Genome Res. 13:2018-2029, 2003
©2003 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/03 $5.00
Letter
Application of DNA Microarrays to Study the Evolutionary Genomics of Yersinia pestis and Yersinia pseudotuberculosis
Stewart J. Hinchliffe1,
Karen E. Isherwood2,
Richard A. Stabler3,
Michael B. Prentice4,
Alexander Rakin5,
Richard A. Nichols6,
Petra C.F. Oyston2,
Jason Hinds3,
Richard W. Titball2 and
Brendan W. Wren1,7
1 The London School of Hygiene and Tropical Medicine, London, WC1E 7HT,
UK
2 Defence Science and Technology Laboratories, Salisbury, Wiltshire, SP4
0JQ, UK
3 Department of Medical Microbiology, St. George's Hospital Medical School,
London, SW17 0RE, UK
4 Department of Medical Microbiology, St. Bartholomew's and the Royal London
School of Medicine and Dentistry, London, E1 2AD, UK
5 Max von Pettenkofer-Institut für Hygiene und Medizinische
Mikrobiologie, 80336 München, Germany
6 School of Biological Sciences, Queen Mary, University of London, London,
E1 4NS UK
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ABSTRACT
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Yersinia pestis, the causative agent of plague, diverged from
Yersinia pseudotuberculosis, an enteric pathogen, an estimated
1500–20,000 years ago. Genetic characterization of these closely related
organisms represents a useful model to study the rapid emergence of bacterial
pathogens that threaten mankind. To this end, we undertook genome-wide DNA
microarray analysis of 22 strains of Y. pestis and 10 strains of
Y. pseudotuberculosis of diverse origin. Eleven Y. pestis
DNA loci were deemed absent or highly divergent in all strains of Y.
pseudotuberculosis. Four were regions of phage origin, whereas the other
seven included genes encoding a vitamin B12 receptor and the insect toxin
sepC. Sixteen differences were identified between Y. pestis
strains, with biovar Antiqua and Mediaevalis strains showing most divergence
from the arrayed CO92 Orientalis strain. Fifty-eight Y. pestis
regions were specific to a limited number of Y. pseudotuberculosis
strains, including the high pathogenicity island, three putative
autotransporters, and several possible insecticidal toxins and hemolysins. The
O-antigen gene cluster and one of two possible flagellar operons had high
levels of divergence between Y. pseudotuberculosis strains. This
study reports chromosomal differences between species, biovars, serotypes, and
strains of Y. pestis and Y. pseudotuberculosis that may
relate to the evolution of these species in their respective niches.
Yersinia pestis is a Gram-negative bacterium that is the causative
agent of the systemic invasive infectious disease classically referred to as
plague (Perry and Fetherston
1997 ). There have been three recorded human plague pandemics; the
Justinian plague (6th to 8th centuries), the Black Death
(14th to 19th centuries), and modern plague
(19th century to present day;
Perry and Fetherston 1997).
The Black Death alone is estimated to have claimed one-third of the European
population, and this catastrophic event shaped the development of modern
civilization. The recent identification of multidrug-resistant strains
(Galimand et al. 1997), and
the possible use of Y. pestis as an agent of biological warfare means
that plague still poses a significant threat to human health.
Multi-locus sequence typing (MLST) of housekeeping genes suggests that
Y. pestis is a clone of the enteropathogen Yersinia
pseudotuberculosis (Achtman et al.
1999). Current Y. pestis strains form a homogeneous group
that is estimated to have emerged 1500–20,000 years ago
(Achtman et al. 1999). In this
short period of evolutionary time, Y. pestis has evolved the ability
to colonize an insect vector (the flea) and establish a transmission cycle
between mammalian hosts by novel subcutaneous and pneumonic routes of
infection. Genetic analysis of these two Yersinia species provides an
excellent opportunity to study how new and highly virulent pathogens evolve.
To date, the acquisition of two plasmids (pMT1/pFra and pPCP1/pPla) and some
small regions of chromosomal DNA have been identified as specific for the
Y. pestis subspecies of Y. pseudotuberculosis
(Ferber and Brubaker 1981;
Parkhill et al. 2001;
Deng et al. 2002;
Radnedge et al. 2002). Apart
from the plasmid-encoded Ymt protein on pMT1 that has been shown to be
necessary for the colonization of fleas
(Hinnebusch et al. 2002),
little else is known about the genetic basis of the recent change in host
adaptation and virulence.
Y. pestis strains can be divided into three biovars, Antiqua,
Mediaevalis, and Orientalis, that are biochemically distinguished by their
abilities to ferment glycerol and to reduce nitrate
(Devignat 1951). They can also
be differentiated by ribotyping (Guiyoule
et al. 1994) and by PFGE analysis of SpeI fragments
(Lucier and Brubaker 1992).
The initial ribotyping studies, in conjunction with data on the geographical
sources of the strains, were used to correlate the different biovars with the
three plague pandemics (Guiyoule et al.
1994). This has been further validated by restriction fragment
length polymorphism (RFLP) analysis of IS100 element insertions from
strains of the three biovars (Achtman et
al. 1999) and PCR-based IS100 genotyping by use of the
Y. pestis CO-92 genome sequence as a reference
(Motin et al. 2002). Y.
pseudotuberculosis strains are grouped in 21 serotypes, on the basis of
differences in their lipopolysaccharide (LPS) content. From these studies, it
has been proposed that Y. pestis originated from a Y.
pseudotuberculosis serotype 0:1b strain
(Achtman et al. 1999;
Skurnik et al. 2000).
The recent sequencing of the entire genome of a Y. pestis
Orientalis strain, CO-92 (Parkhill et al.
2001) and a Mediaevalis strain, KIM10+
(Deng et al. 2002), has
provided an opportunity for the detailed genetic comparisons of Y.
pestis strains of different biovars. However, although multiple genetic
rearrangements affecting gene order were apparent on comparing the two
strains, (Deng et al. 2002) and
some rearrangements were seen during laboratory culture of Y. pestis
CO-92 (Parkhill et al.), >98% of the genome sequence was shared between
Y. pestis KIM and CO-92 (Deng et
al. 2002). In many cases, the boundaries of genome rearrangements
were formed by insertion sequences. A 102-kb region of Y. pestis DNA,
incorporating the pigmentation (pgm) and yersiniabactin loci, flanked
by insertion sequences, is known to be unstable. Although necessary for full
virulence and flea colonization in vivo, this region is lost during in vitro
growth at a high frequency (2 x 10–3;
Hare and McDonough 1999),
probably due to recombination between flanking IS100 elements
(Fetherston et al. 1992;
Fetherston and Perry 1994;
Buchrieser et al. 1998a). It is
possible, therefore, that recombination between these sequence elements is
responsible for other deletions in the Yersinia chromosome with less
obvious in vitro phenotypes. A recent subtractive hybridization study by
Radnedge et al. (2002)
identified six chromosomal regions of Y. pestis that varied between
strains. Four of these six difference regions (DFRs) were associated with
flanking-insertion sequences or other repeats. This study showed that the six
DFRs could be used to form profiles of Y. pestis strains that can be
correlated to biovar and the evolution of individual strains
(Radnedge et al. 2002).
Traditional phylogenetic classification of bacteria to study evolutionary
relatedness is based on the variation in a limited number of conserved genes,
rRNA/rDNA, or signature sequences. However, due to the acquisition of DNA
through lateral gene transfer, the differences between closely related
bacterial strains, particularly members of the enterobacteriaceae such as the
yersiniae, can be significant. Genome comparisons between pathogenic and
nonpathogenic strains within a species are particularly useful for identifying
determinants important in virulence, transmission, and host specificity. DNA
microarray analysis has been utilized recently to investigate genome-wide
analysis of several bacterial pathogens including Helicobacter pylori
(Salama et al. 2000),
Campylobacter jejuni (Dorrell et
al. 2001), Mycobacterium tuberculosis
(Kato-Maeda et al. 2001),
Staphylococcus aureus (Fitzgerald
et al. 2001), Vibrio cholerae
(Dziejman et al. 2002), and
Salmonella enterica (Porwollik et
al. 2002). Because Y. pestis and Y.
pseudotuberculosis are highly clonal and closely related
(Achtman et al. 1999),
microarray analysis represents an ideal methodology for full genome
comparisons of the two species.
To further elucidate the genetic differences between Y. pestis and
Y. pseudotuberculosis since their divergence, we designed a Y.
pestis CO-92 biovar Orientalis gene-specific microarray to probe full
genome comparisons of chromosomal DNA between different strains, biovars,
serotypes, and species. The data confirms and extends previous studies and
identified new loci specifically present in Y. pestis and absent in
Y. pseudotuberculosis strains.
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RESULTS AND DISCUSSION
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DNA sequences representing all 4221 predicted coding sequences (4012
chromosomal and 209 plasmid encoded) from Y. pestis CO-92 (biovar
Orientalis) were amplified and spotted onto glass microscope slides to produce
a CO-92 gene-specific microarray (see Methods). Twenty-two strains of Y.
pestis and ten strains of Y. pseudotuberculosis were chosen to
be compared at a genomic level with the CO-92 strain by competitive
hybridization to the array. Y. pestis strains were chosen to cover
all three biovars and included a previously sequenced strain (KIM10+) and a
strain that has been referred to previously as Y. pestoides (G-8786).
Y. pseudotuberculosis strains were chosen in order to allow a genomic
comparison of different serotypes with Y. pestis, and also to aid
investigation into their differing phenotypes, the results of which are not
discussed here. A comparison of the relative levels of hybridization signal
across the entire CO-92 chromosome for all strains is shown in
Figure 1. Y. pestis
strains showed relatively few differences compared with the CO-92 array
strain, whereas Y. pseudotuberculosis strains revealed greater
variation.
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Figure 1 Chromosomal comparison of 22 strains of Y. pestis and 10 strains
of Y. pseudotuberculosis to the sequenced CO-92 strain. This data was
generated by Genespring software. Y. pestis strains are grouped
according to biovar, whereas Y. pseudotuberculosis strains are
grouped according to serotype. Gene status is color coded according to the
Genespring software default colors with reference to the control strain
(CO-92). Thus, in this comparison, yellow indicates presence, blue indicates
absence or high divergence, and orange/red indicates a duplication.
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Microarray Validation
Data obtained from microarray analysis was validated by comparison with
limited genomic analysis of the species reported in previous studies.
Microarray data for the KIM10+ strain of Y. pestis was compared with
the published genome sequence (Deng et al.
2002). Our microarray data concur with the genome sequence data,
in that four multiple gene regions (YPO0738–YPO0754,
YPO1165–YPO1172, YPO2271–YPO2281, and YPO2375–YPO2376) and
the sepC gene (YPO2380) are absent from the KIM10+ genome.
A genomic signature tag (GST) approach for determining differences between
CO-92 and EV76 (biovar Orientalis) was reported recently, and suggested the
potential absence of six regions in the EV76 genome
(Dunn et al. 2002). Our data
reveals that only two of the regions appear to be absent from the EV76 genome,
both of which were predicted, but not fully defined, by the GST method. The
GST study predicted nucleotides 2,172,627–2,254,447 of the CO-92
sequence, corresponding to YPO1919–YPO1985 in the 102-kb unstable
region, to have a high probability of being absent in EV76. Our data reveals
that YPO1902–YPO1967 is absent in EV76. A second region predicted to be
absent, in the GST study, is nucleotides 1,307,243–1,316,087 of the
CO-92 sequence, corresponding to YPO1159–YPO1168. Our data shows that
YPO1165–YPO1172 is absent in EV76, as well as F361–F366 and
KIM10+. We noted no absences in any of the other four predicted regions. Only
one other absence was revealed by our data, that of a single gene, YPO0599,
encoding a potential adhesin. These discrepancies in boundaries of deleted
regions may be due to the effect of extensive genomic rearrangements in gene
order on the probability functions used in the GST method.
To further validate the microarray studies, we also compared our results
with the subtractive hybridization data published recently by Radnedge et al.
(2002). Thirteen of the
strains analyzed by the Radnedge study were tested on the microarray. Our
microarray data identified all of the deletions that were reported previously
by Radnedge et al. (2002)
along with other deletions not reported previously. From the microarray data,
a total of 16 differences were identified between the Y. pestis
strains tested when compared with CO-92. These differences range from a single
gene to a region spanning 41 genes. Of the 16 regions, 6 corresponded with the
DFRs defined by Radnedge et al.
(2002). This finding suggests
that subtractive hybridization is a relatively insensitive method for
analyzing strain-to-strain variation. In summary, our microarray data concurs
with previous Yersinia genetic comparisons, validating the use of
this methodology for genome-wide comparisons. In comparison with subtractive
hybridization or GST analysis of multiple strains, the microarray analysis
appeared more comprehensive and identified additional gene and locus
differences.
Y. pestis/Y. pseudotuberculosis Strain Comparisons
The most obvious difference between Y. pestis and Y.
pseudotuberculosis was the number of IS elements. The total number of
insertion sequences reported in the CO-92 genome exceeds that reported for
most other bacterial pathogens (comprising 3.7% of the genome). Of these,
IS1541 elements were the most abundant, with IS100,
IS285, and IS1661 elements making up the majority of the
remainder (Parkhill et al.
2001). All of the Y. pestis strains were nearly identical
in their complement of IS elements apart from IS100. Copy numbers of
IS100 elements ranged from half that of CO-92 in strains G-8786 and
735, to approximately three times the CO-92 level in strains KUMA and ZE94.
Elevated copy numbers of IS100 were also noted in strains 195-P and
PEXU2 (two and one-half times) and A1122 (one and one-half times), compared
with CO-92. All 10 Y. pseudotuberculosis strains appeared to contain
single copies of IS903 and IS1400 elements identified in the
Y. pestis CO-92 chromosome. However, they all contained fewer copies
of IS1541 and IS285 elements compared with Y.
pestis CO-92, and virtually no IS100 or IS1661
elements. The high number of IS100 elements in the Y. pestis
genome has been suggested to have played a role in its evolution, as regions
flanked by IS elements are often unstable as a result of recombination events.
A 102-kb region of Y. pestis DNA, incorporating the pigmentation
(pgm) and yersiniabactin loci flanked by insertion sequences is known
to be unstable due to recombination between flanking IS100 elements
(Fetherston et al. 1992;
Fetherston and Perry 1994;
Buchrieser et al. 1998a). Our
data confirmed this instability as the entire 102-kb yersiniabactin,
hms/pgm locus (encompassing YPO1902–YPO1967) was absent from
many of the Y. pestis strains. However, passage of three strains,
ZE94, 195-9, and Java9 (all biovar Orientalis) through mice enriched for
bacteria retaining the 102-kb region on repeat hybridization with the array
(P. Oyston and R. Titball, unpubl.). Strain PB6 (biovar Orientalis) was the
only strain to have lost the yersiniabactin locus, yet retained most of the
hms and pgm loci, with a deletion spanning
YPO1902–YPO1931. Despite the large number of IS elements apparent in
Y. pestis genomes, no other unstable regions in in vitro cultured
bacteria that could be stabilized by in vivo growth were identified.
The yersiniabactin locus of Y. pseudotuberculosis spans the region
YPO1898–YPO1917, and has been reported to be present in only a small
number of Y. pseudotuberculosis strains
(Buchrieser et al. 1998b). Only
1 Y. pseudotuberculosis strain SP93422 (serotype O: 15) hybridized to
the equivalent locus in Y. pestis CO-92, whereas all 10 Y.
pseudotuberculosis strains contained the hemin storage (hms) and
pigmentation (pgm) loci (YPO1918–YPO1967).
Aside from IS element differences, 11 regions of the CO-92 chromosome were
absent from all 10 strains of Y. pseudotuberculosis
(Table 1); of these, only 2
were deemed absent from any of the Y. pestis strains tested. Four of
these Y. pestis-specific regions consisted almost entirely of
phage-related coding sequences. The prophage encoded by YPO2271–YPO2281
is absent from all 10 strains of Y. pseudotuberculosis, and is also
absent from all Antiqua and Mediaevalis strains of Y. pestis. These
prophage genes have high similarity to a filamentous prophage found in
virulent K1 strains of Escherichia coli
(Gonzalez et al. 2002). One of
the genes in this locus, puvA (equivalent to YPO2277), was identified
in E. coli 018:K1:H7 strain RS218 as a potential virulence factor
during a signature-tagged mutagenesis (STM) screen of an infant rat model of
meningitis (Gonzalez et al.
2001).
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Table 1. Regions of the CO-92 Genome That Are Absent or Highly Divergent in All
10 Y. pseudotuberculosis Strains Tested
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Two of the other Y. pestis-specific phage regions
YPO1085–YPO1088 and YPO1094–YPO1098 flanked another
phage-associated region YPO1089–YPO1092a, found in only one of the
Y. pseudotuberculosis strains tested (strain 141, serotype 0:7). The
final Y. pestis species-specific phage region YPO2084–YPO2119
adjoined another phage region YPO2120–YPO2135, which is absent from 5 of
the 10 Y. pseudotuberculosis strains. Most of the putative coding
sequences in the region YPO2120–YPO2135 were also absent in the other
five Y. pseudotuberculosis strains, but a few of the sequences
hybridized to the array. This indicates that this region may also be absent in
these strains, and the presence of other, as yet unidentified phage sequences
in these strains may have resulted in cross-hybridization.
The remaining 7 Y. pestis-specific regions contained 21 predicted
coding sequences, 14 of which are of unknown function. The other seven encode
a putative membrane protein, a YjgF-family lipoprotein, a putative DNA-binding
protein, the putative transcriptional activator RegC, a methylase enzyme, the
vitamin B12 receptor BtuB, and the putative insecticidal toxin SepC. No
obvious role in virulence or host adaptation can be ascribed to any of these
genes, with the exception of SepC, which could be important in the
colonization of the flea. However, this gene is also absent in the KIM10+
genome sequence, a strain that is capable of colonizing the flea
(Hinnebusch et al. 2002). The
apparent absence of the vitamin B12 receptor was unexpected, as Y.
pseudotuberculosis is vitamin B12 dependent and uptake is known to occur
(M. Prentice, unpubl.). A BLAST search of the Y. pseudotuberculosis
IP32953 currently being sequenced at The Lawrence Livermore Institute
(http://bbrp.llnl.gov/bbrp/bin/y.pseudotuberculosis_blast)
using the CO-92 btuB gene sequence reveals that the gene is present,
but contains regions of diversity separated by regions of near perfect
identity. PCR and sequence analysis revealed that btuB in all 10
Y. pseudotuberculosis strains tested was identical to the Y.
pseudotuberculosis IP32953 strain (data not shown). Y. pestis
and Y. pseudotuberculosis are so closely related at the genetic level
that Y. pestis is sometimes classified as a subspecies of Y.
pseudotuberculosis rather than a distinct species. Thus, the divergence
of the btuB gene between Y. pestis and Y.
pseudotuberculosis is surprising, as, to date, there have been no reports
of variation in btuB between strains of the same species. The vitamin
B12 receptor may act as a receptor for colicins and bacteriophages (for
review, see James et al.
1996), thus, there is the potential for variation of the
btuB gene due to selective pressure. Y. pseudotuberculosis
is found widely in the soil environment and only causes human disease when
ingested. Thus, it is likely to be in contact with other bacterial species, a
source of colicins and bacteriophages. To survive in these habitats, variation
in the btuB gene may be advantageous. Y. pestis, on the
other hand, is an obligate blood-borne pathogen of mammals with an insect
vector. In these niches, Y. pestis would not be in close contact with
other bacterial species, possibly resulting in a lack of selective pressure
for variation of the btuB gene.
Y. pestis Interstrain Comparison
A schematic comparison of the deletions found in all of the Y.
pestis strains tested, excluding the unstable 102-kb
yersiniabactin/pgm/hms locus, is shown in
Figure 2. Overall, strains
belonging to biovars Antiqua or Mediaevalis showed the greatest divergence
from Y. pestis strain CO-92. Conversely, the other biovar Orientalis
strains showed the least divergence from strain CO-92. Three separate regions
were identified as being present in all Orientalis strains, but absent in the
majority of Antiqua and Mediaevalis strains. Only the filamentous CUS-2
prophage, YPO2271–YPO2281 was deemed absent from all seven Antiqua and
Mediaevalis strains, but two further regions were absent from both Mediaevalis
strains and four of the five Antiqua strains. These two regions may be
considered biovar specific as the only Antiqua strain that encodes these
regions, G-8786, has also been referred to as Y. pestoides, and thus,
is considered an atypical Antiqua strain. Strain G-8786 also contains four
strain-specific regions of deletion, perhaps reflecting the remote origin of
this strain (a rodent from the Caucasian High Mountainous region in Georgia),
and further indicating that this is not a typical Antiqua strain. These
regions encoded a putative aldo/keto reductase and hypothetical protein,
YPO2375–YPO2376, and one of the two cryptic flagella gene clusters,
YPO0738–YPO0747, along with the adjacent genes YPO748–YPO754.
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Figure 2 Schematic of chromosomal comparison of 22 strains of Y. pestis
detailing all of the regions of divergence from CO-92. Strains are grouped by
biovar. The 102-kb unstable region (YPO1902–YPO1967) has not been
included in this comparison. Gene status is color coded as in
Figure 1 for ease of
comparison, with yellow indicating presence, blue indicating absence, and red
indicating a duplication.
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In common with some of the Y. pseudotuberculosis strains, eight
Orientalis strains and one of the Antiqua strains (Nepal 516) did not contain
YPO0599 (Fig. 2). This putative
adhesin was not enriched by passage through mice in Y. pestis strain
ZE94 (P. Oyston and R. Titball, unpubl.). Three strains, one Mediaevalis and
two Orientalis, had deletions of YPO1165–YPO1172 encoding proteins
involved in the choline-glycine betaine pathway, whereas two Antiqua strains
had deletions of YPO1943–YPO1944, encoding two putative membrane
proteins. All other deletions were strain specific, with the Antiqua strain
G-8786 appearing to be the most divergent strain, with four strain-specific
deletions. A phylogenetic tree based on the Y. pestis differences
clearly shows the divergence of strain G-8786 from the other strains
(Fig. 3). A clear divide can
also be seen between the Orientalis strains and strains from the other two
biovars. The 15 Y. pestis Orientalis strains are all very similar
genetically, and thus, are closely related. The Antiqua and Mediaevalis
strains also share a great deal of genetic similarity with each other, but
also show several strain-specific variations; thus these two biovars are
unable to be easily distinguished by their genetic differences.
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Figure 3 Parsimony analysis of Y. pestis strain microarray data. Bootstrap
50% majority-rule consensus tree with bootstrap values (1000 replicates)
overlying branch points. A total of 22 characters were used, of which 8 were
parsimony informative and given equal weight. Six equally parsimonious trees
were found (see Supplemental Data, available at
www.genome.org).
Strain names are followed by their biovar in parentheses.
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There are five regions of the CO-92 chromosome that appear to have been
duplicated in some Y. pestis strains. One of these regions,
YPO2274–YPO2281, corresponds to the invariant part of the E.
coli CUS-2 prophage, which is identical to CUS-1
(Gonzalez et al. 2001), and a
possible explanation for this apparent partial duplication is the presence of
a CUS-1 or similar phage in addition to CUS-2. This duplication is seen in 4
of the 15 Orientalis strains. The other four duplications appear to be strain
specific and are all flanked by insertion sequences.
Y. pseudotuberculosis Strain Comparison
As found previously for housekeeping gene sequences
(Achtman et al. 1999), gene
complement and some gene sequences are more divergent when compared across
Y. pseudotuberculosis strains than within the Y. pestis
group. Microarray analysis revealed high levels of variation throughout the
entire genome of Y. pseudotuberculosis strains compared with Y.
pestis CO-92 and with each other (Fig.
1). Analysis of this data revealed that between 6.3%–10.4%
of predicted coding sequences, identified from the sequencing of Y.
pestis strain CO-92, were absent or divergent in individual Y.
pseudotuberculosis strains. Many of these encode proteins that may be
involved in the successful colonization of mammalian and insect hosts
(Table 2).
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Table 2. Potential Virulence Determinants Which Are Absent or Highly Divergent in
Some of the 10 Y. pseudotuberculosis Strains Tested
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Two regions showed high levels of divergence from the Y. pestis
CO-92 genome in all 10 of the Y. pseudotuberculosis strains. These
both contain genes responsible for the structure of potentially antigenic
surface structures, the flagella and lipopolysaccharide (LPS), and thus, they
may be under selective pressure, for example, from the mammalian immune
system, resulting in high levels of divergence.
All Y. pseudotuberculosis strains produce LPS, an important
virulence determinant, mediating resistance to complement-mediated and
phagocyte killing (Makela et al.
1988; Darwin and Miller
1999; Karlyshev et al.
2001). In contrast, Y. pestis strains produce rough LPS,
lacking the O-antigen, due to a number of mutations in the O-antigen gene
cluster (Skurnik et al. 2000;
Parkhill et al. 2001;
Deng et al. 2002). Variance in
LPS structure is the basis for typing Y. pseudotuberculosis strains,
with 21 sero-groups identified. Microarray analysis revealed significant
variation within the O-antigen gene cluster between serotypes, with a high
level of divergence of most genes, as has been demonstrated previously by PCR
and sequencing (Skurnik et al.
2000). This data provided evidence that Y. pestis
originated from a serotype O:1b strain of Y. pseudotuberculosis. Our
results concur with these findings and are summarized in
Table 3. The serotype O:7
strains, for example, have no genes within this region with sufficient
similarity to CO-92 to hybridize to the array, with the exception of the
O-antigen chain-length determinant wzz. This was also the only gene
within this region that Skurnik et al.
(2000) was able to identify
using PCR, with the serotype O:7 strains. The 01b serotypes, on the other
hand, contain CO-92-homologous counterparts for all of these genes with the
exception of wzx, the O-unit flippase gene YPO3110, which appears to
be divergent. Sequence data on the O-antigen gene cluster of the serotype O:1b
strain Pa3606 (GenBank accession no. AJ251712
[GenBank]
) showed that all genes within
this cluster are 98%–100% identical to their CO-92 array PCR products at
the nucleotide level, with the exception of wzx, which is only 84.5%
identical. Many genes in this cluster are apparently absent in some serotypes
due to their diversity from the CO-92 sequence. The absence of key genes such
as the O-unit flippase and O-unit polymerase would mean that these strains
lack high molecular weight LPS, as they are unable to transport O-units to the
periplasmic face, or polymerize them into O-antigen. However, these serotypes
express LPS, indicating that the genes are present, but are divergent enough
not to hybridize to the microarray. This particular region of the genome is
important in the virulence of the organism in a murine infection model.
Signature-tagged mutagenesis of the serotype O:3 strain YPIII pIB1 revealed
that mutation in any of five of the LPS biosynthesis genes caused a decrease
in virulence (Karlyshev et al.
2001). Thus, it is possible that the differences in LPS
composition between serotypes may account for differences in the virulence of
certain strains of Y. pseudotuberculosis. The lack of variation in
Y. pestis may be due to the absence of the selective pressure,
because these surface structures are no longer expressed. It has been shown
that Y. pestis contains mutations in five of the O-antigen cluster
genes, and thus produces lipo-oligosaccharide (LOS) lacking O-antigen
(Skurnik et al. 2000;
Parkhill et al. 2001;
Prior et al. 2001).
The other region that showed high levels of divergence from the Y.
pestis CO-92 genome in all 10 of the Y. pseudotuberculosis
strains is part of 1 of the 2 cryptic flagella operons
(Table 4). In contrast to
Y. pestis, Y. pseudotuberculosis strains are highly motile. However,
the CO-92 genome sequence revealed the presence of two cryptic flagella
operons, YPO0704&ndashYPO0747 and YPO1790–YPO1842. The
YPO0704–YPO0747 operon is highly unlikely to be functional in Y.
pestis due to multiple frameshift mutations
(Parkhill et al. 2001;
Deng et al. 2002). A large part
of this operon (YPO0714–YPO0749) shows high levels of divergence in all
Y. pseudotuberculosis strains tested
(Table 4). The serotype O:3
strain, YPIII pIB1, is the only strain that appears to show little divergence
from Y. pestis CO-92 in this region, with only the fliF,
fliG, and fliA genes exhibiting divergence. The other operon
(YPO1790–YPO1842) is potentially fully functional in CO-92 and is also
present in all of the Y. pestis and Y. pseudotuberculosis
strains tested, with no apparent divergence from CO-92 in any strain. This
indicates that this operon is not under any selective pressure to vary and may
not be expressed in either species.
Variable Determinants That May Be Important in Host Adaptation and
Virulence
To elucidate the mechanisms of bacterial virulence and the evolution of
virulence, we focused our analysis on those sequences that encode known or
putative virulence factors. These are grouped into three categories, those
potentially involved in the colonization of insects, those involved in
adhesion and invasion of mammalian cells, and other bacterial toxins.
Insect Colonization
There are seven potential insecticidal toxins encoded by the CO-92
chromosome, which are similar to those produced by the insect pathogen
Photorhabdus luminescens
(Waterfield et al. 2001).
Three are members of the tca gene family (tcaA, tcaB, and
tcaC), and four are paralogs of the tccC genes. The function
of these genes in the pathogenic yersiniae has yet to be elucidated. The
putative insect toxins (Tcc) encoded by Y. pseudotuberculosis show
significant variation between strains. The tccC paralog,
sepC (YPO2380), is absent from all Y. pseudotuberculosis
strains tested, whereas two of the serotype O:3 strains are also deficient in
another tccC paralog (YPO3673). Due to the high level of identity of
YPO3673 with the adjacent YPO3674, lower levels of hybridization signal were
observed for both genes (Table
2). PCR analysis was used to confirm the absence of YPO3673 rather
than YPO3674 (data not shown).
The second insect toxin complex tcaABC, encoded by genes
YPO3678–YPO3681, also showed divergence within Y.
pseudotuberculosis strains (Table
2). The tcaA gene appeared highly conserved in all 10
Y. pseudotuberculosis strains. However, only two serotype O:3 strains
possessed DNA sequences with high similarity to the CO-92 tcaB gene.
The other 8 strains give a weakened signal for tcaB, whereas all 10
strains give a weakened signal for tcaC. PCR analysis confirmed the
presence of tcaB and tcaC paralogs within these Y.
pseudotuberculosis strains (data not shown). One explanation for this
divergence may be that the expression of the tcaB and tcaC
paralogs encoded by Y. pseudotuberculosis may be more toxic for the
flea vector. Y. pestis has to persist in the flea gut for relatively
long periods, thus potent insect toxins may be detrimental to its life cycle.
The tcaB gene has been shown to have a frameshift mutation and
tcaC an internal deletion in the CO-92 genome sequence
(Parkhill et al. 2001).
Another sequence that may be related to the parasitism of insects, YPO0339,
a putative enhancin encoded by baculoviral pathogens of insects
(Parkhill et al. 2001),
appeared conserved among all Y. pestis and Y.
pseudotuberculosis strains.
Adhesion and Invasion of Mammalian Cells
Three putative autotransporter genes, yapA, yapB, and
yapH show variation among Y. pseudotuberculosis strains.
Eight of the ten strains were positive for yapA, the two exceptions
were both serotype O:3. All strains hybridized to yapB, although the
hybridization signal was reduced in seven strains, suggesting that this gene
may be slightly divergent from the CO-92 ortholog. Attempts at amplifying
genomic DNA from strain YPIII pIB1, with two primer sets specific for CO-92
yapB failed in any of the four possible combinations, suggesting that
the yapB gene has diverged sufficiently to affect primer annealing
(data not shown). Six of the ten strains hybridized to yapH,
including the two strains that lacked yapA. The other four strains
amplified a truncated PCR product with yapH-specific primers,
indicating a deletion within the gene (data not shown). Autotransporter
proteins are capable of being secreted from the bacterial outer membrane
without the aid of secretion pathways, or other proteins, and have been
implicated in the virulence of bacterial pathogens
(Henderson et al. 1998). The
E. coli autotransporter TibA has strong amino acid similarity to both
YapA and YapB and acts as an adhesin and invasin of human epithelial cells
(Lindenthal and Elsinghorst
2001). It has been shown that noninvasive E. coli can be
induced to invade epithelial cells by the expression of TibA. It is possible
that divergence of yapA, yapB, and yapH in Y.
pseudotuberculosis may be important in the spectrum of disease caused by
this pathogen.
The putative adhesin, YPO0599, is absent from 7 of the 10 Y.
pseudotuberculosis strains, along with 3 adjacent genes of unknown
function. It appears to be a relatively recent loss from the Y.
pestis genome, being absent from 8 of the 15 Orientalis strains, and only
1 of the more ancient strains. This indicates that YPO0599 was acquired by
Y. pseudotuberculosis along with the three adjacent genes, but has
been subsequently lost by some of the Y. pestis strains. Other
proteins involved in attachment and invasion of mammalian cells such as
inv (YPO1793), ail (YPO1860 and YPO2905), hmwA
(YPO3247), and the putative invasin encoded by YPO3944 were all present in all
strains of Y. pseudotuberculosis. Also present was a coding sequence
similar to virK from Shigella flexneri, which is required
for intercellular spreading in this organism.
Other Toxins
Several coding regions with similarity to known pore-forming toxins were
identified by the sequencing of the two Y. pestis strains, CO-92 and
KIM10+. These include four putative hemolysins and activator proteins with
similarity to ShlA and ShlB from Serratia marcescens. The ShlA
hemolysins from S. marcescens require phosphatidylethanolamine as a
cofactor and can form pores in fibroblasts and epithelial cells as well as
erythrocytes (for review, see Hertle
2000). Two of these putative hemolysins and two associated
activators showed divergence in Y. pseudotuberculosis strains
(Table 2). Divergence was also
seen in a coding region with similarity to the pore-forming protein RtxA of
V. cholerae (YPO0947). However, no divergence was apparent in genes
encoding the putative RTX toxin transporters, RtxB (YPO2249) and RtxD
(YPO2250). Thus, the toxin itself shows divergence and, therefore, possible
variation in activity between strains, whereas the toxin delivery system
remains constant. The RTX toxin of V. cholerae requires an activator
protein RtxC, a RTX activator has yet to be identified in Yersinia
species. The other pore-forming toxin encoded by Yersinia species is
the antimicrobial toxin colicin E8 along with its immunity protein. These
appear to be absent from 7 of the 10 strains.
Nonpore-forming toxins identified in the sequenced Y. pestis
strains include an enterotoxin-like protein similar to the iron-regulated
shET2 enterotoxin SenA from S. flexineri and E. coli. These
toxins cause diarrhea by interfering with the signal-transduction pathways
involved in regulating water and electrolyte fluxes across intestinal mucosa
(Fasano et al. 1995). The
cytotoxic necrotizing factor (YPO1449) shows no apparent divergence between
the 10 Y. pseudotuberculosis strains tested, yet this toxin has been
shown to be expressed in only a limited number of strains
(Lockman et al. 2002).
Y. pestis and Y. pseudotuberculosis Evolution
Y. pestis seems to have adapted rapidly from being a mammalian
enteropathogen, widely found in the environment, to a blood-borne pathogen of
mammals that is able to parasitize insects, and has a limited capability for
survival outside these hosts. Some of these changes may be a result of the
genome differences identified in this study, in addition to the known
importance of the pMT1 and pPCP1 plasmids. However, genome rearrangements,
particularly as a result of the recombination of IS elements, and the
accumulation of pseudogenes may also have played a significant role in the
rapid evolution of Y. pestis. These types of genetic differences are
difficult to identify by microarray analysis. Analysis of the acquired
chromosomal regions has provided no clear explanation as to the differences
between the two species, but the acquisition of phage-related sequences may be
significant. However, the data from this study has shown that sequences that
may be related to the parasitism of insects (insecticidal toxins and
baculovirus enhancin) in Y. pestis are found in a wide range of
Y. pseudotuberculosis strains. This finding suggests that Y.
pestis did not adapt to the flea gut in a single evolutionary event, but
rather that Y. pseudotuberculosis had been associated with insect
hosts or insect pathogens for a considerable time. Y. pestis has had
to adapt to colonize, but not kill, the flea in order to be transferred
between mammalian hosts.
The microarray data was also used to construct a phylogenetic tree for
Y. pseudotuberculosis (Fig.
4). Due to differences in scale, all of the Y. pestis and
Y. pseudotuberculosis strains could not be shown on the same figure,
but reference Y. pestis strains were included in the Y.
pseudotuberculosis analysis. The phylogenetic tree, on the basis of the
genetic differences between Y. pestis strains, shows a clear
divergence between the Orientalis strains and strains from the other two
biovars (Fig. 3). However, no
obvious evolutionary pathway from Y. pseudotuberculosis can be
determined. Only 2 of the 10 regions determined to be absent from all Y.
pseudotuberculosis strains were absent from any of the Y. pestis
strains. Thus, it appears that the ancestral Y. pestis strain
acquired at least eight of these regions before the three biovars diverged.
The progenitor of the Antiqua and Mediaevalis biovars subsequently lost
regions YPO0738–YPO0754 and YPO2375–YPO2376 before the individual
strains diverged by the loss of strain-specific regions, whereas the
Orientalis biovar acquired the filamentous CUS-2 prophage
(YPO2271–YPO2281). Strain G-8786 probably diverged from the Antiqua and
Mediaevalis progenitor before the loss of YPO0738–YPO0754 and
YPO2375–YPO2376.
View larger version (13K):
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Figure 4 Parsimony analysis of Y. pseudotuberculosis and representative
Y. pestis strain microarray data. The two most parsimonious trees are
shown. Of 87 variable characters, 63 were parsimony informative and equally
weighted. Y. pseudotuberculosis strain names are followed by their
serotype in parentheses. Three Y. pestis strains are shown, one from
each of the main clades in Fig.
3 and are followed by their biovar in parentheses. Horizontal
scale bar indicates number of character changes (Reanalysis omitting all
characters pertaining to the O-antigen locus gives a similar result, see
Supplemental Data).
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|
Although Y. pestis does not produce LPS with an O-antigen, the
vestigial O-antigen cluster is still present, and this has been used to infer
the evolution of Y. pestis. Previous sequence analysis of the
O-antigen gene clusters from different strains of Y.
pseudotuberculosis has indicated that Y. pestis evolved from a
serotype O1b strain of Y. pseudotuberculosis
(Skurnik et al. 2000).
Although our studies confirm this finding
(Table 3), looking at overall
gene complement, we found that, whereas one O:1b strain (Pa3606) was closer to
representative Y. pestis strains than other Y.
pseudotuberculosis strains tested, the other O:1b strain (354) was as
different as other Y. pseudotuberculosis strains from a range of
serotypes (Tables 2 and
4;
Fig. 4). Thus, although it is
likely that Y. pestis evolved from a single O:1b strain, the O:1b
strains as a whole appear to be no more similar to Y. pestis than any
other serotype.
Our results provide the first detailed comparisons of different strains of
Y. pestis and Y. pseudotuberculosis at a genome-wide level.
We show that the genes specifying serotype form a small minority of the
chromosomal gene complement differences between Y. pestis and Y.
pseudotuberculosis, and that an approach identifying all Y.
pseudotuberculosis serotype O:1b strains as immediately ancestral to
Y. pestis is potentially misleading. Our findings also provide
further evidence that biovar typing Y. pestis strains based on the
ability to reduce nitrate and ferment glycerol consistently correlates with
true genetic differences for Orientalis biovar strains, but may not always
show correct genetic groupings for Medievalis and Antiqua biovars. Genes that
are unique to Y. pestis should now be characterized in more detail to
determine whether they play a specific role in colonization of the flea, or in
disease in mammals. Additionally, genes that are unique to Y. pestis
might form the basis of alternative tests for the identification and molecular
typing of Y. pestis.
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METHODS
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|---|
Construction of the Y. pestis CO-92 Microarray
The Y. pestis CO-92 microarray was constructed from spotted PCR
products designed and printed at the Bacterial Microarray facility at St.
George's Hospital Medical School. Gene-specific primers were designed using
Microarray Design (MAD) software (Hinds et
al. 2002). This utilizes an algorithm to select a single PCR
product for each gene that was unique and only self-detected in BLAST analysis
of the whole genome. If a unique PCR product was not possible for a particular
gene, then the algorithm selected a PCR product from the BLAST analysis that
demonstrated minimal cross-hybridization with other nontarget genes. PCR
products were amplified in a 96-well plate format using an MWG Biotech RoboAmp
4200. The PCR products were gridded at high density on poly-L-lysine coated
glass microscope slides using a BioRobotics MicroGrid II robot.
The finished array consisted of spotted PCR products representing each of
the 4011 predicted coding sequences from the CO-92 chromosome together with
the 209 predicted coding sequences from the three plasmids found in CO-92 (9
from pPCP1, 97 from pCD1, and 103 from pMT1).
Strains Used in Study
Y. pestis strains were provided by D. Tsereteli (National Centre
for Disease Control, Tiblisi, Georgia), A. Rakin (Max von Pettenkofer-Institut
für Hygiene und Medizinische Mikrobiologie, Munich, Germany), M. Chu
(Centers for Disease Control and Prevention, Atlanta, USA), and R. Titball
(DSTL, Porton Down, UK). The Y. pseudotuberculosis strain YPIII pIB1
was obtained from H. Wolf-Watz (Umea University, Sweden). All other Y.
pseudotuberculosis strains were obtained from H. Fukushima (Shimane
Prefectural Institute of Public Health and Environmental Science, Matsue,
Japan).
Hybridization of Genomic DNA
Genomic DNA extraction and microarray hybridization procedures were
performed as described previously (Dorrell
et al. 2001). Test strains were labeled with Cy5-dCTP. CO-92
genomic DNA was used as a control on all hybridizations and was labeled with
Cy3-dCTP. Labeling of from 2 to 3 µg of denatured genomic DNA was performed
using the Klenow fragment in the presence of 5 mM dATP, 5 mM dGTP, 5 mM dTTP,
2 mM dCTP, and 750 pM Cy5-dCTP or Cy3-dCTP. Klenow fragment was obtained from
Invitrogen, and Cy-labeled dCTP was obtained from Amersham Pharmacia. After
incubation at 37°C in the dark for 90 min, control and test strain-labeled
DNA were mixed and purified together using a single QIAGEN mini-elute column.
Microarray slides were prehybridized in 3.5 x SSC, 0.1%SDS, 10 mg/mL BSA
at 65°C. The denatured DNA was applied to the microarray slide in
hybridization solution at a final concentration of 4 x SSC, 0.3% SDS.
Hybridization was for 18 h at 65°C, prior to washing the slides once in 1
x SSC, 0.05% SDS at 65°C, and twice in 0.06 x SSC for 2 min.
All hybridizations were performed in duplicate.
Microarray Analysis
All 4221 of the CO-92 predicted coding sequences (4012 chromosomal and 209
plasmid encoded) are represented on the DNA microarray. Extensive analysis of
the three plasmids was not undertaken, due to the frequent loss of these
plasmids from many isolates. All gene numbers refer to the published CO-92
annotated chromosomal sequence (EMBL Accession No. AL590842
[GenBank]
). During the
construction of the DNA microarray, those predicted coding sequences that are
disrupted by IS elements in CO-92 were treated as two separate genetic
elements and represented on the array by two spots, one for each part of the
sequence. Thus 4042 spots represent the 4012 predicted coding sequences in the
CO-92 chromosome. Of these, a total of 73 spots were poor, failing to
hybridize with CO-92 genomic DNA. The remaining 3969 genetic elements
hybridized with the CO-92 genomic DNA, and were therefore used for the
analysis.
Microarray slides were scanned using an Affymetrix 418 scanner (MWG
Biotech), and images analyzed using Imagene (BioDiscovery) and Genespring
(Silicon Genetics) software. Samples were normalized using the following
conditions: All values <0.0 were set to 0.0. All sample signals were
divided by the corresponding control signal from the same hybridization. The
entire array was normalized to the 50th percentile of all
measurements from that array.
Analysis of genomic DNA from multiple strains results in varied signal
intensity due to sequence divergence between strains. Previous hybridization
studies have shown that there is a sigmoidal relationship between the
percentage nucleotide identity of a gene to the PCR product on the microarray,
and the signal produced (Dong et al.
2001; Wu et al.
2001). The relative strength of the hybridization signal produced
by genes of known percentage identity from genome sequence data can be used to
estimate the relative divergence of genes from nonsequenced strains. To
estimate the levels of divergence in our study, we analyzed the signal
intensity generated by Y. pseudotuberculosis strains with previously
sequenced O-antigen gene clusters. A signal ratio of 0.82 was observed for
YPO3096 (wzz) from strain 197, which is 96.8% identical at the
nucleotide level to the array PCR product for this gene. Similarly, signal
ratios of 0.6 and 0.4 were observed for the YPO3110 (wzx) genes of
strains SP93422 and Pa3606, which are 94.6% and 84.5% identical at the
nucleotide level to the PCR product, respectively. Thus after normalization,
genes were determined to be absent if the signal to control ratio was <0.2.
Genes with a ratio of above 0.8 were presumed to be present and highly similar
to the CO-92 gene. Genes that gave a ratio of between 0.2 and 0.8 were deemed
to be present, but slightly divergent from the CO-92 gene. Genes that gave a
signal intensity of approximately twice the fluorescence intensity in the Cy5
channel compared with the Cy3 CO-92 control were deemed to have been
duplicated.
PCR Analysis
The presence of certain genes deemed absent or divergent was verified by
PCR analysis. Gene-specific PCR primers, designed for the array construction,
were used to amplify specific products from the genomic DNA of relevant
strains, using Promega Taq DNA polymerase and standard conditions. If
no PCR product was obtained, then a second set of gene-specific primers were
designed and PCR amplification was repeated.
Phylogenetic Analysis
Y. pestis
Genes found present by the above microarray analysis were scored as two,
absent genes were scored as zero, divergent genes were scored as one, and
duplicated genes as D. IS elements were excluded from analysis. For
interstrain comparison of Y. pestis isolates, all genes were present,
absent, or duplicated with no divergent ORFs. Characters adjacent in the
Y. pestis CO-92 genome sequence showing the same pattern of absence
(signal 0) or duplication (D) in one or more of the tested strains compared
with Y. pestis C0-92 were combined into single characters, which were
incorporated into the PAUP* software program version 4.0b10 (PPC;
http:paup.csit.fsu.edu).
Maximum parsimony trees with equal weighting of characters were drawn
following a heuristic search.
Y. pestis/Y. pseudotuberculosis
No duplicated genes were found in Y. pseudotuberculosis strains,
and many genes were divergent and difficult to score as characters for
phylogenetic analysis. Therefore, only loci for which at least one Y.
pseudotuberculosis strain scored zero were incorporated in the dataset.
The loci in each strain were scored as either present or absent. The genome
contains several large operons (O-antigen, flagella, etc.), with shared
patterns of absence, divergence, or presence of multiple ORFs in different
strains, and which might be subject to strong selection. To deal with the
possible non-independence of adjacent loci, characters that are adjacent in
the Y. pestis CO-92 genome sequence and show the same pattern of
presence and absence across all Y. pseudotuberculosis and Y.
pestis strains were combined into a single character for the analysis. To
check for possible biases introduced by selection (e.g., convergent evolution)
the analysis was repeated with the omission of loci thought to be candidates
for strong selection. Maximum parsimony trees were constructed using PAUP*
(4.0b10) with data from the Y.pseudotuberculosis strains and three
Y. pestis strains (one from each main clade).
|
Acknowledgements
|
|---|
We acknowledge DSTL for funding this research, and the Wellcome Trust
funded Bacterial Microarray facility at St. George's Hospital for the
construction of the array. We thank D. Tsereteli, M. Chu, H. Wolf-Watz, and H.
Fukushima for their generous gifts of purified genomic DNA or bacterial
strains.
The publication costs of this article were defrayed in part by payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 USC section 1734 solely to
indicate this fact.
|
Footnotes
|
|---|
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.1507303.
[Supplemental material is available online at www.genome.org. The following
individuals kindly provided reagents, samples, or unpublished information as
indicated in the paper: D. Tsereteli, M. Chu, H. Wolf-Watz, and H.
Fukushima.]
7 Corresponding author.
E-MAIL
Brendan.Wren{at}lshtm.ac.uk;
FAX 44-207-637-4314. 
|
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ABSTRACT
RESULTS AND DISCUSSION