0.1%), pH 7.2 to 7.5 (25). Two nonpathogenic rickettsial isolates, R. rickettsii
Hlp#2, originally cultured from rabbit ticks (Haemaphysalis leporis-palustris)
(33), and MOAa, an isolate from the lone star tick (Amblyomma americanum),
were grown in I. scapularis cell lines ISE6, IDE2, and IDE8 (45, 52).
To extract genomic DNA, rickettsiae were recovered (38) and lysed in 300 ?l
of buffer containing 10 mM Tris-HCl (pH 7.8), 0.5 mM EDTA, 0.5% sodium
dodecyl sulfate (SDS), 40 ?g of RNase A (Promega, Madison, Wis.)/ml, and 250
?g of proteinase K (Fisher, Pittsburgh, Pa.)/ml at 55°C for 2 to 16 h. Lysates were
phenol-chloroform extracted, and DNA was ethanol precipitated and resus-
pended in nuclease-free H2O (Life Technologies, Rockville, Md.).
Ticks and DNA extraction. We collected adult female D. andersoni ticks on the
north fork of Crestone Creek 4 miles upstream of Crestone, Colo. (200 miles
south of Rustic, Colo.) in May 2002. We also obtained adult female D. andersoni
ticks collected on the east side (Skalkaho Mountain and Burnt Fork) of the
Bitterroot Valley, Ravalli County, Mont., or on the west side (near Como Lake)
in May 2003 (we thank Tom Schwan, Rocky Mountain Laboratories, Hamilton,
Mont., for providing us with these ticks).
To harvest DNA, ticks were surface disinfected by 5 min of agitation in 0.5%
bleach followed by 5 min in 70% ethanol and two rinses in sterile H2O. Ticks
were dissected individually in sterile Hanks balanced salt solution, and internal
tissues were separated from the integument and transferred to 300 ?l of lysis
buffer (Puregene; Gentra Systems, Minneapolis, Minn.) with 0.2 ?g of proteinase
K (Sigma, St. Louis, Mo.)/?l and incubated overnight at 55°C. Recovered DNA
was resuspended in nuclease-free H2O.
Strain designations. The R. peacockii strains that we analyzed, whether they
were in culture (i.e., DAE100R isolated from ticks originally collected near
Rustic, Colo. [47]) or from field-collected ticks (i.e., Skalkaho Mountain, Mont.,
or Crestone, Colo.) are designated according to the site or locale where the ticks
were originally collected.
Primers and PCR amplification. The primers that we used are listed in Table
1. Gene positions and primer designations are based on those given for R.
rickettsii (2, 15, 36, 39, 41, 42) unless stated otherwise. Lyophilized primers were
purchased from Invitrogen (Carlsbad, Calif.) or Integrated DNA Technologies
(Coralville, Iowa) and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA;
pH 8.0). All PCRs were performed in a RoboCycler thermocycler (Stratagene,
La Jolla, Calif.), with 0.5 ?g, or less, of template DNA and 2.5 U of Taq
polymerase (Promega), unless stated otherwise, in a 50-?l reaction volume.
Standard buffer, MgCl2, and deoxynucleoside triphosphate concentrations were
as recommended by the enzyme manufacturers.
RFLP analysis of the 5? end of rickettsial ompA. The SFGR-specific primer
pair 190-70p and 190-602n was used to amplify a 532-bp fragment at the 5? end
of ompA (cycling parameters of 95°C for 5 min; 35 cycles of 95°C for 20 s, 48°C
for 30 s, and 60°C for 2 min, followed by a final 60°C 5-min elongation step) (39)
for restriction fragment length polymorphism (RFLP) analysis. Fifty to 250 ng of
the gel-purified (see below) ompA PCR fragment was digested for 2 h at 37°C
with restriction enzyme PstI (Promega) or RsaI (Life Technologies) in the
manufacturer’s buffer in 10- to 30-?l volumes. Digests were electrophoresed
through 4% agarose gels (3% Gene Pure LE agarose, 1% NuSieve GTG agarose;
ISC BioExpress, Kaysville, Utah) and stained with ethidium bromide. RFLP
patterns were compared to those described for SFGR (39) and R. peacockii (28).
PCR amplification and sequencing of rickettsial ompA. We used primers
designed by Fournier et al. (15) to amplify the 5? and 3? ends of the ompA gene
(Table 1). Figure 1 provides a map of the ompA gene, indicating the regions
targeted by the primers. We amplified the PCR products for sequencing using
Pfu Turbo Hotstart DNA polymerase or cloned Pfu DNA polymerase (Strat-
agene) at 5 U per reaction mixture of 50 ?l. Cycling parameters for the cloned
polymerase were 94°C for 45 s followed by 35 cycles of 94°C for 45 s, X°C (X ?
annealing temperature specified below for each primer pair) for 2 min, and 72°C
for 1 min followed by a final 10-min extension step at 72°C. For Turbo Hotstart
polymerase, denaturation temperatures were increased to 95°C and lengthened
by 15 s, and total cycles were reduced to 30. The 5? end of the ompA gene,
including the promoter, was amplified as a 381-bp fragment with primer pair
190-(?110) and 190-271 (X ? 50°C). A 631-bp fragment overlapping the 532-bp
product described above was amplified with primer pair 190-70p and 190-701 (X
? 48°C). The 3?-end region of the gene was amplified with primer pairs that
produced five overlapping fragments as follows: 818 bp with 190-3588 and 190-
4406 (X ? 42°C); 850 bp with 190-4388 and 190-5238 (X ? 46°C); 888 bp with
190-5125 and 190-6013 (X ? 46°C); 891 bp with 190-5917 and 190-6808 (X ?
42°C); 399 bp with 190-6585 and 190-6984 (X ? 46°C). PCR products were
purified by electrophoresis (1% agarose), recovered with QIAquick gel extrac-
tion spin columns (QIAGEN, Valencia, Calif.), and directly sequenced in for-
ward and reverse directions two to four times with an ABI 377 automated
sequencer (Advanced Genetic Analysis Center, University of Minnesota). Con-
sensus sequences were obtained by alignment using the ClustalX multiple-se-
quence alignment program (20).
Phylogenetic analysis. Nucleotide sequences of R. peacockii and R. rickettsii
Hlp#2 corresponding to R. rickettsii (R strain) ompA positions 91 to 680 and
3635 to 6789 (2) were joined (the tandem repeat region was excluded) so that
3,764 nucleotides were included in the analysis (15). However, because of a
premature stop codon in the 5? prerepeat region of the R. peacockii ompA gene,
translated amino acid sequences were not used for phylogenetic analysis (28, 47).
The ompA sequences of R. peacockii, R. rickettsii Hlp#2, and other Rickettsia
species (Table 2) were manually edited in a word processing file with ClustalX.
FIG. 1. PCR amplification of the rickettsial ompA gene. The
7,088-bp R. rickettsii R strain ompA sequence (GenBank accession no.
M31277) (2) is represented by the solid line, with a numerical nucle-
otide scale. The protein-encoding portion of the gene is represented by
the open rectangle, with the tandem repeat region indicated by vertical
bars. Overlapping PCR fragments (primer positions in Table 1) are
indicated by lines above the open rectangle. The left- and right-most
fragments contain the promoter and transcription terminator regions,
respectively. Amplified sequences were assembled into a 4,032-bp
composite sequence excluding the primer binding sites at the termini.
VOL. 70, 2004R. PEACOCKII ompA GENE ANALYSIS 6629

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Alignments were imported into PAUP? 4.0b10 (51) for construction of phylo-
genetic trees, and distance matrices were determined using Kimura’s two-pa-
rameter option (21). Relationships were analyzed with neighbor-joining (43) and
maximum parsimony (48) methods. Node stability of dendrograms was estimated
using bootstrap analysis (12) of values obtained from 1,000 trees generated
randomly.
PCR and sequence analysis of 16S rRNA, 17-kDa antigen, and gltA (citrate
synthase) genes. 16S rRNA gene targets were amplified with the general bacte-
rial primers E45 and E1242 (32) (1,197 bp; cycling parameters of 95°C for 2 min,
30 cycles of 95°C for 45 s, 42°C for 45 s, and 72°C for 1 min, and a final 72°C
10-min extension), Francisella-specific primers F5 and F11 (13) (1,141 bp; cycling
parameters of 95°C for 2 min, 30 cycles of 95°C for 45 s, 48°C for 45 s, and 72°C
for 90 s and a final 72°C 10-min step), and rickettsial primers Rs16S354 and
Rs16S647 (this report) (249 bp; cycling conditions of 95°C for 1 min, then 30
cycles of 95°C for 1 min, 54°C for 1 min, and 72°C for 1 min, and a final 10 min
at 72°C). We used primer pair RpCS.877p and RpCS.1273r (42) to amplify a
381-bp citrate synthase gene fragment with cycling parameters of 95°C for 5 min,
35 cycles of 95°C for 20 s, 48°C for 30 s, and 60°C for 2 min followed by a final
60°C 5-min step. A 432-bp fragment of the 17-kDa genus-common rickettsial
antigen gene was amplified using primer pair Rr17kDA1 and Rr17kDA2 and the
cycling parameters of Williams et al. (53).
RT-PCR assays. RNA was purified from infected and uninfected tick DAE100
cells with the SV total RNA isolation system (Promega). DNase I-treated RNA
samples were stored in H2O at ?70°C. RT-PCR amplifications were performed
using the Access RT-PCR system (Promega) and temperature profiles recom-
mended by the manufacturer. Transcripts of the ompA, gltA, and 17-kDa antigen
genes were amplified with the primers listed in Table 1, electrophoresed through
1.5% agarose gels, and stained with ethidium bromide for visualization by UV
illumination.
SDS-PAGE and Western blot analyses. Rickettsiae were gradient purified by
centrifugation (20,000 ? g for 40 min at 4°C) through 30% diatrizoate (Hypaque
76; Nycomed Inc., Princeton, N.J.). Rickettsiae were washed in Hank’s balanced
salt solution and resuspended in 0.5 M Tris-HCl (pH 6.8), and approximate
protein concentrations were determined by UV spectrophotometry. Sixty-five
micrograms of denatured proteins per well was separated by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) through 7.5% mini-gels and stained with Rapid
Coomassie blue (Diversified Biotech, Boston, Mass.). For Western blot analyses,
proteins were transferred to an Immobilon-P membrane (Millipore Corporation,
Bedford, Mass.) (30) and reacted with mouse monoclonal antibody (MAb) 13-5
to rickettsial OmpA (1) diluted 500-fold in phosphate-buffered saline with 3%
bovine serum albumin or hamster polyclonal anti-Rickettsia monacensis serum
diluted 300-fold (45). Bound primary antibodies were detected using horseradish
peroxidase-conjugated goat anti-mouse or anti-hamster immunoglobulin G di-
luted 1:1,000 and the 4CN membrane peroxidase system (Kirkegaard & Perry
Laboratories, Gaithersburg, Md.).
TABLE 1. Primers used for PCR amplification and sequencing
Gene and primerNucleotide sequence (5?-3?) Gene positionc
Reference
ompA
190–(?110)a
190–70pa
190–271a
190–485b
190–602nb
190–701a
190–3588a
190–3817b
190–3968b
190–4388a
190–4406a
190–4710b
190–4859b
190–5125a
190–5238a
190–5561b
190–5768b
190–5917a
190–6013a
190–6427b
190–6585b
190–6808a
190–6984a
GCAACAAGGCCAACACCGC
ATGGCGAATATTTCTCCAAAA
CTGCAGCCGTTATCTCATTCC
GCAAAAGCTTAACTTTAAA
AGTGCAGCATTCGCTCCCCCT
GTTCCGTTAATGGCAGCATCT
AACAGTGAATGTAGGAGCAG
CTGCAACTGTTCCTATAG
TAGCAGCTGATTTAGTAGCT
TTCAGGAAACGACCGTACG
ACTATACCCTCATCGTCATT
AACATTTACATTGTTATTTA
GCGAAATCCAAGGTACAGG
GCGGTTACTTTAGCCAAAGG
ACTATTAAAGGCTAGGCTATT
CGGTTGTCATTATTAATAG
CACCGCTACAGGAAGCAGAT
TCAGGGAATAAAGGTCCTG
TCTTCTGCGTTGCATTACCG
ATCTAAGCCCAGCTAGCGGT
CGCAATGGTCGATTATGC
CACGAACTTTCACACTACC
TGCAAGGAAATTACGAAGTAAGTG
(?110)–(?92)d
70–90
271–251
485–503
602–582
701–681
3588–3607
3817–3800
3968–3986
4338–4356
4406–4387
4710–4690
4859–4877
5125–5144
5238–5218
5561–5543
5768–5787
5917–5935
6013–5994
6427–6408
6585–6602
6808–6790
6984–6961
36
39
This report
This report
39
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
This report
17-kDa antigen
Rr17kDa1a
Rr17kDa2a
GCTCTTGCAACTTCTATGTT
CATTGTTCGTCAGGTTGGCG
31–51
463–444
3
3
Citrate synthase
RpCS.877pa
RpCS.1273ra
GGGGACCTGCTCACGGCGG
CATAACCAGTGTAAAGCTG
877–895
1273–1255
42
42
16S rRNA
Rs16S354a
Rs16S647a
E45a
E1242a
F5a
F11a
CAGCAATACCGAGTGAGTGATGAAG
AGCGTCAGTTGTAGCCCAGATG
GCTTAACACATGCAAG
CCATTGTAGCACGTGT
CCTTTTTGAGTTTCGCTCC
TACCAGTTGGAAACGACTGT
357–381
697–676
45–60
1242–1227
1290–1272
149–168
This report
This report
32
32
13
13
aPrimers used for PCR amplification and sequencing of amplicons.
bPrimers used only for sequencing.
cGene positions are relative to those reported for R. rickettsii (see text).
dSequence within putative promoter region.
6630 BALDRIDGE ET AL.APPL. ENVIRON. MICROBIOL.

Page 4

Double fluorescence staining to assess actin tail formation. IDE8 tick cells
(ATCC CRL-11974) grown to confluency on glass coverslips in 24-well plates
were inoculated with dilutions of host cell-free R. rickettsii Hlp#2 or DAE100
cells infected with R. peacockii and incubated in a candle jar at 34°C for 7 days
(45). Coverslips were fixed, and rickettsiae were labeled with mouse MAb 13-2 to
the rickettsial 120-kDa surface antigen OmpB (1), diluted 1:100, and anti-mouse
immunoglobulin G conjugated to fluorescein isothiocyanate (Pierce, Rockford,
Ill.), while tick cell F-actin was labeled with rhodamine-conjugated phalloidin
(Molecular Probes, Eugene, Oreg.) as described elsewhere (19, 24). Coverslips
were mounted on microscope slides in phosphate-buffered saline containing 3%
bovine serum albumin, 10% glycerol, and 10% (wt/vol) 1,4-diamino-bicyclo
(2,2,2)octane (Sigma) and viewed using a Nikon E400 microscope fitted with
dual fluorescence illumination and a Nikon DX1200 digital camera.
Nucleotide sequence accession numbers. GenBank accession numbers for the
rickettsial ompA sequences reported in this paper are as follows: R. peacockii
Skalkaho, AY357765 and AY357764; R. peacockii Crestone, AY357766 and
AY357763; R. peacockii Rustic, AY319292 and AY319291; R. rickettsii Hlp#2,
AY319293 and AY319290. Accession numbers for the partial sequences of the
16S rRNA gene for R. peacockii Crestone and Skalkaho strains are AY360093
and AY360094, respectively; for Hlp#2 the partial 16S rRNA sequence acces-
sion number is AY573599. The accession number for partial 17-kDa antigen
gene sequences of R. rickettsii strain Hlp#2 is AY189818, and those for R.
peacockii Crestone and Skalkaho are AY576905 and AY590153, respectively.
The partial gltA sequence accession numbers for R. peacockii strains from Cre-
stone and Skalkaho are AY576904 and AY590152, respectively, and for R.
rickettsii strain Hlp#2 it is AY189819.
RESULTS
R. peacockii is present in Colorado and Montana tick pop-
ulations. We detected R. peacockii in D. andersoni collected in
Crestone, Colo., and the Bitterroot Valley of Montana by PCR
and RFLP analysis (Table 3). Amplification of a 1,157-bp frag-
ment with eubacterial 16S rRNA gene-specific primers indi-
cated the presence of bacteria in all ticks, and all samples
tested with Francisella 16S rRNA gene primers generated the
expected 1,123-bp product (data not shown), suggesting they
harbored the DAS endosymbiont (26). We sequenced 341 nu-
cleotides of the 16S rRNA gene (nucleotides 357 to 697, using
the numbering of Roux and Raoult for R. rickettsii [41]). Se-
quence alignment of the three R. peacockii strains (Skalkaho,
Crestone, and DAE100R) with those of R. rickettsii strains
Hlp#2 and R showed that all were identical. Seventy-eight
percent of Crestone tick DNA samples (Table 3) yielded
TABLE 2. GenBank accession numbers of Rickettsia ompA sequences used in the phylogenetic analysis
Species Strain
GenBank accession no.
Prerepeat (5?)Postrepeat (3?)
R. rickettsii
R. rickettsii
R. peacockii
R. peacockii
R. peacockii
R. japonica
R. massiliae
R. montanensis
R. rhipicephali
Bar29
R. aeschlimanii
“R. slovaca”
R. honei
“R. mongolotimonae”
R. parkeri
R. africae
Strain S
Israeli tick typhus
Astrakhan fever rickettsia
R. conorii
R. heilongjiangii
R. hulinii
R. sibirica
R. felis
R. australis
Ixodes ricinus rickettsia
Ixodes ricinus rickettsia
RT
HIp2
Skalkaho
Crestone
Rustic
YM
MtulT
ATCC VR-611 (M5/6)
3-7-6
Bar-29
MC-16T
13-B
TT-118
HA-91
Maculatum 20
ESF-5 (2500-1)
S
ISTT CDC1
A-167
Malish ATCC VR-613T
HLJ-054
HL-93
246, ATCC VR-151T
URRWXCal2
PHS
IRS3
IRS4
M31227
AY319293
AY357765
AY357766
AY319292
U43795
U43799
U43801
U43803
U43792
U43800
U43808
U43809
U43796
U43802
U43790
U43805
U43797
U43791
U43806
AF179362
AF179364
U43807
AF210694
AF149108
AF141909
AF14911
M31227
AY319290
AY357764
AY357763
AY319291
U83442
U83445
U83447
U83450
U83438
U83446
U83454
U83456
U83439
U83449
U83436
U83452
U83441
U83437
U83453
AF179363
AF179366
U83455
AF231136
AF149108
AF141910
AF141912
T
TABLE 3. D. andersoni females PCR or RFLP positive for bacteria, Rickettsia, and Francisella species
Site
% Positive (no. positive/no. tested)
PCRRFLP
Bacteria (16S) Rickettsia spp. (ompA) Francisella sp. (16S)R. peacockii R. rickettsii
Crestone
East side
West side
100 (9/9)
100 (13/13)
100 (16/16)
77.8 (7/9)
68.8 (11/16)
12.5 (2/16)
100 (6/6)
100 (6/6)
100 (6/6)
77.8 (7/9)
66.7 (10/15)
6.3 (1/16)
0 (0/9)
0 (0/9)
6.3 (1/16)
VOL. 70, 2004 R. PEACOCKII ompA GENE ANALYSIS6631

Page 5

532-bp ompA products with primers Rr190.70p and 190.602n
(data not shown), indicating infection with SFGR. The RFLP
analysis, as shown for ticks DaC 5 and 6 (Fig. 2A), produced
the pattern (RsaI, uncut; PstI, 120, 160, and 250 bp) typical of
the ompA fragment from R. peacockii strains Skalkaho (28)
and DaE100R (46). Similarly, 69% of D. andersoni ticks col-
lected on the east side of the Bitterroot Valley carried R.
peacockii based on PCR and RFLP analyses, as shown for
extracts DaEs 6, 8, and 10 (Fig. 2B), whereas only two D.
andersoni ticks, DaWs 9 and 12, from the west side of the
Bitterroot Valley tested PCR positive for ompA. DaWs9 had
an RFLP pattern identical to that of R. rickettsii Hlp#2 (RsaI,
220, 220, and 106 bp; PstI, 266, 205, and 80 bp) (Fig. 2C) but
an AluI digest that was indicative of an R. rickettsii R-like strain
rather than Hlp#2 (data not shown) (16). The RFLP pattern
of the DaWs12 and R. peacockii ompA PCR products were
identical (Fig. 2D).
We sequenced R. peacockii ompA PCR products, including
the ompA promoter (bp 1 to 69), the protein coding region
upstream of the tandem repeats (bp 70 to 680), and the coding
region beginning just downstream of the repeats and continu-
ing through the transcription terminator (bp 3608 to 6960)
(Fig. 1). Sequences from R. peacockii Rustic (DaE100R) (46)
and ticks collected in Montana (east side, Skalkaho ticks
DaEs1, 3, and 4) and in Colorado (Crestone ticks DaC3 and 8)
were identical except that one sample (DaEs1) had a G-to-A
transition at bp 455. In contrast to R. rickettsii, R. peacockii
Rustic (DaE100R) and tick-derived R. peacockii sequences all
contained a deletion of G at bp 403 and insertions of GT and
A following bp 4872 and 5828, respectively, resulting in pre-
mature stop codons.
DNA and translated sequence comparisons of R. peacockii
and R. rickettsii ompA PCR products. We compared the R.
rickettsii R and Hlp#2 ompA sequences to those of R. peacockii
DaE100R and tick extracts. Sequences of R. rickettsii Hlp#2
and R. peacockii (all strains) were 99.7 and 98.1% identical to
R. rickettsii R and 98.3% identical to each other over the total
of 4,032 bp. In order to compare the R. peacockii OmpA
protein sequence to those of R. rickettsii R and Hlp#2, we
reconstructed the open reading frame by restoring G at bp 403
and removing the insertions at bp 4872 and 5828. The recon-
structed R. peacockii OmpA sequence was 95.9 and 96.1%
identical to those of R. rickettsii R and Hlp#2, which were
99.5% identical.
Phylogeny of SFGR ompA sequences. Both the neighbor-
joining and maximum parsimony phylogenetic analyses re-
vealed four major branches among the SFGR. Three of these
branches, as shown in the unrooted phylogram (Fig. 3), were
comprised of R. felis, R. australis, and the IRS3 and IRS4
rickettsiae, demonstrating how divergent their ompA se-
quences are. The remaining SFGR clustered together on the
fourth branch, a direct consequence of their more-conserved
ompA sequences (the inset phylogram in the figure is an ex-
panded view of the fourth branch using R. felis as outgroup).
Within this cluster, five distinct monophyletic groupings with
bootstrap supports of 96% or better were obtained (Fig. 3,
inset phylogram). One group contained R. rickettsii R and
Hlp#2 and R. peacockii (98% bootstrap support), a second
comprised R. rhipicephali, R. aeschlimannii, Bar 29, and R.
massilae (100%), while R. japonica, R. hulinii, and R. hei-
longjiangii clustered together in a third group (100%). R. mon-
golotimoniae, R. sibirica strain S, R. africae, and R. parkeri
formed another group (99%). A fifth group contained R. cono-
rii, the Israeli tick typhus, and the Astrakhan fever rickettsiae
(98%). R. montanensis, R. slovaca, and R. honei did not cluster
with any other rickettsiae. Maximum parsimony and neighbor-
joining analyses using the IRS as the sister outgroup and de-
leting R. felis and R. australis gave the same groupings with
similar bootstrap values (data not shown).
Analysis of gltA and 17-kDa antigen PCR products. Analysis
of the gltA and 17-kDa antigen gene sequences corroborated a
close relationship of R. peacockii and R. rickettsii based on
ompA and 16S rRNA gene sequences. Partial gltA gene se-
quences of all three R. peacockii strains were identical. All
strains of R. peacockii had gltA sequences that differed from R.
rickettsii strain Hlp#2 by 1 nucleotide (at position 998, based
on the numbering of Roux et al. [42] for strain R) and from
strain R by an insertion of 3 nucleotides (between nucleotides
1024 and 1025) and a point mutation (at nucleotide 1026).
Partial 17-kDa antigen gene sequences of all R. peacockii
strains were identical to each other and those for R. rickettsii
strains Hlp#2 and Sheila Smith.
Transcriptional regulatory element sequence comparison
and RT-PCR assays. The R. rickettsii R and Hlp#2 promoter
sequences were identical, but those from R. peacockii and ticks
DaEs1 (Montana) and DaC3 and -8 (Colorado) contained a
T-to-C transition at bp 12 and a G-to-T transversion at bp 54
(also present in the R. conorii sequence; GenBank accession
FIG. 2. RFLP analysis of the rickettsial ompA gene 532-bp PCR
product amplified from purified rickettsiae (DAE100R, Rustic) and D.
andersoni tick extracts. PCR products were left uncut (U) or were
digested with RsaI (R) or PstI (P). A 100-bp DNA size marker ladder
is shown on the left of each panel. (A) R. peacockii strain DaE100R
(Rpea) and Crestone tick 5 and 6 extracts (DaC5 and 6). (B) R.
peacockii (Rpea) and Montana Bitterroot Valley east side tick extracts
(DaEs 6, 8, and 10). (C) R. rickettsii Hlp#2 (HlP#2), R. peacockii
(Rpea), and Bitterroot Valley west side tick 9 extract (DaWs9).
(D) Bitterroot Valley west side tick 12 extract (DaWs12) and R. pea-
cockii (Rpea).
6632BALDRIDGE ET AL.APPL. ENVIRON. MICROBIOL.