Comparative genomics reveals mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae

Abstract

Vibrio cholerae, the causative agent of cholera, is a bacterium autochthonous to the aquatic environment, and a serious public health threat. V. cholerae serogroup O1 is responsible for the previous two cholera pandemics, in which classical and El Tor biotypes were dominant in the sixth and the current seventh pandemics, respectively. Cholera researchers continually face newly emerging and reemerging pathogenic clones carrying diverse combinations of phenotypic and genotypic properties, which significantly hampered control of the disease. To elucidate evolutionary mechanisms governing genetic diversity of pandemic V. cholerae, we compared the genome sequences of 23 V. cholerae strains isolated from a variety of sources over the past 98 years. The genome-based phylogeny revealed 12 distinct V. cholerae lineages, of which one comprises both O1 classical and El Tor biotypes. All seventh pandemic clones share nearly identical gene content. Using analogy to influenza virology, we define the transition from sixth to seventh pandemic strains as a "shift" between pathogenic clones belonging to the same O1 serogroup, but from significantly different phyletic lineages. In contrast, transition among clones during the present pandemic period is characterized as a "drift" between clones, differentiated mainly by varying composition of laterally transferred genomic islands, resulting in emergence of variants, exemplified by V. cholerae O139 and V. cholerae O1 El Tor hybrid clones. Based on the comparative genomics it is concluded that V. cholerae undergoes extensive genetic recombination via lateral gene transfer, and, therefore, genome assortment, not serogroup, should be used to define pathogenic V. cholerae clones.

Full text

Comparative genomics reveals mechanism for
short-term and long-term clonal transitions
in pandemic
Vibrio cholerae
Jongsik Chun
a,b,c
, Christopher J. Grim
b
, Nur A. Hasan
d,e
, Je Hee Lee
a,c
, Seon Young Choi
a,c
, Bradd J. Haley
d
, Elisa Taviani
d
,
Yoon-Seong Jeon
c
, Dong Wook Kim
c
, Jae-Hak Lee
a
, Thomas S. Brettin
f
, David C. Bruce
f
, Jean F. Challacombe
f
,
J. Chris Detter
f
, Cliff S. Han
f
, A. Christine Munk
f
, Olga Chertkov
f
, Linda Meincke
f
, Elizabeth Saunders
f
,
Ronald A. Walters
g
, Anwar Huq
d
, G. Balakrish Nair
h
, and Rita R. Colwell
b,d,i,1
aSchool of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 151-742, Republic of Korea; bCenter for Bioinformatics and
Computational Biology, University of Maryland Institute for Advanced Computer Studies, and dMaryland Pathogen Research Institute, University of
Maryland, College Park, MD 20742; cInternational Vaccine Institute, Seoul 151-818, Republic of Korea; eInternational Center for Diarrheal Disease
Research, Bangladesh, Dhaka-1000, Bangladesh; fBioscience Division, Department of Energy Joint Genome Institute, Los Alamos National Laboratory,
Los Alamos, NM 87545; gPacific Northwest National Laboratory, Richland, WA 99352; hNational Institute of Cholera and Enteric Diseases,
Beliaghata, Kolkata 700 010, India; and iJohns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205
Contributed by Rita R. Colwell, July 21, 2009 (sent for review May 19, 2009)
Vibrio cholerae, the causative agent of cholera, is a bacterium
autochthonous to the aquatic environment, and a serious public
health threat. V. cholerae serogroup O1 is responsible for the
previous two cholera pandemics, in which classical and El Tor
biotypes were dominant in the sixth and the current seventh
pandemics, respectively. Cholera researchers continually face
newly emerging and reemerging pathogenic clones carrying di-
verse combinations of phenotypic and genotypic properties, which
significantly hampered control of the disease. To elucidate evolu-
tionary mechanisms governing genetic diversity of pandemic V.
cholerae, we compared the genome sequences of 23 V. cholerae
strains isolated from a variety of sources over the past 98 years. The
genome-based phylogeny revealed 12 distinct V. cholerae lineages,
of which one comprises both O1 classical and El Tor biotypes. All
seventh pandemic clones share nearly identical gene content.
Using analogy to influenza virology, we define the transition from
sixth to seventh pandemic strains as a ‘‘shift’’ between pathogenic
clones belonging to the same O1 serogroup, but from significantly
different phyletic lineages. In contrast, transition among clones
during the present pandemic period is characterized as a ‘‘drift’’
between clones, differentiated mainly by varying composition of
laterally transferred genomic islands, resulting in emergence of
variants, exemplified by V. cholerae O139 and V. cholerae O1 El Tor
hybrid clones. Based on the comparative genomics it is concluded
that V. cholerae undergoes extensive genetic recombination via
lateral gene transfer, and, therefore, genome assortment, not
serogroup, should be used to define pathogenic V. cholerae clones.
genomic islands 兩cholera toxin prophage 兩lateral gene transfer
Vibrio cholerae, a bacterium autochthonous to the aquatic
environment, is the causative agent of cholera, a severe, water y,
life-threatening diarrheal disease. Historically, cholera bacteria
have been serogrouped based on their somatic O antigens, with
⬎200 serogroups identified to date (1). Although strains from many
of the serogroups of V. cholerae have caused either individual cases
of mild gastroenteritis or local outbreaks of gastroenteritis, only the
toxigenic strains of serogroups O1 and O139 have been identified
as agents of cholera epidemics. Genes coding for cholera toxin,
ctxAB, and other virulence factors have been shown to reside in
bacteriophages and various mobile genetic elements. In addition, V.
cholerae serogroup O1 is differentiated into two biotypes, classical
and El Tor, by a combination of biochemical traits and sensitivity
to specific bacteriophages (2).
Throughout human history cholera pandemics have been re-
corded with seven such pandemics characterized over the past
hundred or more years. Today the disease remains endemic only in
developing countries, even though V. cholerae is native to estuaries
and river systems throughout the world (3). Isolates of the sixth
pandemic were almost exclusively of the O1 classical biotype,
whereas the current (seventh) pandemic is dominated by V. cholerae
O1 El Tor biotype, a transition occurring between 1905 and 1961.
The six pandemics previous to the current pandemic are considered
to have originated in the Indian subcontinent, whereas the seventh
pandemic strain was first isolated in the Indonesian island of
Sulawesi in 1961, and subsequently in Asia, Africa, and Latin
America.
Over the last 20 years, several new epidemic lineages of V.
cholerae O1 El Tor have emerged or reemerged. In 1992, a new
serogroup of V. cholerae, O139, was identified as the cause of
epidemic cholera in India and Bangladesh (4). That is, both V.
cholerae O1 El Tor and O139 consistently have been isolated where
the major cholera epidemics have occurred since 1992, although V.
cholerae O139 appears still to be restricted to Asia. Additionally, V.
cholerae ‘‘hybrid’’ O1 El Tor variants that carry the classical type
CTX prophage, or produce classical type cholera toxin subunit B
have been isolated repeatedly in Bangladesh (5, 6) and Mozam-
bique (7). These new variants have replaced the prototype seventh
pandemic V. cholerae O1 El Tor strains in Asia and Africa, with
respect to frequency of isolation from clinical cases of cholera.
It is clear that the dynamics of V. cholerae, like other enteric
pathogens, involve extensive lateral gene transfer via transduction,
conjugation, and transformation (2, 8, 9). However, the evolution-
ary history of this bacterium remains to be documented. Here, we
compare the genome sequences of 23 V. cholerae strains (Table 1),
representing diverse serogroups isolated at various times over the
past 98 years from a variety of sources and geographical locations.
We conclude that the current pandemic is caused by strains
belonging to a single phyletic line, diversified mainly by lateral gene
transfer occurring in the natural environment.
Results and Discussion
Phylogeny and Gene Content of
Vibrio cholerae
.Phylogenetic anal-
ysis, accomplished using ⬇1.4 million bp of orthologous protein-
coding regions for 23 V. cholerae strains, revealed 12 distinct
Author contributions: J.C., C.J.G., R.A.W., A.H., G.B.N. and R.R.C. designed research; N.A.H.,
T.S.B., D.C.B., J.F.C., J.C.D., C.S.H., A.C.M., O.C., L.M., and E.S. performed research; J.C.,
C.J.G., N.A.H., J.H.L., S.Y.C., B.J.H., E.T., Y.-S.J., D.W.K., and J.-H.L analyzed data; and J.C. and
R.R.C. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: rcolwell@umiacs.umd.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0907787106/DCSupplemental.
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phyletic lineages. Strains belonging to non-O1/non-O139 sero-
groups from various sources showed significant genomic diversity
(Fig. 1A). In fact, each unique phyletic line adds 206 new genes to
the pan-genome of V. cholerae, on average (See SI Text and Fig. S1).
In contrast, all V. cholerae serogroup O1 strains, except for two,
comprised a monophyletic clade, designated V. cholerae phylocore
genome (PG) clade. Strains of both the sixth and seventh pandemics
are concluded to have evolved from a common ancestor of this PG
clade.
Twelve strains of the PG clade were further divided into two
subgroups, as shown in the phylogenetic tree constructed using
⬇2.6 million bp alignment (Fig. 1 Aand B). The PG-1 subclade is
comprised of most of the V. cholerae O1 El Tor strains and one V.
cholerae O139 strain, whereas the PG-2 subclade contains strains of
V. cholerae O1 classical and O37 serogroups. Interestingly, all
clinical isolates associated with the current seventh cholera pan-
demic formed a very tight, monophyletic clade within the PG-1
subclade, which we have designated the seventh pandemic (7P)
clade (Fig. 1C). V. cholerae O1 El Tor and O139 strains isolated
from the Indian subcontinent and Africa epidemics during 1975 to
2004 are located in the 7P clade. We use the terms shift and drift
in a manner similar in some respects to their use in studies of the
influenza virus. In particular, we use shift to refer to long-term
accumulation of numerous base pair mutations whereas drift to
refer to short-term changes resulting from horizontal acquisition of
genomic islands.
O Serogrouping in the Context of Genome Evolution. The lipopoly-
saccharide (LPS) of V. cholerae consists of three major regions: lipid
A, core oligosaccharide (OS), and O antigen. V. cholerae synthesizes
Table 1. Characteristics of Vibrio cholerae strains analyzed in this study
Strain
Genome
code Serogroup Biotype
Geographical
Origin
Source of
isolation
Year of
isolation
Sequencing
status*
No. of
contigs Accession
N16961 VCN16961 O1 Inaba El Tor Bangladesh Clinical 1975 Complete 2 AE003852/AE003853
RC9 VCRC9 O1 Ogawa El Tor Kenya Clinical 1985 S/4/E 11 ACHX00000000
MJ-1236 VCMJ1236 O1 Inaba El Tor Matlab, Bangladesh Clinical 1994 Complete 2 CP001485/CP001486
B33 VCB33 O1 Ogawa El Tor Beira, Mozambique Clinical 2004 S/4/E 17 ACHZ00000000
CIRS 101 VCCIRS101 O1 Inaba El Tor Dhaka, Bangladesh Clinical 2002 S/4/E 18 ACVW00000000
MO10 VCMO10 O139 Madras, India Clinical 1992 S/4 84 AAKF03000000
2740–80 VC274080 O1 Inaba El Tor US Gulf Coast Water 1980 Sanger 257 AAUT01000000
BX330286 VCBX330286 O1 Inaba El Tor Australia Water 1986 Complete 8 ACIA00000000
MAK757 VCMAK757 O1 Ogawa El Tor Celebes Islands Clinical 1937 Sanger 206 AAUS00000000
NCTC 8457 VC8457 O1 Inaba El Tor Saudi Arabia Clinical 1910 Sanger 390 AAWD01000000
O395 VCO395 O1 Ogawa Classical India Clinical 1965 Complete 2 CP000626/CP000627
V52 VCV52 O37 Sudan Clinical 1968 Sanger 268 AAKJ02000000
12129(1) VC12129 O1 Inaba El Tor Australia Water 1985 S/4/E 12 ACFQ00000000
TM 11079-80 VCTM11079 O1 Ogawa El Tor Brazil Sewage 1980 S/4/E 35 ACHW00000000
VL426 VCVL426 non-O1/O139 Albensis Maidstone, Kent, UK Water Unknown Complete 5 ACHV00000000
TMA21 VCTMA21 non-O1/O139 Brazil Seawater 1982 S/4/E 20 ACHY00000000
1587 VC1587 O12 Lima, Peru Clinical 1994 Sanger 254 AAUR01000000
RC385 VCRC385 O135 Chesapeake Bay Plankton 1998 Sanger 550 AAKH02000000
MZO-2 VCMZO2 O14 Bangladesh Clinical 2001 Sanger 162 AAWF01000000
V51 VCV51 O141 USA Clinical 1987 Sanger 360 AAKI02000000
MZO-3 VCMZO3 O37 Bangladesh Clinical 2001 Sanger 292 AAUU01000000
AM-19226 VCAM19226 O39 Bangladesh Clinical 2001 Sanger 154 AATY01000000
623–39 VC62339 non-O1/O139 Bangladesh Water 2002 Sanger 314 AAWG00000000
*Sanger, Draft assemblies by Sanger sequencing; S/4, Sanger sequencing and 454 pyrosequencing were combined; S/4/E, S/4 followed by quality improvement
by standard genome sequencing procedures.
O135 RC385
biovar albensis
VL426
O141
V51
non-O1/O139
623-39
O1 El Tor
12129(1)
O14 MZO-2
O12 1587
O37 MZO-3
Non-O1/O139
TMA21
O39
AM-19226
O1 El Tor
TM11079-80
Vibrio cholerae
Phylocore Genome (PG)
clade
PG-1
subclade
PG-2
subclade
O37
V52
O1 classical
O395
0.002
O37 V52
O1 classical O395
O1 El Tor 2740-80
O1 El Tor MAK757
O1 El Tor BX330286
O1 El Tor NCTC 8457
7th Pandemic (7P)
clade
0.0005
100
100
100
100
Genome “Shift”
c
Genome “Drift”
A
O1 El Tor RC9
O1 El Tor N16961
O139 MO10
O1 El Tor B33
O1 El Tor MJ-1236
100
100
99
0.000005
O1 El Tor CIRS 101
100
B
C
Fig. 1. Neighbor-joining trees showing phylogenetic relationships of 23 V. cholerae strains representing diverse serogroups. (A) All V. cholerae strains based
on 1,676 genes (1,370,469 bp). (B) Phylocore genome (PG) clade based on 2,663 genes (2,567,393 bp). (C) Seventh pandemic (7P) clade based on 3,364 genes
(3,291,577 bp). Bootstrap supports, as percentage, are indicated at the branching points. Bars represent the numbers of substitution per site, respectively. Only
orthologous genes showing ⬎95% nucleotide sequence similarity to those of V. cholerae N16961 were selected. The tree was rooted using Vibrio vulnificus YJ016
and Vibrio parahaemolyticus RIMD 2210633.
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core OS and O antigen using wav and wb* gene clusters, respectively
(10, 11). Molecular phylogeny and genetic organization of the wav
and wb* gene clusters are summarized in SI Text, and Figs. S2 and
S3.
In contrast to the limited diversity observed in the wav gene
cluster (5 major types), 11 different types of wb* gene clusters were
observed among the 23 strains. Phylogeny and genetic organization,
based on the whole genome (Fig. 1), core OS, and O antigen gene
clusters (Fig. S2A), clearly indicate both core OS and O antigen
gene clusters have been horizontally transferred. The relatively
stable gene order (synteny) of the core OS gene cluster suggests that
it transfers as an entity. In contrast, the region coding for the O
antigen is comprised of combinations of several smaller gene sets
of different origin, leading to a remarkable diversity of the various
O antigens seen in nature (Fig. S2B). This finding is in good
agreement with the study in ref. 12 showing that the gene cluster
coding for the O139 antigen is similar to that of V. cholerae
serogroup O22, where substitution of a part of the cluster occurred,
but not a deletion.
Genome phylogeny (Fig. 1A) revealed that strains of O1 sero-
group are found in three different phyletic lineages, namely the PG
clade, and the V. cholerae O1 El Tor 12129 (1) and TM11079-80
strains, in which the coding region for the O1 antigen is nearly
identical. It is concluded that the O1 antigen phenotype arose by
lateral gene exchange at least three times in the evolution of V.
cholerae presented here. Furthermore, we hypothesize that the
ancestor of the PG clade possessed a combination of the type 1 core
OS and the O1 antigen gene clusters, giving rise to the present 12
V. cholerae PG strains, including the two V. cholerae non-O1 strains
(V52 and MO10). The latter two became different serogroups by
gene replacement, via lateral gene transfer, with strain V52 receiv-
ing both type 1 core OS and O37 antigen gene clusters from a V.
cholerae O37 strain and V. cholerae MO10 receiving only the V.
cholerae O139 antigen gene cluster from an unknown source, most
likely a variant of the V. cholerae O22 serogroup (12).
The V. cholerae O1 strains not belonging to the PG group, V.
cholerae 12129 (1) and TM11079-80, are environmental isolates
from Australia isolated in 1985 (13), and from Brazil, isolated in
1980 (14). They showed the typical El Tor phenotype, but unlike
other V. cholerae O1 El Tor strains in the PG-1 subclade, lack the
two major virulence-related genomic islands, i.e., CTX prophage
containing ctxAB and Vibrio pathogenicity island-1 (VPI-1) con-
taining genes for biosynthesis of the toxin coregulated pilin (TCP).
By comparing genome phylogenies based on the whole genome
(Fig. 1) and gene clusters coding for the core OS (Fig. S2A) and O1
antigen (Fig. S2C), it is clear that genesis of these nontoxigenic V.
cholerae O1 El Tor strains can be attributed to independent lateral
gene transfer events, most probably transfer of only the O1 antigen
gene cluster, but not the core OS region.
Four O serogroup conversions, from non-O1 to O1 (twice), O1
to O139, and O1 to O37, were detected among the 23 V. cholerae.
Several previous studies suggested such conversions take place in
nature (11, 14, 15), and chitin-induced natural transformation has
been proposed as the mechanism in the natural environment (16).
V. cholerae O1 to O139 serogroup conversion by a single-step
exchange of large fragments of DNA was demonstrated in a
microcosm experiment (9), and is supported by the conclusion of
this study that O serogroup conversion occurs frequently in nature.
Mobility of the O phenotype in V. cholerae was first proposed by
Colwell et al. (17), and the cumulative results of both in vivo and in
vitro experiments are compelling. Given the inconsistency between
O serogroup typing and genome-based phylogeny, we conclude
that, at the very least, the term ‘‘O1 El Tor’’ is both misleading and
inaccurate for describing a set of phylogenetically coherent V.
cholerae strains, in light of the frequency of serogroup conversion.
Therefore, we propose a new terminology based on genome
sequence; namely the phylocore genome (PG) clade, PG-1 and
PG-2 subclades, and seventh pandemic (7P) clade, to describe
homologous intraspecific groups of V. cholerae (Fig. 1).
Virulence-Associated Prophage and Genomic Islands Within the Con-
text of the Genome. V. cholerae possesses several known virulence
factors, of which the cholera toxin (CT) and TCP are considered the
most significant. Genes coding for CT (ctxAB) are part of a
temperate filamentous bacteriophage CTX
␾
(8) that can be incor-
porated into both chromosomes of V. cholerae at specific positions.
The CTX
␾
genes were found to be present in members of the PG
clade, except for V. cholerae NCTC 8457 and 2740–80. Among
non-PG strains, only V. cholerae serogroup O141 (V51) contains
this prophage.
The CTX
␾
found in classical and El Tor biotypes differs in the
sequence of their repressor gene, rstR, and are classified as
CTX
␾
Class
and CTX
␾
El Tor
, according to the biotype of the original
hosts in which they were described (18). From the genome se-
quences, we found that CTX
␾
Class
is not restricted to the classical
biotype, but is also widely distributed in V. cholerae O1 El Tor and
O141 strains (Fig. 2). Given that V. cholerae O1 El Tor MAK 757,
a clinical strain isolated in 1937, has this type of prophage, corre-
lation of host biotype and prophage type is not considered signif-
icant. A recent study(19) showing the infection of CTX
␾
Class
to V.
cholerae non-O1 supports this finding.
Chromosomal attachment sites for CTX
␾
are known to harbor
Large chromosome Small chromosome
O1 El Tor
MJ-1236/B33 CTXClass CTXClass
O1 El Tor
RC9 TLC RS1 RS1 CTXEl Tor CTXEl Tor RS1
O1 El Tor
N16961 RS1
CTXEl Tor
TLC
Classical type CT B
O1 El Tor
NCTC 8457 GI-19
O39 AM-19226
O12 1587 GI-19 VSK
623-39 GI-33
GI-19
O39
MZO-3 GI-48
GI-19
TMA2 1
O135 RC385 GI-43
GI-33
O1 El Tor
12129(1) GI-19
O14
MZO-2 GI-19
O1 El Tor
TM11079-80 GI-33
VL426 GI-19
Truncated prophage
O1 El Tor
CIRS101 RS1
TLC CTXEl Tor
O139
MO10 CTXEl Tor
TLC VSK
O1 El Tor
BX330286 RS1Env
CTXClass CTXClass
TLC
O1 El Tor
MAK757 GI-22
TLC CTXClass
O1 El Tor
2740-80 TLC
O1 classical
O395 TLC CTXClass CTXClass CTXClass
O37 V52 *TLC CTXEl Tor RS1
O141 V51 †GI-33 CTXClass RS1
Fig. 2. Schematicrepresentation of various prophages and genetic elements
present in the target regions of CTX
␾
insetion. *, TLC, El Tor type CTX
␾
, RS1
element are found, but no positional information can be obtained from
genome assemblies. †, classical type CTX
␾
and RS1 are present, but no posi-
tional information can be obtained.
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other genetic elements, including toxin-linked cryptic (TLC), RS1
elements, and VSK(⫽pre-CTX) prophages (20, 21). We have
discovered five genomic islands (GI-19, GI-22, GI-33, GI-43, GI-48;
for details see Table S1) in the region of the CTX
␾
attachment sites
on both chromosomes. In total, nine distinct genetic elements were
found in these regions, where they appear in different combinations
(Fig. 2). Seven strains possess GI-19 in either chromosome, which
is similar but not identical to KSF-1
␾
, previously discovered in an
environmental V. cholerae strain (22). It is evident that more
bacteriophages/genetic elements are located in the CTX
␾
attach-
ment regions of PG strains than non-PG strains. The ability to
harbor more, especially toxigenic, bacteriophage-like elements in
these regions of the PG strains might explain why only PG strains
have been the agents of the pandemics. We found no two toxigenic
(CTX
␾
-harboring) strains with identical GI organization and com-
bination, with the exception of two hybrid strains (the only 7P
members harboring CTX
␾
Class
). It is evident from Fig. 2 that the
two CTX
␾
attachment sites serve as an engine of genetic diversity
for the V. cholerae PG clade.
Genes coding for TCP are part of a genomic island, VPI-1
present in all PG strains. Among the non-PG strains, only V.
cholerae O141 V51 contained VPI-1 but with less sequence simi-
larity. Because TCP serves as a receptor for CTX
␾
(8), it explains
why only this strain, of all non-PG strains, possesses CTX
␾
. Results
of phylogenetic analysis using the 24 genes of VPI-1 indicate that
the original GI of V. cholerae NCTC 8457 was replaced by VPI-1 of
a non-PG strain (Fig. S4). Interestingly, GI-47, but not VPI-1, was
found in strains MZO-3, 1587, MZO-2, and VL426 in the same
genomic region. This cassette-like property of GI mobility was also
observed for the other known pathogenicity islands, including
VPI-2, VSP-1, and VSP-2 (Table S2).
Extensive Lateral Gene Transfer in
V. cholerae
.Because it is generally
accepted that lateral gene transfer plays an important role in the
evolution of many pathogenic bacteria, V. cholerae serves as a useful
paradigm. For purposes of this study, a GI is defined as a genomic
region containing five or more ORFs, where transfer, but not
deletion, is obvious from comparison of genome phylogeny and its
presence/absence among test strains. A total of 73 GIs were
identified (Table S1) and their chromosomal locations are shown in
Fig. 3. As discussed above, with respect to GIs associated with O
antigen biosynthesis, CTX
␾
, VPI-1,2 and VSP-2, a total of 13
genomic regions (eight in the large and five in the small chromo-
some) were found to have a cassette-like property, whereby differ-
ent GIs occupy the same or a similar region (Table S2). Most GIs
were singletons in a given genome, although two (GI-12, GI-21)
were present as four and two copies, respectively. Thus, we con-
clude that genetic diversity of V. cholerae derives most significantly
from lateral gene transfer, of which several transfers are cassettes.
Genomic Definition of the
V. cholerae
Phylocore Genome (PG) Clade
and Pandemic Strains. The V. cholerae PG clade, with both sixth and
seventh pandemic strains, is defined by gene content. Twenty-seven
genes are present exclusively in the genomes of the PG strains, but
only five genes are unique to the PG-1 subclade. Four of these
(VCA0198 –VCA0201) comprise a genomic island (GI-5) on the
small chromosome, including genes coding for cytosine-specific
DNA methyltransferase (23) and hypothetical proteins, adjacently
located to the IS1004 transposase gene. The 7P strains are differ-
entiated in harboring two unique GIs, the Vibrio seventh pandemic
island-1 (VSP-1) and VSP-2, first discovered by microarray analysis
(24). In addition to the 7P strains, a variant of VSP-1 was found in
V. cholerae biovar albensis VL426 (Fig. S5). Similarly, VSP-2 like
GIs were detected in three non-PG strains (TMA21, O39 MZO-3,
O135 RC385). Interestingly, a similar GI was also detected in Vibrio
vulnificus YJ016 and Vibr io splendidus 12B01, suggesting that VSP-2
may be widespread among vibrios (Fig. S6). It should be noted that
the stability of these well known pathogenicity islands among 7P
members is questionable, because most of VPI-2 and VSP-2 were
deleted in MO10 and CIRS 101, respectively.
V. cholerae contains a superintegron, a large integron island
(gene capture system), in the small chromosome (⬇120 Kbp),
comprising predominantly hypothetical genes and proposed as a
source of genetic variation (25). All V. cholerae strains examined in
this study have this integron, a source of much of the variation in
gene content (Fig. S7). Interestingly, if this region is excluded, all six
members of the 7P clade have an identical gene content, with the
exception of a few genomic islands, including those found in the
CTX
␾
attachment region. An SXT element belonging to a family
of conjugative transposon-like mobile genetic elements encodes
multiple antibiotic resistance genes and is present only in V. cholerae
MO10, CIRS 101, MJ-1236, and B33, but not in the other V.
cholerae strains. V. cholerae O139 MO10 differs from other mem-
bers of the 7P clade in having an O139 antigen specific genomic
island, a finding strongly supporting the conclusion of several
previous studies that V. cholerae O139 derives from a seventh
Fig. 3. Genomic representation of genomic islands of both V. cholerae chromosomes. The two circles in the middle represent the genes in V.cholerae O1 El
Tor N16961. The inner circle indicates genomic islands found in strain N16961, whereas the outer circles are those absent in strain N16961.
Chun et al. PNAS
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pandemic V. cholerae O1 El Tor strain (26). No other V. cholerae
O139-specific genes were found in V. cholerae MO10.
The hybrid strains, possessing an El Tor biotype phenotype, but
classical biotype CTX
␾
, were isolated during current cholera
epidemics in Asia and Africa (6, 7). Two hybrid strains (B33 and
MJ-1236) share a virtually identical genome backbone. Among
3,587,239 bp of orthologous protein-coding regions, only 106 nu-
cleotide positions are different and the only significant difference
is the presence of a V. cholerae MJ-1236 specific 19,729 bp genomic
island (GI-12). This GI occurs four times as an almost identical
sequence in the large chromosome, with 14 genes including those
coding for the putative phage integrase and type I restriction-
modification system, probably a recently introduced temperate
bacteriophage. It is not clear why the hybrid strains outcompete V.
cholerae O1 El Tor/O139 in the clinical setting, but a key to this
puzzle surely lies in differences among closely related strains, i.e.,
tandem copies of CTX
␾
Class
, GI-14 and single nucleotide polymor-
phisms. In addition to these hybrid clones, V. cholerae O1 El Tor
strains producing the classical type of cholera toxin B repeatedly
have been isolated from patients in Asia and Africa (6). The
genome sequence of a representative of this newly emerged group,
i.e., V. cholerae CIRS 101, reveals that these strains also have a
typical 7P gene content, but with CTX
␾
El Tor
, not CTX
␾
Class
, albeit
expressing the classical type subunit B protein (Fig. 2).
The comparative genomics of phylogenetically diverse strains has
permitted analysis of the mechanism by which current seventh
pandemic clones may have arisen. An highly conserved gene
content, synteny, and significant similarity among the six strains of
the 7P clade indicate that these V. cholerae strains share an almost
identical genome ‘‘backbone,’’ having evolved very recently from a
common ancestral strain. An hypothetical evolutionary pathway
proposed for V. cholerae (Fig. 4), with GI migration matched to a
genome-based phylogenetic tree, allows the conclusion that the
ancestor for the 7P clade was a V. cholerae O1 El Tor strain
containing several GIs (VPI-1,2, GI-1 to GI-10), receiving VSP-1,
VSP-2 and GI-11 by lateral gene transfer, and finally giving rise to
the contemporary V. cholerae O1 El Tor and O139 strains. Inter-
estingly, such an hypothetical ancestral strain shows a gene content
similar to V. cholerae O1 El Tor BX330286, isolated from a water
sample collected in Australia in 1986, a geographic location near
Indonesia where the first seventh pandemic V. cholerae O1 El Tor
was reported in 1961.
Mechanism of
V. cholerae
Evolution. There are only a few human
pathogens for which the complete sequences of many isolates are
available (27, 28, 29). Because V. cholerae is both highly pathogenic
for humans and an autochthonous inhabitant of estuaries world-
wide, it provides a unique opportunity to elucidate evolutionary
mechanisms. Furthermore, it is the natural inhabitant of the
estuarine environment of both cholera epidemic and nonepidemic
countries (3).
Unlike Salmonella enterica serovar Typhi and Bacillus anthracis,
bacterial species showing clonal properties, V. cholerae,withStrep-
tococcus agalactiae and Escherichia coli, offers a prime example of
the important role of lateral gene transfer in the evolution of a
bacterial species. The transition from sixth to seventh cholera
pandemic genome type is concluded to result from a change from
V. cholerae O1 classical to O1 El Tor biotype. We propose the term
shift for the event occurring between two distinct phyletic lineages
(Fig. 1B). It should be noted that only one genome of O1 classical
biotype was included in this study, therefore more isolates of this
biotype should be examined to determine its population structure.
In contrast, the present cholera global pandemic is ascribed to a
change among 7P strains, e.g., emergence of V. cholerae O139, V.
cholerae O1 El Tor hybrid, and V. cholerae O1 El Tor with altered
cholera toxin subunit B. These represent transitions among genet-
ically nearly identical clones, with a few different GIs, for which we
propose the term drift. Much as in the case of inf luenza viruses,
cholera bacteria undergo a shift/drift cycle over time, although the
drift in V. cholerae is derived mainly from lateral gene transfer, most
likely occurring in the natural environment in association with its
plankton hosts (3, 30).
Fig. 4. Proposed hypothetical evolutionary pathway of the V. cholerae species. Probable insertions and deletions of genomic islands (Table S1) found in 23 V.
cholerae strains are indicated by black and red arrows, respectively, along the phylogenetic tree based on genome sequence data. Hypothetical ancestral strains
are indicated by open circles.
15446
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0907787106 Chun et al.

The present cholera global pandemic is concluded to have been
initiated by multiple descendants of a V. cholerae O1 El Tor
ancestor, diversified and continuously rapidly evolving, mainly via
lateral gene transfer and most likely driven by environmental
factors. Most importantly, the common genome backbone and
variable genomic islands of the 7P clade of V. cholerae require that
a reevaluation be done of the epidemiological practice that employs
serogroups as the primary marker for V. cholerae. The so-called
pandemic clones, identified by serogroup, instead, should be de-
fined by gene content, the description of which offers significantly
greater potential for development of reliable and useful diagnostics,
vaccines, and therapeutics for cholera. Without doubt, more vari-
ants of the 7P clade, as a result of drift, will be encountered in the
future, yielding new serogroups (other than O1 and O139) and
phenotypic combinations. Public health workers will be unprepared
if the evolution of this species remains unappreciated as an ongoing
process in the natural environment, where V. cholerae is autoch-
thonous and plays an important role in the nutrient cycles of the
natural aquatic ecosystem.
Materials and Methods
Genome Sequencing. Draft sequences were obtained from a blend of Sanger and
454 sequences and involved paired end Sanger sequencing on 8-kb plasmid
libraries to 5⫻coverage, 20⫻coverage of 454 data, and optional paired end
Sanger sequencing on 35-kb fosmid libraries to 1–2⫻coverage (depending on
repeat complexity). To finish the genomes, a collection of custom software and
targeted reaction types were used. In addition to targeted sequencing strategies,
Solexa/Illumina data in an untargeted strategy were used to improve low quality
regions and to assist gap closure. Repeat resolution was performed using in-
house custom software. Targeted finishing reactions included transposon bombs
(31), primer walks on clones, primer walks on PCR products, and adapter PCR
reactions. Gene-finding and annotation were achieved using the RAST server (32)
and details are given in Table S3.
Comparative Genomics. Genome to genome comparison was performed using
three approaches, because completeness and quality of nucleotide sequences
varied from strain to strain (Table 1). First, ORFs of a given pair of genomes were
reciprocally compared each other, using the BLASTN, BLASTP and TBLASTX
programs (ORF-dependent comparison). Second, a bioinformatic pipeline was
constructed to identify homologous regions of a given query ORF. Initially, a
segment on target contig, which is homologous to a query ORF, was identified
using the BLASTN program. This potentially homologous region was expanded in
both directions by 2,000 bp. Nucleotide sequences of the query ORF and selected
target homologous region were then aligned using a pairwise global alignment
algorithm (33), and the resultant matched region in the subject contig was
extracted and saved as a homolog (ORF-independent comparison). Orthologs
and paralogs were differentiated by reciprocal comparison. In most cases, both
ORF-dependent and -independent comparisons yielded the same orthologs,
although ORF-independent method performed better for draft sequences of low
quality, in which sequencing errors, albeit rare, hampered identification of
correct ORFs.
Identification and Annotation of Genomic Islands. In this study, we defined
genomic islands (GIs) as a continuous array of five or more ORFs that were found
to be discontinuously distributed among genomes of test strains. Correct transfer
or insertion of GIs was readily differentiated from deletion event by comparing
genome-based phylogenetic tree and full matrices showing pairwise detection of
orthologous genes between test strains. Identified GIs were designated, and
annotated using the BLASTP search of its member ORFs against GenBank NR
database.
Phylogenetic Analyses Based on Genome Sequences. A set of orthologues for
each ORF of V. cholerae N16961 was obtained for different sets of strains, and
then aligned using the CLUSTALW2 (34) program. The resultant multiple align-
ments were concatenated to generate genome scale alignments, which were
subsequently used to reconstruct the neighbor-joining phylogenetic tree (35).
The evolutionary model of Kimura (36) was used to generate the distance matrix.
The program MEGA (37) was used for phylogenetic analysis.
ACKNOWLEDGMENTS. This work was supported in part by Korea Science and
Engineering Foundation National Research Laboratory Program Grant R0A-
2005-000-10110-0 (to J.C.); National Institutes of Health Grant 1RO1A139129-01
(to R.R.C.); National Oceanic and Atmospheric Administration, Oceans and Hu-
man Health Initiative Grant S0660009 (to R.R.C.); Department of Homeland
Security Grant NBCH2070002 (to R.R.C.); Intelligence Community Post-Doctoral
Fellowship Program (to C.J.G.); and the Korean and Swedish governments (to
I.V.I.). Funding for genome sequencing was provided by the Office of the Chief
Scientist and National Institute of Allergy and Infectious Diseases Microbial
Sequencing Centers Grants N01-AI-30001 and N01-AI-40001.
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no. 36
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