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Comparative genomic analysis of
Vibrio cholerae
:
Genes that correlate with cholera endemic and
pandemic disease
Michelle Dziejman*, Emmy Balon*, Dana Boyd*, Clare M. Fraser
†
, John F. Heidelberg
†
, and John J. Mekalanos*
‡
*Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115; and
†
The Institute for Genomic Research, Rockville, MD 20850
Contributed by John J. Mekalanos, December 13, 2001
Historically, the first six recorded cholera pandemics occurred be-
tween 1817 and 1923 and were caused by Vibrio cholerae O1 sero-
group strains of the classical biotype. Although strains of the El Tor
biotype caused sporadic infections and cholera epidemics as early as
1910, it was not until 1961 that this biotype emerged to cause the 7th
pandemic, eventually resulting in the global elimination of classical
biotype strains as a cause of disease. The completed genome se-
quence of 7th pandemic El Tor O1 strain N16961 has provided an
important tool to begin addressing questions about the evolution of
V. cholerae as a human pathogen and environmental organism. To
facilitate such studies, we constructed a V. cholerae genomic microar-
ray that displays over 93% of the predicted genes of strain N16961 as
spotted features. Hybridization of labeled genomic DNA from differ-
ent strains to this microarray allowed us to compare the gene content
of N16961 to that of other V. cholerae isolates. Surprisingly, the
results reveal a high degree of conservation among the strains tested.
However, genes unique to all pandemic strains as well as genes
specific to 7th pandemic El Tor and related O139 serogroup strains
were identified. These latter genes may encode gain-of-function
traits specifically associated with displacement of the preexisting
classical strains in South Asia and may also promote the establishment
of endemic disease in previously cholera-free locations.
T
here have been seven pandemics of cholera, a severe diarrheal
disease caused by Vibrio cholerae, a bacterium whose genomic
sequence has recently been reported (1). The first six pandemics
were caused by the classical biotype of V. cholerae, which spread
from the Indian subcontinent to most other areas of the world
between 1817 and 1923 (2–5). However, by 1900, cholera had
disappeared from the Western hemisphere as an epidemic and
endemic disease. A major epidemic in the Celebes Islands (present
day Indonesia) was caused by an ‘‘El Tor’’ biotype strain in 1935, but
cholera failed to become endemic in this locale and did not spread
elsewhere in Asia at this time (2, 6). The 7th pandemic of cholera
technically began in 1961, when pathogenic El Tor biotype strains
established endemicity in Indonesia. These strains then rapidly
spread to cause disease on the Asian mainland and in Africa,
replacing classical strains as the cause of endemic cholera (7). In
1991, V. cholerae El Tor caused a massive epidemic in Lima, Peru.
Since then, cholera has spread to virtually all neighboring countries
and, as a result, is now endemic in much of Latin America.
Molecular typing methods have suggested that isolates from the
1991 Latin American epidemic are clonal and closely related to
Asian and African 7th pandemic strains (3, 7).
In 1992, a newly identified serogroup, O139, was recognized
as the cause of epidemic cholera in South Asia (8). O139 strains
initially displaced serogroup O1 El Tor strains as the main cause
of cholera in India and Bangladesh. However, over the last few
years, El Tor O1 strains have reestablished their prominence and
now share this locale with O139 strains.
Much research has been focused on understanding the basis of
V. cholerae pathogenicity and its pandemic potential. Numerous
studies have highlighted the requirement of three essential
components in pathogenic V. cholerae strains: the filamentous
CTX bacteriophage (CTX
) that encodes cholera toxin, the
TCP pathogenicity island encoding the TCP pili, a colonization
factor and receptor for CTX
, and toxR, an essential virulence
regulatory gene (7). Investigators have also sought to understand
the evolution of 7th pandemic El Tor and O139 strains by using
various molecular typing techniques, such as ribotyping, restric-
tion fragment length polymorphisms, and sequence analysis of
housekeeping and virulence genes (9–12). Data from these
methods support the hypothesis that O139 strains evolved from
recent 7th pandemic El Tor isolates (3, 13). Furthermore,
although they are closely related, classical biotype strains and
nonpathogenic El Tor environmental strains have evolved from
a separate lineage from 7th pandemic El Tor isolates (14, 15).
In comparison to classical strains that caused the 6th pan-
demic, the El Tor strains responsible for the 7th pandemic show
clear differences in regard to their epidemic and endemic
behavior. It has been suggested that El Tor strains demonstrate
increased persistence in aquatic ecosystems (16, 17). Further-
more, El Tor strains can be distinguished from classical strains
by properties such as their hemolytic activity, agglutination
reactions with erythrocytes, and polymyxin B resistance. How-
ever, there is no genetic evidence that these phenotypic prop-
erties correlate with the pandemic and endemic potential of V.
cholerae strains, or with their ability to replace predecessor
strains as observed during the 7th pandemic.
To begin to address these issues, we sought to identify genes
unique to El Tor strains of the 7th pandemic. Here, we report the
construction of a V. cholerae genomic microarray based on the
sequenced O1 El Tor strain N16961 from the 7th pandemic of
cholera (1). This array has been used to compare the gene
content of classical, prepandemic El Tor, pandemic El Tor, and
two nontoxigenic strains to that of strain N16961. Although these
comparative genomic analyses revealed a high degree of genetic
similarity among strains isolated over the past century, we were
able to identify genes unique to pandemic El Tor and O139
strains. These genes potentially encode key properties that have
led to the global success of 7th pandemic strains as agents of
endemic and pandemic cholera.
Materials and Methods
Strains and Media. V. cholerae strains used for this study are
described in Table 1 and were obtained from either the Amer-
ican Type Culture Collection or laboratory stocks. All strains
were grown in Luria–Bertani broth and stored as frozen stocks
in Luria–Bertani broth with 20% glycerol.
Primer Design and PCR Amplification of ORFs. PCR primers were
designed by using
PRIMER3 software (http://www.genome.wi.mit.
edu/genome_software/other/primer3.html). The 5⬘ primer for each
ORF began with the start codon and the 3⬘ primer began with the
nucleotide preceding the stop codon. PCR amplification of each
‡
To whom reprint requests should be addressed. E-mail: jmekalanos@hms.harvard.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
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ORF was performed by using Takara Taq polymerase (Intergen,
Purchase, NY) according to manufacturer’s instructions and in-
cluded 5% dimethyl sulfoxide. After an initial 5-min denaturation
at 95°C, 30 PCR cycles were run as follows: 95°C for 30 s, 54°Cfor
30 s, and 72°C for 3 min. Products were purified by using the
Multiscreen 96-well PCR unit according to the manufacturer’s
directions (Millipore). After elution with 3⫻ SSC, Sarkosyl was
added to a final concentration of 0.03%. Two-microliter aliquots of
each purified product were run on 1% agarose gels to confirm the
presence of a product migrating at the expected size for each ORF.
Array Printing. Glass slides were coated with polylysine according
to the protocol of Eisen and Brown (18). PCR products were
spotted by using a GMS 417 arrayer (Affymetrix) or a Q arrayer
(Genetix). DNA corresponding to the ORF of an unrelated
gene, gfp from Aequorea victoria, was included to serve as a
control for spotting and hybridization conditions.
Preparation and Hybridization of Fluorescently Labeled DNA.
Genomic DNA (gDNA) was prepared from each strain by using
the Easy-DNA kit (Invitrogen) according to the manufacturer’s
instructions. For a 50-
l reaction, 2–3
g of gDNA was com-
bined with 3
g of random hexamer, heated to ⬎95°C for 5 min,
and chilled on ice. Remaining components were added to final
concentrations as follows: 0.05 mM dA/G/TTP, 0.5 mM Cy3 or
Cy5-dCTP (NEN), 1⫻ Eco Pol buffer, and 15 units of the
Klenow fragment of Escherichia coli polymerase (NEB, Beverly,
MA). The reaction was placed at 37°C for 2 h, and labeled DNA
was purified by using the Qiagen (Chatsworth, CA) PCR puri-
fication kit according to manufacturer’s instructions. Cy3- and
Cy5-labeled DNAs were combined and precipitated according to
standard protocols after addition of 50
g兾ml sheared salmon
sperm DNA. DNA was recovered by centrifugation, and the
pellet was washed with 70% EtOH and recentrifuged. The pellet
was briefly air dried, then resuspended in hybridization buffer
containing 50% formamide兾6⫻ SSC兾5⫻ Denhardt’s solution兾
0.5% SDS兾5mMKH
2
PO
4
. Labeled DNA was heated to ⬎95°C
for 5 min and chilled on ice before use in hybridization.
Printed slides were crosslinked in a UV Stratalinker 2400 (Strat-
agene) at 1,000 ⫻ 100
joules after brief hydration over boiling
dH
2
O. Crosslinked slides were washed briefly in 0.1% SDS, rinsed
twiceindH
2
O, then placed in a boiling dH
2
O bath for 3–5min.
Slides were immersed in ice-cold 95% EtOH, dried by centrifuga-
tion, then prehybridized at 42°Cforatleast1hin5⫻ SSC兾0.1%
SDS兾10 mg/ml BSA. Slides were washed twice in dH
2
O, twice in
95% EtOH, and dried by centrifugation. Hybridization was carried
out under glass cover slips (VWR) in a sealed wet box at 42°C
overnight. In at least one case for each test strain, the array was
hybridized under the exact same conditions, except at 44°C. Fol-
lowing hybridization, slides were washed for 10 min in each of the
following solutions: one time in 2⫻ SSC兾0.1% SDS heated to 55°C;
one time in 0.2⫻ SSC兾0.1% SDS at room temperature two times
in 0.2⫻ SSC, and were then dried by centrifugation.
Data Generation and Analysis. In a single array experiment, the
genomic content from one of the test strains was compared with
that of N16961. For each of the nine test strains, data were
compiled from at least three array experiments. For each test
strain, two independent gDNA preparations were used as tem-
plate, and both Cy3 and Cy5-dCTP were used in independent
labeling and hybridization experiments to account for any dif-
ferences in DNA preparation or dye incorporation.
Hybridized, washed slides were scanned for Cy5 and Cy3
fluorescence intensities by using a ScanArray 5000 (Packard
Instruments). Laser power and兾or PMT were adjusted such that
the two channels were balanced. The resulting files were ana-
lyzed using
GENEPIX 3.0 software (Axon Instruments, Foster City,
CA). Spots were excluded from analysis because of high local
background fluorescence, slide abnormalities, or weak intensity
as determined primarily by the background signal observed for
hybridization to the gfp spot. For graphical representation of the
results, gene absence or presence was converted to 1 for absent,
0 for present.
CLUSTER and TREEVIEW (19) were then used to
compile and visualize the data (Fig. 1) by binary analysis.
Verification of Absent Genes. Confirmation of absent genes in-
cluded verification by Southern analysis, PCR analysis, or both
methods. Standard 50-
l PCR reactions using Taq DNA poly-
merase were performed according to the manufacturer’s instruc-
tions (Invitrogen). Southern analysis was performed by using
standard protocols and the ECL method of probe labeling and
detection (Amersham Pharmacia).
Results
Array Construction and Evaluation. The V. cholerae microarray we
constructed was composed of gene-length PCR products. Primer
design was based on the initial release of the V. cholerae N16961
genomic sequence by TIGR, which reported 3,890 ORFs (com-
pared with 3,885 ORFs in the final, published sequence). Of the
3,890 ORFs, gel electrophoresis revealed successful amplifica-
tion of 3,632 ORFs, representing 93.5% of the genome. Seventy-
two percent of PCR reactions classified as ‘‘failed’’ were from
ORFs predicted to be less than 200 bp in length. Because it can
be difficult to visualize short PCR products by agarose gel
electrophoresis, it is possible that for some of these small ORFs,
a significant amount of PCR product was actually generated and
spotted on the array. In support of this possibility, significant
signal was observed for some of the spots corresponding to these
reactions when the array was hybridized with labeled N16961
DNA. In addition, a number of the ‘‘failed’’ reactions corre-
sponding to ORFs of greater than 500 bp resulted in spots having
significant fluorescent signal (greater than 5,000 units). On
Table 1. Strains used for comparative analysis
Strain Origin Year isolated Biotype, serogroup
No. genes
absent*
N16961 Bangladesh 1971 El Tor, O1 (7th pandemic) N兾A
2740-80 Gulf Coast, United States 1980 environmental, nontoxigenic El Tor, O1 49
NCTC 8457 Saudi Arabia 1910 El Tor, O1 (prepandemic) 39
MAK 757 Celebes Islands 1937 El Tor, O1 (prepandemic) 49
569B India 1948 Classical, O1 46
O395 India 1965 Classical, O1 36
NIH 41 India 1940 Classical, O1 48
HK1 Hong Kong 1961 El Tor, O1 (7th pandemic) 0
C6709 Peru 1991 El Tor, O1 (7th pandemic) 1
MO10 India 1992 El Tor, O139 47
*As determined by microarray, PCR, and兾or Southern analysis.
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further inspection, many of the PCR reactions originally classi-
fied as ‘‘failed’’ actually produced weak bands that were barely
visible by gel electrophoresis. Therefore, we believe that greater
than 93.5% of the genome is truly represented by the array.
Classification of Genes from Test Strains as Absent or Present. The
background-corrected fluorescence ratio of N16961-labeled
DNA divided by that of the test strain (N16961兾test strain) was
determined for each spot on the microarray by using the
GENEPIX
3.0
software. Microarray results for ⬇30 genes were chosen over
a range of ratios, and the presence or absence of a gene was
confirmed by PCR analysis using original ORF primers. This led
to the following empirical metric used to report genes as absent
or present for any given spot: if the N16961兾test strain fluores-
cence ratio was ⱖ3.0, we had high confidence (90–100%) that
the gene is absent in the test strain.
Conservation of the Genome Between N16961 and Test Strains. Array
analysis combined with PCR and Southern analysis identified 143
genes as absent from eight of the nine test strains that were
compared with N16961 (Fig. 1, Table 1). Strain HK1 (an early 7th
pandemic isolate) seems to be identical to strain N16961. Strain
C6709 (a late 7th pandemic isolate) is missing only a single gene
(described below). The remaining seven test strains are each
missing between 36 and 49 genes (Table 1). Surprisingly, this
represents only a ⬇1% difference between the genomes of most test
strains and the sequenced strain N16961. This is in contrast to
results reported for Staphylococcus aureus and Helicobacter pylori
(20, 21). Comparative studies using clinical isolates of these bacteria
identified approximately a 12% difference between S. aureus
clinical strains and the reference strain, and a 6% difference
between two sequenced strains of H. pylori. Our results suggest a
remarkable conservation of genomic information among the V.
cholerae strains described here, despite their isolation over the past
century.
For a number of cases, we found that the genes absent from test
strains are clustered in the N16961 genome. These loci may
correspond to multigene insertions or chromosomal ‘‘islands’’ (see
below). However, the microarray data did not always indicate that
every gene within that region was absent. In some cases, PCR
and兾or Southern analysis was conducted to confirm the presence or
absence of genes within a suspected island insertion.
Genes we identified as absent fell into four main groups (Table
2). We were most interested in three of these groups (Fig. 2).
First, we found genes that could differentiate classical biotype
strains from El Tor biotype strains. These genes would be absent
only from classical strains and present in all other strains selected
for analysis (group I). Because the array was constructed by using
the N16961 genome (a 7th pandemic isolate), we could not
identify genes present only in classical strains and absent in all
other strains. Although the ‘‘nonclassical’’ strains selected for
this analysis include El Tor strains of diverse origins, all are
TCP⫹ and therefore potentially pathogenic. Second, we iden-
tified genes present only in pandemic strains, including classical
isolates (group II). Third, we found genes specific to 7th
pandemic strains, including epidemic and endemic El Tor O1
strains and the closely related O139 strain MO10 (group III).
Group I: Genes Present in All El Tor Strains but Not Classical Strains.
Because classical and El Tor strains are believed to have evolved
from separate lineages (12, 15), we sought to identify genes that
would uniquely define strains of the El Tor biotype. Only seven
genes were absent solely in classical strains but present in all
other strains (Fig. 2). Interestingly, five of the seven are located
on the small chromosome. Of these five, three are hypothetical
Fig. 1. Representation of absent兾present genes in nine test strains compared
with N16961. Final absent兾present calls for each gene were translated to
binary code and analyzed using the
CLUSTER兾TREEVIEW software of Eisen et al.
(19). For each strain, a black line indicates the presence of a gene, whereas a
white line indicates its absence. Black lines represent all genes from strain
N16961, as this strain’s sequence was used to construct the array.
Table 2. Classification of absent genes
Group
Strains where
genes are
absent
No. of
genes
absent*
Group I O395 7
Genes present in El Tor but not classical strains. 569B 7
NIH41 7
Group II 2740-80 14
Genes present only in strains able to cause
epidemic disease (absent from environmental
and prepandemic El Tor).
NCTC8457
MAK757
14
2
Group III 2740-80 22
Genes present only in 7th pandemic strains HK1, NCTC8457 22
C6709, and MO10. MAK757 22
O395 22
569B 22
NIH41 22
Group IV MO10 42
Genes uniquely absent from a single strain. NIH41 14
MAK757 15
*As identified by array, PCR, and兾or Southern analysis.
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proteins, for which we have no further information (VCA0728–
VCA0730). The other two, VCA0316 and VCA0417, are iden-
tical genes predicted to encode a putative acetyltransferase. Both
of these ORFs reside on the integron island, a gene capture
system located on the small chromosome (22). PCR analysis
using primers for neighboring genes combined with an ORF
primer revealed that strain 2740-80 carries VCA0417 only,
whereas strain MO10 carries only VCA0316. The conserved
physical location of the VCA0417 and VCA0316 genes in these
two strains suggests that each strain suffered an independent
deletion of one copy of this particular gene. Strains NCTC8457,
MAK757, HK1, and C6709 carry both copies of the gene.
The remaining two group I genes are located on the large
chromosome in the region encoding the RTX toxin. Lin et al.
previously showed that in classical strains, there is a deletion in this
region that includes the rtxC gene (VC1450), which likely encodes
an acylation enzyme that activates the toxin (23). Array analysis
confirmed the absence of the rtxC gene in classical strains, as well
as the absence of an adjoining gene predicted to encode a hypo-
thetical protein (VC1449). In classical strains, the RTX deletion
extends into the flanking genes that encode RtxA (VC1451) and
RtxB (VC1448). However, there is sufficient sequence remaining
for each of these two genes to provide hybridization on the array
with labeled DNA from classical strains.
We were surprised that so few genes uniquely define the El
Tor biotype. These data suggest that classical biotype strains may
be derived from a primordial environmental strain that was more
El Tor-like than previously thought.
Group II: Genes Present Only in Pandemic Strains. This group repre-
sents genes that differentiate classical and 7th pandemic El Tor
strains from environmental (2740-80) and prepandemic El Tor
strains (MAK757 and NCTC8457, Fig. 2). NCTC8457 was isolated
from individuals in an El Tor quarantine camp in 1910, and
MAK757 was a clinical isolate from the Celebes Islands in 1935 (2,
6). Consistent with their isolation from human stools, both of these
early El Tor, pre-7th pandemic strains were positive for TCP island
genes (and thus likely encode the pilus colonization factor TCP).
Strain 2740-80 is a nontoxigenic United States Gulf Coast envi-
ronmental isolate (3, 5). Work by Goldberg and Murphy (24) has
provided strong evidence that this strain is clonal with toxigenic
CTX⫹ strains isolated from patients and Gulf Coast environmental
sources. As expected, strain 2740-80 contains the TCP pathogenic-
ity island but not the CTX
genome. One additional absent gene
was common to strain 2740-80 and NCTC8457: VC0510, whose
product is similar to the E. coli RadC protein involved in DNA
repair (25). Five absent genes were common to 2740-80 and
MAK757, all encoding hypothetical proteins (data not shown).
Strain MAK757 seems to have all of the CTX
genes except
for VC1455 and VC1464, which are identical genes encoding the
phage transcriptional regulator RstR. Waldor et al. have re-
ported that RstR represses the transcription of rstA2, whose gene
product is required for CTX
replication (26). Although not
identified by our array analysis, sequence divergence has been
shown for RstR, consistent with its role in the formation of
heteroimmune temperate phages (27, 28). We therefore predict
that the CTX
genome present in MAK757 possesses highly
divergent copies of the rstR gene that are not detectable by
microarray analysis. If so, this result is consistent with the
independent emergence of MAK757 as a toxigenic clone distinct
from earlier classical strains and the future 7th pandemic clone.
Group III: Genes Present Only in 7th Pandemic El Tor O1 Strains. A
total of 22 genes is missing from classical isolates (O395, 569B,
and NIH41) and prepandemic TCP⫹ El Tor strains (NCTC8457
and MAK757). These genes are also absent from the environ-
mental isolate 2740-80. Array analysis identified 15 of these
genes, and Southern and PCR analysis assigned an additional 7
genes to this group (Fig. 2).
Seven genes identified by the array span a 16-kb region from
VC0175 to VC0185, and Southern analysis confirmed that four
additional genes within this region are also absent. This region
therefore represents a block of 11 genes in 7th pandemic strains
that has an uncharacteristically low GC content (40% vs. 47% for
the entire genome), suggesting that it was likely acquired by
horizontal transfer. Thus, we have designated this region ‘‘Vibrio
seventh pandemic island-I’’ or VSP-I.
Seven of the genes in VSP-I encode hypothetical or conserved
hypothetical proteins with no known function. The annotation of
three other genes further suggests that this region may have been
derived from a mobile genetic element. VC0185, which defines the
3⬘ end of the region, is predicted to encode a putative transposase,
similar to that identified in the S. aureus transposon Tn554 (29).
VC0175 shows similarity in its C-terminal domain to deoxycytidy-
late deaminase-related proteins. Deaminase proteins from a num-
ber of other species perform a variety of functions and can be
Fig. 2. Group I–III genes identified by array, Southern, and PCR analysis. Absent genes were categorized based on criteria listed in Table 2. Black squares indicate
genes identified as absent by array analysis and confirmed as absent by PCR or Southern analysis; gray squares indicate genes identified as absent by array analysis
alone; and white squares indicate genes identified as absent by PCR or Southern analysis. The two regions of group III genes comprising newly identified Vibrio
seventh pandemic islands are labeled VSP-I and VSP-II.
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involved in nucleotide scavenging or DNA uptake during compe-
tence (30, 31). Phage T2 and T4 also possess dCMP deaminase
proteins (32, 33), although the similarity to VC0175 at the amino
acid level is poor. VC0176 is homologous to the xre transcriptional
regulator from the defective PBSX prophage of Bacillus subtilis (34)
and contains a helix-turn-helix motif. Interestingly, VC0176 en-
codes a product belonging to a paralogous family that includes the
lysogeny repressor protein for CTX
, RstR. Thus, the product of
VC0176 may serve as a regulator of genes within this region.
Another gene in this region, VC0178, shows significant homology
to a variety of phospholipases and is annotated as a ‘‘patatin-related
protein.’’ Patatin is a major storage protein found in potato tubers
and roots and has recently been shown to encode a novel phos-
pholipase A activity (35).
We sequenced the chromosomal region corresponding to the
‘‘empty site’’ for insertion of the VSP-I island in the strains
missing this island. The sequence was identical in these strains
and shows that the flanking genes (VC0174 and VC0186) are
intact. The junction lies within the intergenic region between
VC0174 and VC0175 and encompasses 100 base pairs down-
stream of the VC0174 stop codon. This 100-bp region has a lower
GC content than the rest of the genome (41%) and contains both
inverted and direct repeats as well as a 14-bp palindromic
sequence. The junction abuts the 3⬘ end of VC0185, which is
transcribed on the negative DNA strand. Three additional
nucleotides, predicted to encode an arginine residue, are in-
serted before the stop codon of VC0185 (data not shown).
Another region unique to 7th pandemic strains encompasses
eight genes spanning VC0490–VC0497. Array analysis identified
VC0490, VC0493, VC0494, and VC0496, and PCR analysis dem-
onstrated that VC0491, VC0492, and VC0497 were also present
only in 7th pandemic strains. Although PCR analysis of VC0495 was
inconclusive, we believe that other methods will show it is a group
III gene based on its location within this block of genes. Like VSP-I,
the average GC content of genes VC490–VC0497 is lower than
average (41% vs. 47% for the rest of the chromosome), suggesting
that this region was also acquired by horizontal transfer. We
therefore propose that this region likely represents a second
chromosomal island, which we now designate as ‘‘Vibrio seventh
pandemic island-II’’ or VSP-II. The boundaries of VSP-II are not
precisely defined because of primer-related PCR technical prob-
lems. For example, we have not yet definitively confirmed the
absence or presence of VC0498–VC0502 in nonepidemic兾
pandemic strains. If missing, the size of VSP-II would increase from
7.5kbto⬇13.5 kb. Like VSP-I, VSP-II is composed of genes
predicted to encode hypothetical and conserved hypothetical pro-
teins. However, VC0497 is predicted to encode a transcriptional
regulator, similar to a bacteriophage P4 protein (36).
Three other genes outside of VSP-I and VSP-II fit the group III
pattern of distribution. Genes VC0514, encoding a methyl-
accepting chemotaxis protein, and VC0516, encoding a putative
phage integrase, were also located in regions of uncharacteristically
low GC content. However, the absence of flanking genes that may
contribute to a larger deletion awaits confirmation by another
method. VC0514 is one of 42 methyl-accepting chemotaxis proteins
identified by the sequencing project, all of which belong to a single
paralogous family. VC0514 was the sole gene identified as absent
from 7th pandemic strain C6709. Finally, a single, isolated gene
located on the integron island (VCA0300) belongs to group III and
is predicted to encode a chloramphenicol acetyltransferase.
Group IV: Genes Uniquely Absent from Individual Strains. Based on
array data, MO10 is missing two large regions of genes, which for
simplicity are not shown in Fig. 2 (see Fig. 1). The first is from
VC0241 to VC0270 and includes genes that encode enzymes
involved in O antigen capsule and O139 lipopolysaccharide
antigen synthesis. Because MO10 is an O139 serogroup strain
and has an O antigen composition that differs from that of O1
strains, this result was expected.
The second region spans genes VC1761–VC1786. It includes
gene VC1776, encoding a putative N-acetylneuraminate lyase,
and VC1784, encoding the NanH neuraminidase. Both genes
encode potential virulence factors, as cleavage of sialic acid
residues from certain host gangliosides increases the sensitivity
of host cells to cholera toxin (37). Nonetheless, PCR results
verified that these two genes are absent in pandemic strain
MO10. The missing region also includes genes VC1765 and
VC1776, encoding a putative type I restriction enzyme and its
cognate DNA methylase. In strain N16961, this region has a
lower than average GC content. Together, these results suggest
that this region might have originally been acquired by horizontal
transfer (probably by the archaic precursor of both classical and
El Tor strains) but was subsequently lost by strain MO10.
There is evidence of other potential strain-specific deletions.
Classical strain NIH41 is apparently missing genes necessary for
citrate metabolism, as 14 genes within a 20-gene region from
VC0790 to VC0809 were called as absent by array analysis.
Pre-7th pandemic El Tor strain MAK757 is missing a number of
genes that likely encompass genes VC1791–VC1817. This region
includes genes encoding a putative transcriptional regulator and
a deoxyribodipyrimidine photolyase, likely involved in DNA
repair. Microarray analysis also detected the previously reported
deletion of the toxS gene in classical strain 569B (38, 39).
Conclusions
Microarray technology is a powerful new tool that allows global
comparative analysis of gene content between different bacterial
isolates of a given species. We have used this method to investigate
the genetic similarity among strains of V. cholerae isolated from
diverse geographical locales and over decades of time. Our analysis
indicates that the nine test strains of V. cholerae we selected show
a remarkably close degree of relatedness to strain N16961. Al-
though the strains varied in biotype, serogroup, and year and site of
isolation, as a group they lacked ⬇1% of N16961 genes. Because of
the asymmetric nature of microarray analysis, we cannot say
whether the test strains carry more genes than N16961.
The close relatedness of the test strains to N16961 suggests
that only a small group of V. cholerae strains might be capable
of evolving to become human pathogens through the acquisition
of the TCP pathogenicity island and the CTX
genome. How-
ever, this conclusion assumes that other nonpathogenic (i.e.,
TCP⫺ and CTX⫺) environmental isolates of V. cholerae (in-
cluding non-O1 and non-O139 serogroup strains) will be quite
different in gene content when compared with N16961. This
analysis remains to be done. If such further analysis reveals that
these nonpathogenic strains are as similar to N16961 as the test
strains we selected, then we must conclude that virtually any
strain of V. cholerae has the capacity to become a human
pathogen simply by acquisition of the TCP island, the CTX
, and
perhaps the genes encoding a few other select properties (such
as production of O1 or O139 O antigens). We are currently
investigating this possibility by analyzing V. cholerae isolates
representing environmentally well adapted, non-O1 and non-
O139 strains. These isolates do not carry virulence genes usually
associated with strains that cause cholera. Such studies should
provide a near-complete evolutionary history of how the envi-
ronmental organism V. cholerae can become a human pathogen.
It is important to appreciate that the genetic changes that have
allowed V. cholerae to become a human pathogen may not be the
sole determinants of its success as an endemic and pandemic
pathogen. For example, at least one strain of pathogenic V.
cholerae El Tor caused an epidemic before the beginning of the
7th pandemic (i.e., MAK757 in Indonesia in 1935), and another
distinct El Tor clone caused sporadic cholera cases decades later
along the Gulf Coast of the United States (2–6). Yet, these
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strains did not spread to cause significant disease outside the
locales where they initially emerged. In contrast, 7th pandemic
El Tor strains have flourished globally over four decades since
their emergence, displacing resident classical strains in South
Asia and establishing their endemic presence virtually every-
where they have been introduced (7).
Investigators using a variety of molecular typing methods have
concluded that 7th pandemic strains represent a globally dis-
tributed clone that is closely related to the more recently
emerged O139 clone (3, 10–15). Because our microarray was
constructed based on the genomic sequence of the 7th pandemic
strain N16961, we believed that comparative analysis would
provide supporting evidence for the clonal nature of 7th pan-
demic strains. As predicted, we found no or only minor differ-
ences between N16961 and the 7th pandemic strains HK1 and
C6709 as well as the O139 strain MO10. We further hypothesized
that 7th pandemic strains would have unique gene content that
might contribute to their apparent fitness as endemic and
pandemic agents of cholera. This prediction was also supported
by the microarray analysis, in that we identified genes specific to
7th pandemic isolates of V. cholerae. Most of these group III
genes are located in two chromosomal gene clusters, which we
have designated VSP-I and VSP-II (Fig. 2).
The VSP-I and VSP-II gene clusters have a lower than
average GC content when compared with the rest of the V.
cholerae chromosome, suggesting that these gene clusters
represent chromosomal ‘‘islands’’ acquired by horizontal
transfer. Although we cannot determine when these islands
were acquired by V. cholerae, two possibilities seem most
probable. The VSP-I and VSP-II islands might have been
acquired by a fully pathogenic (i.e., TCP⫹, CTX⫹) pre-7th
pandemic El Tor O1. Alternatively, a primordial, nonpatho-
genic El Tor O1 strain acquired the VSP islands first, followed
by acquisition of the TCP and CTX genetic elements to
complete its emergence as human pathogen.
We predict that the genes associated with the VSP-I and
VSP-II islands are likely to encode some of the properties that
are responsible for the unique characteristics of 7th pandemic
clones. Although three other genes located outside this region
(VC0514, VC0516, and VCA0300) show a similar group III
pattern and could also contribute to these traits, the VSP
islands encode far more group III genes and thus seem likely
to be the origin of 7th pandemic properties. These might
include adaptive properties that, for example, allow 7th pan-
demic strains to withstand nutrient deprivation or physical兾
chemical stresses and thus survive in aquatic environments
more efficiently than pre-7th pandemic strains. Such environ-
mental adaptation could also include genes that allow colo-
nization of non-human hosts, such as phytoplankton, filamen-
tous algae, or crustaceans.
Alternatively, the genes on the VSP islands might simply
increase adaptation of V. cholerae to the human host in ways
that may or may not manifest themselves as an increase in
pathogenicity per se. For instance, human adaptation genes
might encode functions that allow more efficient infection of
humans by, for example, promoting bacterial resistance to
stomach acid. These genes could also encode properties that
allow more extensive or prolonged intestinal colonization, thus
increasing the shedding of vibrios and seeding of environmen-
tal reservoirs.
If the genes of the VSP islands are, in fact, responsible for
more efficient (or prolonged) infection of human hosts rather
than improved survival in the aquatic ecosystems, we would
favor a conclusion that has recently been unpopular in the
field. The evolutionary success of the 7th pandemic clone of V.
cholerae as an endemic and pandemic pathogen may be more
related to its improved interaction with the human host than
to its improved fitness within environmental reservoirs. Now
that we have identified a number of unique genes that define
the 7th pandemic clone, it will be possible to delete these genes
systematically to address their potential roles in human infec-
tion and in promoting fitness of V. cholerae in environmental
ecosystems.
We thank Catherine Lee, Su Chiang, and James Bina for critically
reading the manuscript, and Christina Mills for generation of preliminary
data. This work was funded by National Institutes of Health Grants
AI18045 (to J.J.M.) and AI53535-02 (to TIGR).
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