Hindawi Publishing Corporation
International Journal of Microbiology
Volume 2010, Article ID 760819, 9 pages
CharacterizationofpPCP1 Plasmids in Yersiniapestis Strains
Isolatedfrom theFormerSoviet Union
1Emerging Pathogens Institute and Department of Molecular Genetics and Microbiology, University of Florida College of Medicine,
Gainesville, FL 32610, USA
2National Center for Disease Control and Public Health, Georgian Ministry of Health, 0177 Tbilisi, Georgia
3Tbilisi State University, 0218 Tbilisi, Georgia
4Naval Medical Center Annex, Rockville, MD 20852, USA
5US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD 21010, USA
Correspondence should be addressed to Chythanya Rajanna, firstname.lastname@example.org
Received 23 July 2010; Revised 26 October 2010; Accepted 15 November 2010
Academic Editor: Todd R. Callaway
Copyright © 2010 Chythanya Rajanna et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
and compared with those of pPCP1 plasmids in three well-characterized, non-FSU Y. pestis strains (KIM, CO92, and 91001). Two
of the FSU plasmids were from strains C2614 and C2944, isolated from plague foci in Russia, and one plasmid was from strain
C790 from Kyrgyzstan. Sequence analyses identified four sequence types amongthe six plasmids. The pPCP1 plasmids in the FSU
strains were most genetically related to the pPCP1 plasmid in the KIM strain and least related to the pPCP1 plasmid in Y. pestis
91001. The FSU strains generally had larger pPCP1 plasmid copy numbers compared to strain CO92. Expression of the plasmid’s
plagenewassignificantly(P ≤ .05)higherinstrainC2944thaninstrainCO92.Given pla’s rolein Y.pestisvirulence, thisdifference
may have important implications for the strain’s virulence.
Yersinia pestis, the causative agent of Black Death, is a highly
virulent bacterium responsible for an estimated 200 million
human deaths throughout recorded history. The bacterium
is believed to have evolved from the much less virulent
Y. pseudotuberculosis relatively recently on the evolutionary
scale, approximately 1,500–20,000 years ago . In most
of the developed world, the genetic organization, virulence
mechanisms, and life cycle of Y. pestis have been extensively
studied using a few Y. pestis strains , some of whose
genomes have been fully sequenced [3, 4]. In contrast, there
remains a striking paucity of data concerning the genetic
organization, virulence traits, prevalence, and epidemiology
ofY. pestis in otherparts ofthe world, includingthe Republic
of Georgia and other republics of the former Soviet Union
The evolution of Y. pestis, and much of its virulence, is
due to its acquisition of plasmids. Three plasmids (pCD1,
pFra, and pPCP1) are typically present in all biovars of
Y. pestis although additional plasmids, many of which are
cryptic, have been reported [8–11] to be present in several
worldwide strains of Y. pestis. pCD1 (also designated pCad,
pLcr, pVW, and pYV) is a 68 to 75kb plasmid found
in all three currently recognized pathogenic species of
Yersinia: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica
[9, 12]. It contains genes encoding several essential virulence
determinants, including the highly conserved low-calcium
2International Journal of Microbiology
and antihost functions and the Yersinia outer membrane
The pFra plasmid (also designated pYT and pMT1) is
unique to Y. pestis. It is typically ca. 100-kb, but its size can
vary drastically among various strains, ranging from 60kb in
the deleted version of the Dodson strain  to 280-kb in
other strains . The plasmid’s role in Y. pestis virulence is
not fully understood, but it is known to contain genes that
encode two putative virulence factors: (i) the F1 protective
antigen associated with increased resistance to phagocytosis
by monocytes and (ii) the Y. pestis murine toxin (YMT),
a phospholipase D encoded by ymt, whose intracellular
activity protects Y. pestis from digestion in the flea gut;
thus, facilitating the bacterium’s ability to colonize the flea’s
midgut and to increase its arthropod-borne transmission
pPCP1 (also designated pPla, pYP, and pPst) is another
plasmid unique to Y. pestis and has a size of approximately
9.6kb. In addition to possessing a few regulatory genes
and genes encoding “hypothetical proteins,” it contains
pla, pst, and pim, which encode a plasminogen activator
(PLA) protease, the bacteriocin pesticin, and a pesticin
immunity protein, respectively . Its size and genetic
organization are usually similar among various strains of
Y. pestis although some isolates of the bacterium may lack
the plasmid or may contain a multimer of the plasmid
[8, 14, 16]. pPCP1 is an important virulence determinant in
protease virulence factor. However, despite the importance
of pPCP1 and other plasmids for Y. pestis virulence, there
is a lack of information about the genetic structure of the
plasmids in FSU strains. During the studies reported in this
communication, we (i) determined the complete sequences
of pPCP1 plasmids in three Y. pestis strains isolated from the
FSU and examined in a recent study , (ii) compared their
nucleotide sequences with those of pPCP1 plasmids in three
well-characterized, non-FSUstrains, and(iii)determinedthe
relative pPCP1 plasmid copy number of three FSU Y. pestis
2.1. Bacterial Strains. pPCP1 plasmids were extracted from
bacteria in a recentlycharacterized FSU Y. pestis strain
collection . The pPCP1 plasmid from strain CO92 was
used as a control. Multiple small specimens of each strain
were stored at −80◦C in 70% Luria-Bertani (LB) broth
supplemented with 30% glycerol (v/v), and each aliquot was
used only once before being discarded.
2.2. Multilocus Variable Number Tandem Repeat (MLVA)
Analysis. Our MLVA analysis used a simple 7-polymorphic
marker (ms01, ms04, ms06, ms07, ms46, ms62, and ms70)
protocol previously described by Pourcel et al. 2004 .
The primers used for PCR amplification are listed in Table 1
(supplementary material). The PCR reactions were per-
formed with aliquots of purified chromosomal DNA (50μL
containing 1 to 2ng) and Choice-Taq Blue DNA polymerase
(Denville Scientific, Metuchen, NJ). The sequential reaction
conditions were (i) 96◦C for 5 minutes, (ii) 34 cycles of
denaturation (96◦C, 20 seconds), (iii) annealing (54◦C, 30
seconds), (iv) elongation (72◦C, 1 minute), and (v) a final
extension step for 5 minutes at 65◦C. The PCR products
were purified with a Qia Quick 96-well Plate Kit (QIA-
GEN, Valencia, CA), and the purified PCR products were
sequenced in both directions with a BigDye 200 Terminator
Cycle Sequencing Kit and a 201ABI 3730xl 202DNA ana-
lyzer (Applied Biosystems, Foster City, CA). The individual
sequences were analyzed with a Tandem Repeat Finder
(TRF) program (http://tandem.bu.edu/trf/trf.html) and the
number of repeats for each marker was determined.
Well-isolated colonies of each strain were obtained by
streaking and incubating (28◦C) the bacteria on brain heart
infusion (BHI) agar. After 48 hours, 5mL aliquots of BHI
broth were inoculated with well-isolated colonies of the
strains and incubated at 28◦C for 24 hours with agitation
(200rpm). DNA was extracted from the bacteria in 3mL
aliquots from each broth culture, and the pla, pim, and
pst genes were PCR-amplified using primers plaF1, plaR1,
pimF, pimR pstF, and pstR (Supplementary Material Table 1
availableatdoi:10.1155/2010/760819),which amplified 470-
, 201-, and 197-bp regions of the respective genes. The
cultures which were PCR-positive for all three genes were
streaked on BHI agar and incubated at 28◦C for 24 hours.
Aliquots of 100mL BHI were inoculated with one loopful
of culture from the streak plates. After incubation with
agitation (200rpm) at 28◦C for 24 hours the bacteria were
collected by centrifugation, and plasmid DNA was extracted
with a QIA filter Plasmid Midi Kit (QIAGEN) according
to the manufacturer’s instructions, and the DNA specimens
were characterized by agarose gel (1%, w/v) electrophoresis.
The reproducibility of the data was confirmed by repeating
the experiments at least twice for each strain.
2.4. Sequence Comparison and Confirmation pMT1-pCD1
Chimera Plasmid. To determine the nature of atypical
plasmid seen in C790, we obtained 454 sequence of the
plasmid and genomic DNA and compared this sequence
to the respective plasmids of CO92 genome (Sozhaman-
nan laboratory, unpublished results). To confirm the
atypical plasmid as a chimera of pMT1 and pCD1, PCR
primers sulk560 (ACTCACGCAGCGTATCTTCC, pMT1)
and sulk561 (ATTCTCTGTCGTTCGGCTTG, pCD1) were
designed to amplify across the junction by PCR.
sequences of the extracted pPCP1 plasmids were determined
by the primer walking approach. The primers used for
sequencing are listed in Table 1 (Supplementary Material).
The plasmids were sequenced, in both directions, with a
BigDye 200 Terminator Cycle Sequencing Kit and a 201 ABI
3730xl 202DNA analyzer (Applied Biosystems). Contigs
were assembled with Phred  and Phrap (available
at http://www.washington.edu/) programs. The contigs
Sequencing the pPCP1 Plasmids. The nucleotide
International Journal of Microbiology3
were viewed with the Consed program , and the
resulting DNA sequences were trimmed by removing
low-quality nucleotide sequences from the end. The
sequences were aligned by the Sequencher 4.7 program. The
final sequences were compared with those of previously
characterized pPCP1 plasmids in three non-FSU Y. pestis
strains (KIM, CO92, and 91001). The latter sequences were
available from the Institute of Genomic Research (TIGR)
database (http://www.tigr.org/). The genetic relatedness
among the plasmids was determined by the neighbor joining
tree method, using the BLOSUM62 of EMBL-EBI ClustalW2
2.6. Quantitative PCR of DNA and RNA. For extracting
genomic DNA and total RNA, cultures were grown over
night in 5ml BHI broth at 28◦C shaking at 200rpm after
inoculating fresh well isolated colony from a BHI agar plate.
For total RNA extraction 5ml of fresh BHI media was
inoculated with 500μl of overnight culture and incubated
shaking (200rpm) at 28◦C for 4h.
Total DNA was extracted with a Genomic DNA Purifi-
cation Kit (Promega, Madison, WI), and the DNA concen-
tration in the extracts was adjusted to 100ng/μL. Primers
for the pla gene (plaF2 and plaR2, Supplementary Mate-
rial Table 1) were used to estimate the copy number of the
pPCP1 plasmid, and glnF and glnR primers (Supplementary
Material Table 1) for the chromosomal, single-copy glnA
gene were used as the control. A standard curve (for
was constructed in order to estimate the copy number of
pla. An iQ Syber Green Kit (BioRad, Hercules, CA) was
used for real-time PCR (RT-PCR), and the ratio of the pla
starting quantitytoglnA starting quantitywascalculatedand
compared among the strains. The experiment was repeated
two times and the average values were estimated.
Total RNAwas extracted with an RNEasy Mini Kit (QIA-
GEN), and contaminating DNA was removed by passing
the extracts through a gel matrix containing bound RNase-
free DNase (QIAGEN). The RNA concentration in the
extracts was adjusted to approximately 50ng/μL, and 100ng
aliquots in 25μL reaction mixtures were reverse-transcribed
with an iScript cDNA Synthesis Kit (BioRad). RT-PCR
was performed with reaction mixtures (25μL) containing a
template composed of 2μL of the cDNA preparations. The
relative expression of pla to the expression of the reference
gene (glnA) was determined using the 2−ΔΔCT method
. Statistical analyses of each set of the pla expression
data were performed separately, with the GraphPad InStat
(version 3.05) program (GraphPad Software, San Diego,
CA). An unpaired t-test was used to determine whether the
differencesobservedin pla expression among thefour strains
analyzed (C790, C2614, C2944, and CO92) were statistically
significant. A P-value <.05indicated a statistically significant
difference between the results.
2.7. Nucleotide Sequence Accession Numbers. Complete
sequences of the three pCP1 plasmids described in this
study (extracted from FSU strains C790, C2944, and C2614)
have been deposited in GenBank, under accession numbers
BankIt1374063 C790 HM807366, BankIt1374063 C2614
HM807367, BankIt1374063 C2944 HM807368, and the
partial sequence of C790 chimera plasmid of pMT1 and
pCD1 BankIt1408393 Chimera HQ612242.
3.1. Genetic Relatedness of FSU Y. pestis Strains. A recent
publication  describing the forty-six FSU Y. pestis strains
isolated from the Republic of Georgia and surrounding
countries used biochemical profiling, pulsed field gel elec-
to determine the genetic relationships between Georgian
Y. pestis strains and Y. pestis strains from neighboring
countries and other parts of the world. It was found that
the Georgian Y. pestis strains were of clonal origin and that
PFGE discriminated the Y. pestis strains better than did
MLST. In the present study, we expanded this investigation
by analyzing the same strain collection with MLVA, which
bacterial pathogens, including Y. pestis [17, 21, 22]. The
results of the overall MLVA analysis were in agreement with
those of the PFGE and MLST analyses: MLVA grouped
the Georgian strains in three major clusters (Figure 1 and
Supplementary Material Table 2), except for strain 771G
which was a clear outlier; this strain also clustered differently
during previous studies . Further, in agreement with the
reports mentioned above, we found that MLVA differen-
tiated the strains with greater sensitivity than PFGE and
MLST. Specifically, MLVA was able to discriminate between
Georgian Y. pestis strains that were grouped in a single
cluster by MLST and PFGE in the previous study by ;
for example, Y. pestis strains 8787G, 3757G, and 1412G were
unresolved by PFGE typing, but they were differentiated by
MLVA (Figure 1), which suggests that MLVA is better suited
for specific identification of FSU Y. pestis strains than PFGE
3.2. Prevalence of the pPCP1 Plasmid in FSU Strains. Some of
the Y. pestis strains isolated from the FSU, including those
isolated in the Transcaucasian and Daghestan Mountains;
that is, two natural foci of plague adjacent to the areas
from which our Y. pestis strains were isolated , have been
reported not to contain the pPCP1 plasmid . Many,
but not all of these strains, are thought to belong to the
so-called Pestoides biovar of rhamnose-fermenting Y. pestis
strains commonly found in enzootic hosts, but not usually
associated with human infections. Also,Yersinia plasmids are
unstable and may beeasily lost during prolongedstorageand
passaging. The Y. pestis strains in our collection were isolated
during 1966–1997 , and they were regularly passaged
during their storage, which could have further facilitated
plasmid loss. Therefore, we first screened the FSU strains
of Y. pestis by pPCP1-specific PCR (using the primers listed
in Supplementary Material Table 1), in order to identify the
strains in which the plasmid was still present. Only three of
the forty-six FSU strains we examined (C2614, C2944, and
4International Journal of Microbiology
Figure 1: Neighbor-joining tree generated from MLVA data based
on 7 loci.
C790) gave strong specific positive PCR signals for all the
three genes (pla, pim, and pst) known to be contained in the
pPCP1 plasmid. Also, the buffer control and Y. enterocolitica
strain ATCC 9610 (both negative controls) did not yield
the PCR amplicons. The results of the pPCP1-targeted PCR
screeningstudysuggestedthatmany oftheFSUstrains inour
collectiondonotcontainthe pPCP1plasmid, an observation
that was further verified by direct plasmid extraction and
other approaches described below. Even though in our
previousreport  we did report all the strains isolated from
Republic of Georgia to posses pla gene, in this study, we find
only 3 strains to posses the plasmid. We are still investigating
the possible cause for this discrepancy and certainly will
address in our future publications. Some possible reasons
why this could have happened are 1. different batches of
strains were used in each of this study, and there could be
difference in plasmid composition of strains depending how
they were handeled and shipped to us 2. there could have
been lowlevelcontamination with apla positive strain/ DNA
in our first batch of strains.
3.3. Plasmid Extraction and Copy Numbers of pPCP1. Efforts
were made to extract the pPCP1 plasmid from all of the
forty-six FSU Y. pestis strains in the collection, including
three strains that gave positive signals for pla, pim, and pst.
The pPCP1 containing, non-FSU strain CO92 was used as
the positive control during plasmid isolation. Plasmid DNA
was obtained from all three PCR-positive strains, but not
from the forty-three Y. pestis strains that were pla, pim,
and pst PCR negative, which supports our PCR screening
data and indicates that PCR-based screening is useful for
detecting the presence of the pPCP1 plasmid in Y. pestis
strains. The presence of the pPCP1 plasmid appeared to
be limited to the C2614-C2944 MLVA cluster and closely
related clusters (Figure 1). It is possible that the strains in
those clustersare inherently more stable in maintaining their
plasmid composition; however, it is also possible that the
strains in the most common cluster never contained that
plasmid. Because of the known laboratory passage histories
of these strains, resolution of this question may be resolved
by ongoing efforts in the FSU to discover new epizootic
strains. Moreover,thedearth ofplasmid-positive strains does
not allow for rigorous analysis of any possible association
between plasmid-containing strains and their distribution
among various MLVA groups or clusters. The plasmid yield
fromthethree PCR-positiveFSUstrains and fromCO92that
varied even though the strains were grown under identical
conditions and their concentrations (CFU/mL) prior to
plasmid extraction were about the same: approximately 250
to 300μg of plasmid DNA was consistently obtained from Y.
pestis strain C790 compared to around 350 to 450μg from
strains C2614, C2944, and CO92. Interestingly, FSU strain
was larger than the typical 96-kb pPMT1 plasmid (Figure 2).
When 454 whole-genome sequence (WGS) output of C790
was compared to CO92 plasmid sequences, the alignment
indicated that the atypical plasmid is in fact a chimera of
pMTI and pCD1 with one junction being at NC 003131.1,
position 65224and NC 003134.1,position 73221.Individual
sequencing reads that spanned this junction were clearly evi-
dent, and this conclusion was verified by PCR amplification
of the expected 659pb PCR product in C790 and but not
in CO92 (Figure 4). We also notice a faint band in strain
C2944 suggesting it may also have a chimera plasmid in low
abundance along with typical pCD1 and pMT1 plasmids.
The idea of the atypical plasmid being a chimera plasmid is
supported by the fact that bothlcrV (encodedon pCD1)and
junction was not identified by WGS. Because of the tendency
we believe that the second junction is likely to lie in one of
International Journal of Microbiology5
LR PFG marker
MR PFG marker
Figure 2: Pulsed field gel electrophoresis of total plasmid content
on 1% agarose gel. Lane 1: low range PFG marker, lane 2: C790,
lane 3: CO92, lane 4: C2614, lane 5: C2944 and lane 6: medium
range PFG marker.
the large, highly repetitive IS100 elements that are found on
all three plasmids and the chromosome. Identification of the
second junction point, the full description of the chimera
and its effect on pathogenesis will be the subject of future
studies. Chimeric plasmids resulting from recombination
between homologous sequences of Y. pestis plasmids have
been documented; for example, the deep-rooted Angola
strain contains a dimeric pPCP1 plasmid that is integrated at
an IS100 element in tandem repeats into the pMT1 plasmid
Quantitative PCR (qPCR) was used to verify the dif-
ference in the pPCP1 plasmid’s copy numbers among the
three FSU strains (C790, C2614, and C2944) and CO92, by
comparing the pla/glnA gene ratios among them as there
is only one copy of glnA in each cell . The mean of
three independent qPCR reactions (Table 1) showed that
the highest copy number of pla in strain C2944, in which
the pla/glnA ratio of 18.16 ± 2.99 suggested that there
were approximately eighteen copies of the pla gene (and of
the pPCP1 plasmid) in that strain. The next highest copy
number was detected in Y. pestis strain C790, in which the
pla/glnA ratio was 15.01 ± 1.14, followed by strain C2614
which had a ratio of 13.21 ± 0.64. Strain CO92, the virulent
non-FSUstrain thatwasused forcomparison, had apla/glnA
ratio of only 6.48 ± 2.07.
Table 1: Ratio ofpla/glnA genecopy numbers determined by qPCR
and pla gene expression by the Livak method.
15.01 ± 1.14
13.21 ± 0.64
18.16 ± 2.99
6.48 ± 2.07
Relative pla gene expression
1.43 ± 1.03
2.24 ± 1.29
3.15 ± 1.31
Although the absolute copy numbers of the pPCP1
plasmids are difficult to calculate based on the qPCR
results (and the validity of the above qPCR-determined copy
numbers must be interpreted with caution), the combined
data suggest that the three PCR-positive FSU strains contain
a larger number of the pPCP1 plasmid than does the non-
FSU Y. pestis CO92 strain. Specifically, when compared to
strain CO92, the FSU strains consistently had: (i) stronger
PCR amplification signals and (ii) higher copy numbers
when examined by qPCR. The underlying mechanism(s) for
the difference in the copy numbers we observed is difficult
to explain at the present time given the rop gene is identical
in all the 4 strains. However, and as discussed below, it may
have some important implications for the strains’ relative
3.4. Genetic Organization of the FSU’s pPCP1 Plasmids. The
pPCP1 plasmids in the three PCR-positive FSU strains
were approximately 9.61-kb in size and had a G + C
content of 45.3%, which is slightly lower than the overall
G + C content (47.6%) of the KIM and CO92 strains’
chromosomes [3, 4] but is similar to that of the pre-
viously characterized pPCP1 plasmid . The plasmids’
predicted genes encode a putative (i) transposase (1.02kb),
(ii) ATP-binding protein (782bp) that together form the
insertion sequence IS100 along with the inverted repeats,
(iii) replication regulation protein (the 195bp rop), (iv)
transcriptional regulator (the 426bp pim), (v) pesticin (the
1,074bp pst), (vi) plasminogen activator(PLA)protease (the
939bp pla), (vii) transcriptional regulator, and (viii and ix)
two hypothetical proteins (Figure 3). Six of the genes-those
(encoding the transposase, ATP-binding protein, replication
regulation protein, transcriptional regulator, PLA protease,
and one of the two hypothetical proteins are transcribed in
the same direction, and the remaining three genes (those
encoding the transcriptional regulator, pesticin, and the
second hypothetical protein) are transcribed in the opposite
direction (Figure 3).
The genetic organization of the pPCP1 plasmids in the
three FSU strains was similar to that of the pPCP1 plasmids
in the previously characterized non-FSU strains KIM and
CO92,and, to a somewhat lesser degree, strain 91001[9, 25].
Similar to the FSU pPCP1 plasmids, the pPCP1 plasmids
in strains CO92 and KIM also contain nine predicted genes
the pPCP1 plasmid in strain 91001 contains ten predicted
genes, consisting of the same nine genes plus one additional
geneencodinga hypotheticalprotein; (http://www.tigr.org/).
All of the FSU pPCP1 plasmids contain insertion sequence
6International Journal of Microbiology
Variable region 1
Variable region 2
Putative transcriptional regulation
StrainVariable region #1 Variable region #2
ATAAAAAAA -CAATAAGTTAAAAAAAA -TAC
TAACGGCAATT -- TGTACGCACCACTGAAAT
TAACGGCAATT -- TGTACGCACCACTGAAAT
TAACGGCAATT -- TGTACGCACCACTGAAAT
TAACGGCAATT -- TGTACGCACCACTGAAAT
TAACGGCAATT -- TGTACGCACCACTGAAAT
TAACGGCAATT -- TGTACGCACCACTGAAAT
Variable region 2
Variable region 1
Y. pestis C790/C2614/
HP: Hypothetical protein
PTP: Putative transcriptional regulator
ST: Sequence type
Figure 3: Structural comparison of the pPCP1 plasmids in the FSU’s Y. pestis strains and a previously characterized strain Y. pestis CO92.
(a) Schematic linear diagram of the pPCP1 plasmid in strain CO92, which shows the locations of variable regions no. 1 and no. 2 present in
the FSU strains’ pPCP1 plasmids. (b) Sequence variations in variable regions no. 1 and no. 2 in the pPCP1 plasmids examined during our
studies. (c) Schematic composition of three pCP1 plasmids in the FSU’s Y. pestis strains, and their genetic relatedness (based on neighbor
joining tree analysis)to the three pCP1 plasmids from non-FSU strains.
IS100, which has been found in all Y. pestis strains and all
serotype Istrains of Y. pseudotuberculosis, and ishomologous
to IS21, IS232 and IS640 [26, 27].
3.5. Genetic Analysis and Comparison of the pPCP1 Plasmids.
We compared the nucleotide sequences of the pPCP1
plasmids in the three FSU strains to the available sequences
of the pPCP1 plasmids in the four, non-FSU strains.
The sequences of the pPCP1 plasmids in all of the seven
strains were similar except for a few nucleotide changes,
which confirm earlier observations [9, 14] about the general
consistency ofthesize andgeneticorganization ofthepPCP1
plasmid in Y. pestis. Based on the minor sequence differences
we detected, five sequence types (STs) were identified
among the plasmids we analyzed (Figure 3): ST1 contained
three plasmids, two of which were isolated from the FSU
strains C2614 and C2944, and the third plasmid from
the KIM strain. ST2 had 1 plasmid from a strain isolated
from China Z176003. ST3 had 1 plasmid from Krygyztan
strain C790. The pPCP1 plasmids in Y. pestis strains CO92
and 91001 had distinct STs (ST4 and ST5, resp.). The
International Journal of Microbiology7
Figure 4: PCR amplification one of the two junctions between
pMT1 and pCD1 in strain C790 which has pMT1-pCD1 chimera
pPCP1 plasmids in the FSU strains were found to be most
genetically related (by neighbor joining tree analysis) to the
pPCP1 plasmid in the KIM strain, and least related to the
pPCP1 plasmid in strain 91001, which is nonpathogenic for
humans . The small variations in plasmid sequences
were found in two intergenic regions designated variable
region 1 and variable region 2 (Figure 3). Though the
sequence variations are minor, variable region 1 is within
promoter sequence of both pesticin and hypothetical
protein as predictedby
.html). At this point, we do not know how this variation will
affect promoter binding and transcription.
3.6. Analysis of pla Transcript Levels. The observed differ-
ences in pPCP1 copy numbers among the strains prompted
usto questionwhether plasmid copynumbercorrelated with
expression levelsof plaA transcript in several Y. pestis strains.
RNA transcript levels of plaA were determined by RT-PCR,
and expression of the chromosomal glnA gene was used as
the reference point to calculate the relative gene expression
of pla. The results were in general agreement with the qPCR
data revealing that pla expression was significantly (P ≤ .05)
higher in strain C2944 than in strain CO92. The second
highest expression of pla was by strain C2614 (1- to 3.5-
fold more than by strain CO92), followed by strain C790
(0.4- to 2.5-fold more than by strain CO92) although the
differences were not statistically significant (P ≥ .05). Our
data suggest that the three FSUstrains containing the pPCP1
plasmid may producelargeramountsofPLA(and in thecase
characterized Western North American CO92 strain.
3.7. pla and Virulence of Y. pestis . Although some wild-type
Y. pestis strains lacking the pPCP1 plasmid have been report-
edly retained their virulence [29, 30], pPCP1 is believed
to play a major role in Y. pestis virulence. In addition to
regulatory genes and genes encoding hypothetical proteins,
the plasmid contains three genes (pst, pim, and pla) that
encode proteins required for several important biochemical
activities of Y. pestis. However, at the present time, the
PLA protease, a 34.6-kDa, multifunctional plasminogen
activatorprotease capableofdegrading fibrin, coagulase,and
the complement component C3 [9, 25], is the only well-
documented virulence factor encoded by the pPCP1 plasmid
The significance of pla in Y. pestis virulence has been
clearly documented in literature [31, 32], and some pla-
negative mutants have been reported to have greatly reduced
virulence when administered subcutaneously . Various
virulence roles for the PLA protease have been proposed
, including (i) cleaving host fibrin deposits that trap the
and (iii) inhibition of interleukin-8 production. More
recently, pla expression has been reported to be essential for
the development of primary pneumonic plague in virulent
CO92 strain, but not in pPCP1-deficient Pestoides F, in
addition to being required for the bubonic form of plague
and increased potential for epidemic spread [30, 31, 33, 34].
Other studies have implicated Pla in resistance to cationic
antimicrobial peptides such as cathelicidin . In view
of these data, our observation that some FSU strains are
capable of producing significantly larger amounts of PLA
than does the highly pathogenic CO92 raises interesting
questions about the pathogenic potential of these strains. In
this context, and to putour findings into further perspective,
several reports in the “Soviet” literature suggest that Y.
pestis virulence for various laboratory animals, including
guinea pigs, varies dramatically depending on its plasmid
composition and the plague focus in the Caucasus from
which the strains were isolated. For example, Y. pestis strains
isolated in the Leninakan focus (in the Shirak Highlands of
Armenia) were found to bemore virulentin guinea pigsthan
are strains obtained from the Zanzegur-Karabakh region
(southeastern range of the Lesser Caucasus mountains) .
Similar observations have been reported for Y. pestis strains
isolated in the Armenian Highlands , the Dagestan-
Highland focus , and the Gissar and Talas regions .
Data comparing the plasmid compositions, the plasmid
copy numbers, and pla-expression in those strains are not
available. However, given the differences we observed in
the pla-expression levels in various FSU strains, additional
studies seem warranted to determine the impact of pPCP1
copy number and PLA production on the virulence of
the FSU Y. pestis strains. The resulting data would help
to advance understanding of the genetic composition and
virulence traits of the Y. pestis strain population in the FSU
(including FSU regions from which such data are either
8International Journal of Microbiology
very scarce or not available), and they may also aid the
development of advanced methods for differentiating highly
virulent and less virulent strains of Y. pestis.
The research described in this paper was made possible by
grant 1R21 AI055660-01A1 (to A. Sulakvelidze) from the
National Institute of Allergy and Infectious Diseases, and
by financial support provided by the U.S. Defense Threat
Reduction Agency (DTRA) projects GG-1 and GG-18 (to A.
Sulakvelidze).The research performed at the USArmy Edge-
wood Chemical Biological Center was supported by DTRA
project AA06TAS025 (to H. Gibbons). The authors thank
Arnold Kreger for his invaluable editorial assistance, Sonya
Narodny and Richard Obiso for their helpful suggestions,
and the honorable Andrew Weber, Kevin O’ Connell, Gavin
Braunstein, James Bartholomew, and Jay Valdes for their
support of the project.
 M.Achtman,K. Zurth, G. Morelli, G.Torrea, A. Guiyoule, and
E. Carniel, “Yersinia pestis, the cause of plague, is a recently
emerged clone of Yersinia pseudotuberculosis,” Proceedings
of the National Academy of Sciences of the United States of
America, vol. 96, no. 24, pp. 14043–14048, 1999.
 R. D. Perry and J. D. Fetherston, “Yersinia pestis—etiologic
agent of plague,” Clinical Microbiology Reviews, vol. 10, no. 1,
pp. 35–66, 1997.
 J. Parkhill, B. W. Wren, N. R. Thomson et al., “Genome
sequence of Yersinia pestis, the causative agent of plague,”
Nature, vol. 413, no. 6855, pp. 523–527, 2001.
 W. Deng, V. Burland, G. Plunkett et al., “Genome sequence of
Yersinia pestis KIM,” Journal of Bacteriology, vol. 184, no. 16,
pp. 4601–4611, 2002.
 A. P. Anisimov, L. E. Lindler, and G. B. Pier, “Intraspecific
diversity of Yersinia pestis,” Clinical Microbiology Reviews, vol.
17, no. 2, pp. 434–464, 2004.
 J. L. Lowell, A. Zhansarina, B. Yockey et al., “Phenotypic and
molecular characterizations of Yersinia pestis isolates from
Kazakhstan and adjacent regions,” Microbiology, vol. 153, no.
1, pp. 169–177, 2007.
 T. Revazishvili, C. Rajanna, L. Bakanidze et al., “Characterisa-
Republic of Georgia, and their relationship to Y. pestis isolates
from other countries,” Clinical Microbiology and Infection, vol.
14, no. 5, pp. 429–436, 2008.
 D. M. Ferber and R. R. Brubaker, “Plasmids in Yersinia pestis,”
Infection and Immunity, vol. 31, no. 2, pp. 839–841, 1981.
 S. W. Bearden, J. D. Fetherston, and R. D. Perry, “Genetic
organization of the yersiniabactin biosynthetic region and
construction of avirulent mutants in Yersinia pestis,” Infection
and Immunity, vol. 65, no. 5, pp. 1659–1668, 1997.
 L. P. Bazanova, M. P. Maevskii, and A. V. Khabarov, “An
experimental study of the possibility for the preservation of
the causative agent of plague in the nest substrate of the
long-tailed suslikEksperimental’noe izuchenie vozmozhnosti
sokhraneniia vozbuditelia chumy v substrate gnezda dlin-
nokhvostogo suslika,” Meditsinskaia parazitologiia i parazi-
tarnye bolezni, no. 4, pp. 37–39, 1997.
 A. I. Aragao, A. C. Seoane, T. C. Leal, N. C. Leal, and A. M.
Almeida, “Surveillance of plague in the State of Ceara: 1990–
1999,”RevistadaSociedadeBrasileira deMedicinaTropical, vol.
35, no. 2, pp. 143–148, 2002.
 A. Sulakvelidze, “Yersiniae other than Y. enterocolitica,
Y. pseudotuberculosis, and Y. pestis: the ignored species,”
Microbes and Infection, vol. 2, no. 5, pp. 497–513, 2000.
 R.D.Perry, M.L.Pendrak,andP.Schuetze,“Identificationand
phenotype of Yersinia pestis,” Journal of Bacteriology, vol. 172,
no. 10, pp. 5929–5937, 1990.
 A. A. Filippov, N. S. Solodovnikov, L. M. Kookleva, and O.
A. Protsenko, “Plasmid content in Yersinia pestis strains of
different origin,” FEMS Microbiology Letters, vol. 55, no. 1-2,
pp. 45–48, 1990.
 S. Felek, A. Muszy´ nski, R. W. Carlson, T. M. Tsang, B. J.
Hinnebusch, and E. S. Krukonis, “Phosphoglucomutase of
Yersinia pestis is required for autoaggregation and polymyxin
B resistance,” Infection and Immunity, vol. 78, no. 3, pp. 1163–
 M. Eppinger, P. L. Worsham, M. P. Nikolich et al., “Genome
sequence of the deep-rooted Yersinia pestis strain angola
reveals new insights into the evolution and pangenome of the
plague bacterium,” Journal of Bacteriology, vol. 192, no. 6, pp.
 C. Pourcel, F. Andr´ e-Mazeaud, H. Neubauer, F. Ramisse, and
G. Vergnaud, “Tandem repeats analysisfor the high resolution
phylogenetic analysis of Yersinia pestis,” BMC Microbiology,
vol. 4, article 22, 2004.
 B. Ewing, L. Hillier, M. C. Wendl, and P. Green, “Base-
callingof automatedsequencer traces usingphred. I. Accuracy
 D. Gordon, C. Abajian, and P. Green, “Consed: a graphical
tool for sequence finishing,” Genome Research, vol. 8, no. 3,
pp. 195–202, 1998.
 K. J. Livak and T. D. Schmittgen, “Analysis of relative gene
expression data using real-time quantitative PCR and the 2T
method,” Methods, vol. 25, no. 4, pp. 402–408, 2001.
 Q. Wang, F. Kong, P. Jelfs, and G. L. Gilbert, “Extended phage
locus typing of Salmonella enterica serovar Typhimurium,
using multiplex PCR-based reverse line blot hybridization,”
Journal of Medical Microbiology, vol. 57, no. 7, pp. 827–838,
 J. J. Kingston, U. Tuteja, M. Kapil, H. S. Murali, and H.
V. Batra, “Genotyping of Indian Yersinia pestis strains by
MLVAand repetitive DNA sequence based PCRs,” Antonie van
Leeuwenhoek, International Journal of General and Molecular
Microbiology, vol. 96, no. 3, pp. 303–312, 2009.
 M. Eppinger, Z. Guo, Y. Sebastian et al., “Draft genome
sequences of Yersinia pestis isolates from natural foci of
endemicplague inChina,”Journal of Bacteriology, vol.191,no.
24, pp. 7628–7629, 2009.
 J. M. Alonso, “Epidemiology and epizootiology of plague :
the main place of supervision of wild rodents reservoir in the
control of permanent focusesEpidemiologie et epizootioiogie
de la peste : le role majeur de la surveillance des populations
reservoirs de rongeurs sauvages dans le controle des foyers
inveteres,” Medecine Tropicale, vol. 58, no. 2, pp. 21–24, 1998.
 O. A. Sodeinde and J. D. Goguen, “Genetic analysis of the
Immunity, vol. 56, no. 10, pp. 2743–2748, 1988.
 R. Helmuth, R. Stephan, and E. Bulling, “R-factor cointegrate
International Journal of Microbiology9
strains,” Journal of Bacteriology, vol. 146, no. 2, pp. 444–452,
 O. N. Podladchikova, G. G. Dikhanov, A. V. Rakin, and
J. Heesemann, “Nucleotide sequence and structural organi-
zation of Yersinia pestis insertion sequence IS100,” FEMS
Microbiology Letters, vol. 121, no. 3, pp. 269–274, 1994.
 Y. Han, D. Zhou, X. Pang et al., “Microarray analysis of
temperature-induced transcriptome of Yersinia pestis,” Micro-
biology and Immunology, vol. 48, no. 11, pp. 791–805, 2004.
 G. P. Aparin, S. V. Balakhonov, L. A. Timofeeva, and A. I.
Logachev, “[Numerical analysis of the phenotypic properties
and total genomic characteristics of strains of Yersinia pestis
related to different subspecies],” Zhurnal Mikrobiologii Epi-
demiologii i Immunobiologii, no. 11, pp. 16–20, 1987.
 S.L.Welkos,A.M.Friedlander, andK.J.Davis,“Studies onthe
role of plasminogen activator in systemicinfection by virulent
4, pp. 211–223, 1997.
 W. W. Lathem, P. A. Price, V. L. Miller, and W. E. Goldman,
“A plasminogen-activating protease specifically controls the
developmentofprimary pneumonicplague,” Science,vol. 315,
no. 5811, pp. 509–513, 2007.
 E. A. Lorange, B. L. Race, F. Sebbane, and B. J. Hinnebusch,
“Poor vector competence of fleas and the evolution of hyper-
virulence in Yersinia pestis,” Journal of Infectious Diseases, vol.
191, no. 11, pp. 1907–1912, 2005.
 P. L. Worshamand C. Roy, “Pestoides F, a Yersinia pestis strain
Advances in Experimental Medicine and Biology, vol. 529, pp.
 F. Sebbane, C. O. Jarrett, D. Gardner, D. Long, and B. J. Hin-
nebusch, “Role of the Yersinia pestis plasminogen activator in
theincidence ofdistinctsepticemic andbubonic formsofflea-
borne plague,” Proceedings of the National Academy of Sciences
of the UnitedStates of America, vol.103, no.14, pp. 5526–5530,
 E. M. Galv´ an, M. A. S. Lasaro, and D. M. Schifferli, “Capsular
antigen fraction 1 and Pla modulate the susceptibility of
Yersinia pestis to pulmonary antimicrobial peptides such as
cathelicidin,” Infection and Immunity, vol. 76, no. 4, pp. 1456–
 Y. M. Elkin and P. A. Petrov, “Paleogenesis of Transcaucasian
lence of Yersinia pestis vole’s strains from different landscape-
geographical Caucasus localities,” in Particularly Dangerous
Infections in the Caucasus, pp. 43–45, Stavropol’ Research
Anti-Plague, Stavropol, Russia, 1974.
 G. P. Abgaryan, Characterization of Some Yersinia Pestis Strains
which Were Isolated on Armenian Highland from Common
Voles, All-Union Research Anti-Plague Institute “Microbe”,
Saratovl, Russia, 1966.
 A. I. Diatlov, Evolutional Aspects in Natural Plague Focality,
Stavropol Bookish Press, Stavropol, Russia, 1989.
 A. A. Sludskii, Vole’s Type of Natural Plague Foci (Structure
and Functioning), Russian Research Anti-Plague Institute
“Microbe”, Saratovl, Russia, 1998.