Evidence for the Emergence of Non-O1 and Non-O139 Vibrio cholerae Strains with Pathogenic Potential by Exchange of O-Antigen Biosynthesis Regions

Abstract

The novel epidemic strain Vibrio cholerae O139 Bengal originated from a seventh-pandemic O1 El Tor strain by antigenic shift resulting from homologous recombination-mediated
exchange of O-antigen biosynthesis (wb*) clusters. Conservation of the genetic organization of wb* regions seen in other serogroups raised the possibility of the existence of pathogenic non-O1 and non-O139 V. cholerae strains that emerged by similar events. To test this hypothesis, 300 V. cholerae isolates of non-O1 and non-O139 serogroups were screened for the presence of virulence genes and an epidemic genetic background
by DNA dot blotting, IS1004 fingerprinting, and restriction fragment length polymorphism (RFLP) analysis. We found four non-O1 strains (serogroups O27,
O37, O53, and O65) with an O1 genetic backbone suggesting exchange of wb* clusters. DNA sequence analysis of the O37 wb* region revealed that a novel ∼23.4-kb gene cluster had replaced all but the ∼4.2-kb right junction of the 22-kb O1 wbe region. In sharp contrast to the backbones, the virulence regions of the four strains were quite heterogeneous; the O53 and
O65 strains had the El Tor vibrio pathogenicity island (VPI) cluster, the O37 strain had the classical VPI cluster, and the
O27 strain had a novel VPI cluster. Two of the four strains carried CTXφ; the O27 strain possessed a CTXφ with a recently
reported immune specificity (rstR-4** allele) and a novel ctxB allele, and the O37 strain had an El Tor CTXφ (rstRET allele) and novel ctxAB alleles. Although the O53 and O65 strains lacked the ctxAB genes, they carried a pre-CTXφ (i.e., rstRcla). Identification of non-O1 and non-O139 serogroups with pathogenic potential in epidemic genetic backgrounds means that attention
should be paid to possible future epidemics caused by these serogroups and to the need for new, rapid vaccine development
strategies.

Full text

INFECTION AND IMMUNITY, May 2002, p. 2441–2453 Vol. 70, No. 5
0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.5.2441–2453.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Evidence for the Emergence of Non-O1 and Non-O139 Vibrio cholerae
Strains with Pathogenic Potential by Exchange of
O-Antigen Biosynthesis Regions
Manrong Li,
1,2
† Toshio Shimada,
3
J. Glenn Morris, Jr.,
1,2
Alexander Sulakvelidze,
1
and Shanmuga Sozhamannan
1,2
*
Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine,
1
and
VA Maryland Health Care System,
2
Baltimore, Maryland 21201, and Laboratory of Enteric Infection 1,
National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
3
Received 8 November 2001/Returned for modification 20 December 2001/Accepted 18 January 2002
The novel epidemic strain Vibrio cholerae O139 Bengal originated from a seventh-pandemic O1 El Tor strain
by antigenic shift resulting from homologous recombination-mediated exchange of O-antigen biosynthesis
(wb*) clusters. Conservation of the genetic organization of wb* regions seen in other serogroups raised the
possibility of the existence of pathogenic non-O1 and non-O139 V. cholerae strains that emerged by similar
events. To test this hypothesis, 300 V. cholerae isolates of non-O1 and non-O139 serogroups were screened for
the presence of virulence genes and an epidemic genetic background by DNA dot blotting, IS1004 fingerprint-
ing, and restriction fragment length polymorphism (RFLP) analysis. We found four non-O1 strains (sero-
groups O27, O37, O53, and O65) with an O1 genetic backbone suggesting exchange of wb* clusters. DNA
sequence analysis of the O37 wb* region revealed that a novel ⬃23.4-kb gene cluster had replaced all but the
⬃4.2-kb right junction of the 22-kb O1 wbe region. In sharp contrast to the backbones, the virulence regions
of the four strains were quite heterogeneous; the O53 and O65 strains had the El Tor vibrio pathogenicity
island (VPI) cluster, the O37 strain had the classical VPI cluster, and the O27 strain had a novel VPI cluster.
Two of the four strains carried CTX␾; the O27 strain possessed a CTX␾with a recently reported immune
specificity (rstR-4** allele) and a novel ctxB allele, and the O37 strain had an El Tor CTX␾(rstR
ET
allele) and
novel ctxAB alleles. Although the O53 and O65 strains lacked the ctxAB genes, they carried a pre-CTX␾(i.e.,
rstR
cla
). Identification of non-O1 and non-O139 serogroups with pathogenic potential in epidemic genetic
backgrounds means that attention should be paid to possible future epidemics caused by these serogroups and
to the need for new, rapid vaccine development strategies.
Cholera is a diarrheal disease caused by the gram-negative
bacterium Vibrio cholerae, and an estimated 120,000 deaths
from cholera occur globally every year (58). Cholera is both an
endemic and epidemic disease and is the only bacterial pan-
demic disease known in modern times. One of the etiological
agent’s major protective antigens appears to be the O antigen
(27), and the enormous serological diversity of V. cholerae is
shown by the fact that more than 200 O-antigen serogroups
have been identified (43). Interestingly, only the O1 and O139
serogroups are known to cause epidemic and pandemic dis-
ease, although occasional outbreaks caused by non-O1 and
non-O139 strains have been reported in the past. For example,
strains of serogroup O37 were responsible for localized out-
breaks in 1965 in Czechoslovakia (2) and in 1968 in the Sudan
(26). Seven pandemics have been recorded in the history of
cholera; the sixth pandemic was caused by O1 strains of the
classical biotype, and the seventh pandemic, which started in
1961 and continues to the present time, is caused by O1 El Tor
biotype strains (27). The only other serogroup known to cause
epidemic cholera (O139) emerged in 1992 in the Bay of Bengal
region and has remained endemic to this region (1, 41). The
O139 Bengal strains cause disease with severity comparable to
the severity of the disease caused by O1 strains, and prior
exposure to O1 strains does not provide protection against
O139 infections (7, 37). Molecular epidemiological studies (ri-
botyping, fingerprinting, multilocus enzyme electrophoresis,
etc.) have indicated that the O139 strains have genetic back-
bones very similar to those of the O1 El Tor Asian seventh-
pandemic strains (6, 25, 56).
The O1 O-antigen biosynthesis genes of V. cholerae are
organized in a cluster (wbe cluster) on chromosome I, between
open reading frames (ORFs) VC0240 (gmhD) and VC0264
(rjg) (22). DNA sequence analyses of the wb* clusters of two
other serogroups (O22 and O139) revealed a similar organi-
zation of this region; i.e., serogroup-specific genes are flanked
by gmhD (which encodes D-glycero-D-manno-heptose 1-phos-
phate guanosyltransferase, involved in lipopolysaccharide core
biosynthesis) at the left junction and by rjg (which encodes a
conserved hypothetical protein with similarities to mRNA 3⬘
end processing factor) at the right junction (9, 14, 18, 33, 48,
59). These data led to the idea that the V. cholerae O139
Bengal strain originated from an O1 strain by homologous
recombination-mediated replacement of the wbe region of an
O1 strain with the O139 wbf region (36, 47, 49). However, the
donor or the vehicle for this horizontal transfer event is not yet
known. An O22 serogroup strain has been proposed to be a
* Corresponding author. Present address: Intralytix, Inc., The Co-
lumbus Center, 701 E. Pratt St., Room 4016, Baltimore, MD 21201.
Phone: (410) 625-2422. Fax: (410) 625-2506. E-mail: ssozhamannan
@intralytix.com.
† Present address: Intralytix, Inc., Baltimore, MD 21201.
2441

possible donor since its wb* region shares extensive homology
with the O139 wbf region (59), and a generalized transducing
phage or a conjugative plasmid is the speculated vector (36,
47).
DNA fingerprinting and phylogenetic analyses of V. cholerae
strains have established that there is a lack of correlation
between serogroup and phylogeny (8, 46); i.e., strains belong-
ing to various serogroups appear to fall in the same phyloge-
netic clade, and strains belonging to the same serogroup have
been found in many different clades. These data support the
hypothesis that there are frequent horizontal transfers of O-
antigen clusters among non-O1 and non-O139 V. cholerae
strains. However, such transfers into epidemic strains seem to
have been limited, since O139 is the only known example of
O-antigen transfer into an epidemic strain.
Two critical virulence factors have been associated with ep-
idemic strains. Cholera toxin is the primary virulence factor
responsible for the severe diarhheal symptoms (27), and the
toxin coregulated pilus (TCP) is the primary factor responsible
for efficient colonization of the human intestinal tract (52, 53).
In a landmark study, Waldor and Mekalanos (57) showed that
the cholera toxin genes (ctxAB) are carried on a filamentous,
f1-like, single-stranded DNA phage, designated CTX␾. Also, it
has been demonstrated (42, 57) that TCP serves not only as a
colonization factor but also as the receptor for CTX␾. Re-
cently, the tcpA gene has been shown (28) to be located on a
pathogenicity island designated the vibrio pathogenicity island
(VPI) and has been reported to be predominantly associated
with epidemic and pandemic strains. Interestingly, the VPI has
also been proposed (29) to be a filamentous phage, designated
VPI␾. Despite the lack of further evidence of the existence of
VPI␾, this idea raises the interesting possibility that there is
phage-phage interaction in horizontal gene transfer (54).
Several Vibrio mimicus strains carrying VPI and CTX␾have
been identified (12), and the remarkable identity at the se-
quence level of some of the V. mimicus and El Tor VPI genes
(aldA and toxT) suggested that there was recent interspecies
horizontal transfer of these factors between V. cholerae and V.
mimicus. Identification of several non-O1 and non-O139 sero-
group V. cholerae strains containing the tcpA gene (13, 39, 40)
suggests that these strains represent the environmental reser-
voirs of this virulence factor. Recently, extensive analysis (38)
of the VPI and CTX prophage regions of several environmen-
tal V. cholerae strains has been described, although the mech-
anism(s) of the origin of these strains has not been addressed.
Conservation of the genetic organization of the wb* region
raises the possibility that non-O1 and non-O139 V. cholerae
strains with an epidemic genetic background may have arisen
by exchange of O-antigen biosynthesis regions. In order to
evaluate this hypothesis, we analyzed 300 V. cholerae strains in
all of the 194 known serogroups, and we found several non-O1
and non-O139 strains possessing ctxAB and tcpA genes. Four of
these strains appeared to have a genetic background similar to
that of the epidemic strains. DNA sequencing of the O-antigen
cluster in one of the strains (O37 serogroup) revealed that
most of the O1 wbe region had been replaced by a novel wb*
cluster. Thus, homologous recombination-mediated O-antigen
shift appears to be a general mechanism for the emergence of
novel virulent strains of V. cholerae.
MATERIALS AND METHODS
Bacterial strains, growth conditions, and primers. The bacterial strains used
in this study included strains in the Shimada type culture collection (194 sero-
types) (43), 36 clinical V. cholerae isolates, and 70 clinical and environmental
strains from the Smith collection (44). The strains characterized in this study are
listed in Table 1, and the primers used in various analyses are listed in Table 2.
Media and culture conditions have been described previously (45).
DNA dot blotting and PFGE. DNA dot blot analysis and pulsed-field gel
electrophoresis (PFGE) were carried out as described previously (45).
Long-range PCR. In order to determine the lengths of the wb* regions in
various serogroup strains, long-range PCR was performed with an XL-PCR kit
(Perkin-Elmer Cetus Corp., Foster City, Calif.). PCRs were performed by using
1␮g of genomic DNA as the template, primers J 101 and J 103, and the protocol
and conditions recommended by the manufacturer. The PCR products were
electrophoresed in 0.5% agarose gels and stained with ethidium bromide.
Sequencing of the O37 wb* region. The entire DNA sequence of the region
between the gmhD and rjg genes of an O37 serogroup strain was obtained from
the XL-PCR product. The XL-PCR fragment amplified from an O37 serogroup
TABLE 1. Characterization of non-O1 and non-O139 V. cholerae strains with pathogenic potential
a
Strain Country Year Source Sakazaki
serogroup
CTX␾VPI
ctxA ctxB rstR Cluster
b
tcpA
395 India 1966 Diarrhea O1 cla WT cla cla cla cla
NIH35A3 India 1941 Diarrhea O1 cla WT cla cla cla cla
5011 Unknown H. Smith collection O1 cla (O333)
c
WT cla cla cla cla
N16961 Bangladesh 1975 Diarrhea O1 ET WT ET ET ET ET
E7946 Bahrain 1978 Diarrhea O1 ET WT ET ET ET ET
365-96 Japan 1996 Prawn, import from Thailand O27 WT NT rstR-4ⴱⴱ
d
NT NT
e
1322-69 India 1969 Diarrhea O37 NT
f
NT ET cla cla
8585 Iraq 1966 Diarrhea O53 (O340)
c
cla ET ET
981-75 India 1975 Diarrhea O65 cla ET ET
63-93 (MO45) India 1992 Diarrhea O139 WT ET ET ET ET
AM2 India 1995 Diarrhea O9
AM107 India 1996 Diarrhea O144
NRT36-S Japan 1990 Diarrhea O31
a
Abbreviations: wt, wild type; cla, classical; ET, El Tor; NT, novel type.
b
Presence of the entire VPI cluster based on restriction mapping and hybridization.
c
O333 and O340 are Smith serogroups.
d
rstR-4** ⫽SCE223 (38).
e
Differs from the tcpA-env allele described by Mukhopadhyay et al. (38) at one position (
O27
V9D
env
).
f
There is a single amino acid substitution (
wt
S46N
O37
).
2442 LI ET AL. INFECT.IMMUN.

strain, 1322-69, was digested with PstI, and the resulting fragments were gel
purified, cloned in the pBluescript vector (Stratagene Corp., La Jolla, Calif.), and
sequenced by primer walking. The fragments were aligned using restriction maps
of the XL-PCR fragment. The order of the PstI sites was confirmed by targeted
PCR and sequencing of the PCR fragments. The XL-PCR end fragments were
PCR amplified, cloned in pCR2.1, and sequenced. The final aligned sequence of
the O37 wb* region was analyzed by the DNASIS program (Hitachi Software
Engineering Co., Ltd., South San Francisco, Calif.) in order to identify the
ORFs, and the individual ORFs were searched by using the National Center for
Biotechnology Information Blast program (3) for identifying protein similarities.
Isolation of wb* regions. In order to isolate the entire wb* region on a single
restriction fragment, a unique NotI site in the rjg gene at the right junction of the
wb* region was utilized. A second NotI site was introduced at the left junction.
In order to accomplish this, the genes orf-2 (VC0239) and gmhD (VC0240) were
cloned on either side of the NotI site of the pBluescript vector, and a Kan
r
cassette was introduced between orf-2 and gmhD.ASacI-EcoRV fragment con-
TABLE 2. List of primers
Primer Gene Sequence Reference(s)
J 101 gmhD
1
5⬘-GCCATCCCACTCTGTGGTCGCAGAGCAAGCTCC-3⬘14
J 103 rjg
2
5⬘-CCCGTGACACTCGCCTTCCCTCCGTGATGAACC-3⬘14
M 177 gmhD
1
5⬘-TTACTTACGATTAATCAGCGCCAT-3⬘45
M 178 gmhD
2
5⬘-GGCGGCGCTGGCATGATTGGCAGC-3⬘45
M 179 rjg
1
5⬘-CATGGAAGTGGTTCATCACGGAGG-3⬘45
J 414 rjg
2
5⬘-GTGGACGCGTTCAAAGCACCGAATATCCGAGTT-3⬘45
M 310 orf2
1
5⬘-GGTGACATCAAAGGGACCACTTTTTC 22; this study
M 311 orf2
2
5⬘-GGTGTATGCCACTAGTGTAGGTAAT 22; this study
M 459 IS1004
1
5⬘-CCCCAGCTTTTGACGCTTATTGTGAACGT-3⬘22; this study
M 460 IS1004
2
5⬘-GATCGATATCTTTCTAACTTCTGTATAAGG-3⬘22; this study
M 277 ctxA
1
5⬘-ACAGAGTGAGTACTTTGACC-3⬘22; this study
M 278 ctxA
2
5⬘-ATACCATCCATATATTTGGGAG-3⬘22; this study
S86 ctxAB
1
5⬘-GGCTGTGGGTAGAAGTGAAACGG-3⬘22; this study
S87 ctxAB
2
5⬘-CTAAGGATGTGGAATAAAAACATC-3⬘22; this study
M 279 tcpA
1
5⬘-AAAACCGGTCAAGAGGG-3⬘(same as KAR 24) 28
M 280 tcpA
2
5⬘-CAAAAGCTACTGTGAATGG-3⬘(same as KAR 25) 28
M 281 tcpA
3
5⬘-CAAATGCAACGCCGAATGG-3⬘(same as KAR 82) 28
M 590 tcpA
L1
5⬘-GATCGCATGCCAGAGTTCTATCTTTCGTC-3⬘22; this study
M 591 tcpA
L2
5⬘-GATCGTCGACATAGTGATAAGAGTCTTACCC-3⬘22; this study
M 668 smt
1
(smt-VCA0198) 5⬘-CCGAAATACGGTCATTACTTGGGC-3⬘22; this study
M 669 smt
2
5⬘-CACTTCATTATTCCCGTAAGCAGC-3⬘22; this study
M 680 smt
1.1
(nupC-VCA0179) 5⬘-AATAGCCAATCACGCACCAAG-3⬘22; this study
M 681 smt
1.2
5⬘-TAATCGCACTGCGGCTTTCAG-3⬘22; this study
M 682 smt
2.1
(hmpA-VCA0183) 5⬘-TGACCCACCAGAAAACCGGAC-3⬘22; this study
M 683 smt
2.2
5⬘-GCGCCTTATCCACACCAAGCG-3⬘22; this study
M 684 smt
3.1
(rhlE-VCA0204) 5⬘-CGCTCAATCGCAAATAATTCC-3⬘22; this study
M 685 smt
3.2
(dcuB-VCA0205) 5⬘-TGCTCTCTCTCCCCAAATGAC-3⬘22; this study
M 686 smt
4.1
(VCA0206) 5⬘-GTATTGTCGGATTTCATTTGC-3⬘22; this study
M 687 smt
4.2
(VCA0208) 5⬘-AGTGACGGCCTCTGGCGGAGC-3⬘22; this study
M 688 smt
5.1
(hlyA-VCA0219) 5⬘-GGGTTCCGCGACACCGGATGC-3⬘22; this study
M 689 smt
5.2
5⬘-TGTTTAATGGCTATGTTGACG-3⬘22; this study
M 698 ctxrgn
1.1
(VC1444) 5⬘-TAATCTGCTATTTCACTGAAG-3⬘22; this study
M 699 ctxrgn
1.2
5⬘-TTCCTGAGTGATCCCCAATCC-3⬘22; this study
M 700 ctxrgn
2.1
(VC1451-rtxA)5⬘-GCGGAAAAGCTGAAAGGCACC-3⬘22; this study
M 701 ctxrgn
2.2
5⬘-ACCTTCATGGTGTGAAATCAC-3⬘22; this study
M 702 ctxrgn
3.1
(VC1465) 5⬘-CCGCTGTCTCAATAGAACCTG-3⬘22; this study
M 703 ctxrgn
3.2
5⬘-GGACATCATACAAGAGAAGAC-3⬘22; this study
M 704 ctxrgn
4.1
(VC1470) 5⬘-GAACATGAACCTTAATGCGAG-3⬘22; this study
M 705 ctxrgn
4.2
5⬘-CACGTCATTTATGAATTACGG-3⬘22; this study
M 706 ctxrgn
5.1
(VC1476-VC1477) 5⬘-GGTATCAGCATGAGACTTTTTTG-3⬘22; this study
S 122 ctx core (orfU)5⬘-CGTCACACCAGTTACTTTTCG-3⬘22; this study
S 123 ctx core (zot)5⬘-AACCCCGTTTCACTTCTAC-3⬘22; this study
M 707 ctxrgn
5.2
5⬘-CCAATAGTGATAACTACTTCG-3⬘22; this study
M 452 ald
2
5⬘-TTTTCTTGATTGTTAGGATGC-3⬘12
M 453 ald
1
5⬘-ATTCTTCTGAGGATTGCTGAT-3⬘12
M 644 tagD
1
5⬘-GCGGTGACACTAAAGTAGTGTTTG-3⬘22; this study
M 645 tagD
2
5⬘-GATGGTCAGATAAAAGAACGCAGG-3⬘22; this study
M 664 tcpAdn
1
5⬘-TTCGCAATTACAGTCGGTGGCTTG-3⬘22; this study
M 665 tcpAdn
2
5⬘-AGCCAACTCAGTTAAAACTTGTTC-3⬘22; this study
M 448 toxT
2
5⬘-CTTGGTGCTACATTCATGG-3⬘12
M 449 toxT
1
5⬘-AGGAGATGGAAGTGGTGTG-3⬘12
M 646 vpi0845
1
5⬘-ATCATTCCAGATAAAGTTACGCAGA-3⬘22; this study
M 647 vpi0845
2
5⬘-TCTACTTCCGGCTTCCCTGCCACG-3⬘22; this study
M 407 rstR1 5⬘-GACGTAGCGTGCGGAGTCGCGTTG-3⬘22; this study
M 408 rstR2 5⬘-TGAAGCATAAGGAACCGACCAAGC-3⬘22; this study
M 573 rstA1 5⬘-ACTCGATACAAACGCTTCTC-3⬘22; this study
M 574 rstA2 5⬘-AGAATCTGGAGGTTGAGTG-3⬘22; this study
VOL. 70, 2002 HORIZONTAL TRANSFER OF O-ANTIGEN CLUSTER 2443

taining orf-2–NotI–Kan
r
–gmhD was blunt ended and cloned into SalI-digested
and blunt-ended suicide vector pCVD442. The NotI site in the derivative of
pVCD442 was introduced into the chromosomes of selected strains, as previously
described (17), by the sucrose selection procedure. The resulting strains were
subjected to PFGE after NotI digestion of their DNAs in agarose plugs as
previously described (45). Southern analysis was performed by transfer of the
restriction enzyme-digested chromosomal DNA fragments onto a nitrocellulose
membrane, followed by hybridization with probes prepared by enhanced chemi-
luminescence (Amersham Biosciences, Piscataway, N.J.).
IS1004 fingerprinting. Genomic DNAs were digested with HpaII, transferred
onto a Zeta-probe membrane (Bio-Rad Laboratories, Hercules, Calif.), and
hybridized with an IS1004 probe. Direct amplification of the IS1004 element
from genomic DNA was unsuccessful, and hence, the following procedure was
used to clone the IS1004 element. Primers M 459 and M 460 were used to PCR
amplify a fragment (2.2 kb) encompassing an IS1004 copy and the neighboring
sequences, and the fragment was cloned into the pCR2.1 vector (Invitrogen Life
Technologies, Carlsbad, Calif.). The resulting plasmid was digested with EcoRI,
and this was followed by purification of the EcoRI fragment containing the
genomic sequences and not the plasmid sequences. This fragment was further
digested with HaeIII and AvrII, and a 0.5-kb fragment containing the IS1004
sequences was cloned into an SmaI-XbaI-digested pBluescript vector. An AccI-
SacII fragment of the resulting plasmid containing the insertion (IS) sequences
was used as the probe.
RFLP analysis. (i) smt region. The region analyzed by restriction fragment
length polymorphism (RFLP) spanned 48,759 bp on chromosome I. The left end
of the region was at coordinate 190747 within ORF VCA0174, and the right end
was at coordinate 239506 within ORF VCA0219 (hlyA). This region contains
nine SphI fragments. They are, in the order in which they are arranged on the
chromosome, 5,201, 5,755, 705, 6,993, 11,899, 1,755, 972, 839, and 14,640 bp long.
Five different probes (smtrgn 1 to 5, prepared by PCR using primers M 680 to M
689) were used to detect the various fragments. The probes were designed in
such a way as to detect two fragments with a single probe. For example, the
5,201- and 5,755-bp SphI fragments are adjacent to each other, and a probe
designed centrally at the SphI site could detect these two fragments.
(ii) ctx region. The ctx region spanned a 54,715-bp region starting within ORF
VC1443 (at the 5⬘end of ccoN; coordinate 1539558) and ending with coordinate
1594273 within VC1488 (encoding a hypothetical protein). It included sequences
upstream of RTX, the RTX cassette, RS1, and the CTX core and sequences
downstream of the CTX prophage (cri,tlc, transposase, fabA,rmf, and several
hypothetical proteins). The SphI fragments detected were 1,106, 13,818, 2,862,
13,387, 3,255, 1,365, 2,651, 2,180, 2,651, and 11,400 bp long, in the order in which
they are arranged on the chromosome. Five probes (ctxrgn 1 to 5, prepared by
PCR using primers M 698 to M 707) were used to detect these fragments.
(iii) VPI region. The VPI region analyzed included ORFs VC0815 to VC0850
(coordinates 871612 to 915843; 44,231 bp). The VPI cluster spans coordinates
873020 (between VC0816 and VCO817) to 914296 (between VC0847 and
VC0848). The XmnI fragments that were detected included 11,064-, 3,994-, 837-,
3,273-, 1,070-, 12,332-, 3,976-, and 7,685-bp fragments, and five different probes
(ald,tagD,tcpAdn,toxT,vpi0845) were used.
Nucleotide sequence accession numbers. The nucleotide sequences of the rstR,
tcpA, and ctxAB genes of strain 365-96 (serogroup O27) and the wb* region of
strain 1322-69 (serogroup O37) determined in this study have been deposited in
the GenBank database under accession no. AF390570, AF390571, AF390572,
and AF390573, respectively.
RESULTS
Conservation of the genetic organization of the wb* regions.
The gmhD and rjg genes have previously been reported (45) to
be present in all of the V. cholerae serogroups examined, and
the wb* regions of these genes are believed to be organized in
a cluster. To further investigate the genetic organization of this
region in non-O1 and non-O139 serogroups, long-range PCRs
were performed using primers designed from the gmhD and rjg
genes. In several serogroups (O37, O53, O65, O77, O80, and
O89), the region between gmhD and rjg was amplified, and the
resulting XL-PCR fragments varied in size from 18 to 28 kb
(Fig. 1a). In serogroup O105, amplification of this region was
not possible, perhaps because of limitations of the PCR. Per-
haps the gmhD and rjg genes flank a wb* cluster that is too long
to be PCR amplified, or perhaps they do not flank the wb*
cluster and are present at different locations on the chromo-
some in this strain.
Thus, an alternative strategy was devised in order to inves-
tigate the organization of the wb* region in an O105 strain and
to isolate the entire wb* region on a single restriction frag-
ment. PCR and restriction analyses of the rjg gene in many
serogroup strains indicated that a rare restriction enzyme site,
NotI, was present in the gene. If rjg and gmhD are close to one
another, introduction of a NotI site at the left junction would
yield a unique NotI fragment containing the entire wb* cluster.
Hence, a NotI site was introduced between orf-2 and gmhD at
the left boundary of the wb* region in three strains (serogroups
O1, O139, and O105), as described in Materials and Methods.
Genomic DNAs of the resulting strains were digested with
NotI in agarose plugs, and the fragments were separated by
PFGE. A fragment that was not present in the parent strains
was detected in the strains with an engineered NotI site at the
left boundary (Fig. 1b, lanes 2, 4, and 6). As expected based on
previously published DNA sequence data (9, 14, 18, 33, 48),
the O1 and O139 NotI fragments were estimated to be 22 and
35 kb long, respectively. In the O105 strain, a 45-kb fragment
was released (Fig. 1b, lane 6), indicating that the sizes of the
serogroup-specific regions varied. Digestion of the chromo-
somal DNAs by various restriction enzymes and hybridization
with gmhD and rjg probes revealed fragments of various sizes
in different serogroups, indicating that the wb* regions contain
unique sequences (Fig. 1c). Taken together, these data dem-
onstrated that the wb* region is organized in a cluster.
Identification of the junctions of the O37 wb* region. Based
on the data described above, we hypothesized that the organi-
zation of the wb* regions of the non-O1 and non-O139 strains
is similar to that of epidemic strains; i.e., the gmhD and rjg
genes flank serogroup-specific O-antigen biosynthesis genes. In
order to test this hypothesis, restriction enzyme PstI-digested
O37 XL-PCR fragments (Fig. 1a) were subcloned and se-
quenced to see if these fragments encode polysaccharide bio-
synthesis genes. We chose to sequence the O37 region instead
of the regions of other serogroups because this serogroup has
clinical relevance and strains of this serogroup were implicated
in localized cholera outbreaks in the past (2, 26). The entire
O37 wb* region is 27,552 bp long, and the O37-specific se-
quence is 23,388 bp long. A comparison of this sequence with
the sequences of the previously described wb* clusters (9, 14,
18, 33, 48, 59) revealed that there was an exchange of the wb*
region, since the O37 region had some remnants of the O1 wbe
region (Fig. 2). The homology breakpoints (i.e., the DNA
sequences where the common backbone sequence ends and
the serogroup-specific sequence starts) in O37 strains were
different from those of the O1 and O139 junctions (Fig. 2 and
3). In the O37 serogroup, the left and right junctions are at
gmhD and wbeV, respectively, instead of at gmhD and rjg as
they are in O139 (Fig. 2). The left junction in O37 is at the
gmhD locus, and the O37-specific sequence begins about 120
bp to the right of initiation codon ATG of the gmhD ORF. In
contrast, the left junction in the O139 strain is at the start
codon (ATG) of the gmhD locus (Fig. 3A). In the case of O37,
the right junction is well within the O1 wbeV gene; i.e., part of
the O1 wbe region is retained in the O37 wb* region (Fig. 3B).
Previously, it was shown that the right junction in the O139
2444 LI ET AL. INFECT.IMMUN.

strain is at the rjg gene (right after the wbeW gene and the wbfX
gene of the O1 and O139 strains, respectively) (Fig. 3C); i.e.,
the entire O1 wbe region has been replaced by the wbf cluster
(14).
O37 wb* region. The O37 wb* cluster is unique, since we did
not observe any significant DNA homology between the O37-
specific sequences and the previously published V. cholerae
wb* cluster sequences except at the right junction (see above).
FIG. 1. Genetic organization of the wb* regions. (a) Agarose gel electrophoresis of XL-PCR amplification products of the O-antigen
biosynthesis regions (wb*) from different serogroup strains. The line at the top is a schematic diagram of the wb* region with gmhD and rjg at the
ends; the primers used in XL-PCR (indicated by the small bars below the line) are situated at the 5⬘ends of the gmhD and rjg genes. Lane S
contained a 1-kb ladder, and lane L contained a HindIII digest of ␭DNA. The positions (in kilobases) of the markers are indicated on the left.
(b) PFGE and Southern analysis of the NotI fragments of O1, O139, and O105 serogroup strains. The blot was hybridized with a gmhD probe. The
asterisk indicates the position of unexplained recombination intermediates or partial digestion products. Lanes 1, 3, and 5 contained parent strains
without the NotI site at the left junction, and lanes 2, 4, and 6 contained the strains with the NotI sites flanking the wb* regions. Lanes 2, 4, and
6 also contained partially digested parent fragments of the wb* region in addition to the newly generated wb* fragments. (c) Southern blot analysis
of the gmhD and rjg junction fragments in various strains. The genomic DNAs were digested with EcoRI and were simultaneously hybridized with
the gmhD and rjg probes. The upper and lower bands in each lane are the rjg- and gmhD-specific fragments, respectively.
FIG. 2. Comparison of the O37 wb* region with the O1 wbe and O139 wbf regions. The genetic organization of the wb* region of the O37 strain
is compared to the genetic organizations reported previously (49) for the O1 and O139 strains. The O139, O1, and O37 regions are 35, 22, and
27 kb long, respectively. The ORFs and the directions of transcription are indicated by the arrows, and the common ORFs in the three serogroups
are indicated by the same types of arrows. The common junction genes (gmhD and rjg) of the three serogroups are indicated by solid arrows. While
the left junction is at gmhD in all three serogroups,the right junction in O37 is different from the right junction in O139. The O1 right junction
genes, wbeV through rjg, are conserved in the O37 wb* region.
VOL. 70, 2002 HORIZONTAL TRANSFER OF O-ANTIGEN CLUSTER 2445

FIG. 3. Analyses of the left junctions (LJ) and the right junctions (RJ) of O1, O139, and O37 wb* regions performed using the ClustalW
program (http://www.ebi.ac.uk/index.html). The asterisks indicate identical bases in the sequences compared. (A) Left junctions of the O1, O37,
and O139 wb* regions. The solid arrowhead and its orientation indicate the gmhD ORF start codon and the direction of transcription, respectively.
The homology breakpoints between O1 and O37 are ca. 120 bp upstream of the start codon of gmhD since the sequences diverge near this position.
The homology breakpoints of the O1 and O139 sequences are at the start codon of the gmhD ORF, since the sequences after gmhD completely
diverge. (B) Right junctions of the O1 and O37 sequences. The homology breakpoint is in the wbeV ORF, which is preceded by wbeU in O1 and
2446 LI ET AL. INFECT.IMMUN.

Twenty-three ORFs were identified in the O37 wb* region, and
as expected, many of the orf genes (orf-1to -13 and orf-18)
(Fig. 2) encode enzymes involved in polysaccharide biosynthe-
sis. orf-14 and orf-15 encode hypothetical proteins of unknown
functions. orf-14 has very weak homology (26% identical and
46% positive in a 64-amino-acid region) to yhfO (which en-
codes a hypothetical protein) of Bacillus subtilis. A 1,549-bp
promoter region separates gmhD and orf-1, and this region
contains a putative promoter and ops elements found in known
polysaccharide biosynthesis regions (4, 23). As seen in other
wb* regions, there is an IS element in the interval between the
region that is unique to O37 and the right junction, and this
element, which appears to contain an insertion of another
fragment encoding a transposase, spans 3,580 bp and has an
18-bp inverted repeat at its ends. The original IS element is
1,054 bp long and is virtually identical (95% identical at the
DNA level; the transposase is 90% identical [278 of 306 amino
acids]) to the IS elements found in Vibrio parahaemolyticus
(ISV-3L, ISV-5R, ISV-5L, ISV-4R) (55). Interestingly, the tnp
gene of the V. cholerae element is interrupted after the 76th
amino acid residue by a 2,527-bp DNA fragment. This insert
encodes an ORF (orf-17) that is transcribed in the opposite
direction and has extensive homology to transposases of vari-
ous IS elements found in other bacterial species. The region
downstream of the IS element has three ORFs almost identical
to the O1 wbe cluster: wbeV,galE, and wbeW followed by rjg.
The wbeV gene in the O37 strain appears to consist of three
small ORFs. Thus, these data clearly demonstrate that (i) the
region between gmhD and rjg in the O37 serogroup encodes
O-antigen biosynthesis genes and (ii) the O37 wb* region orig-
inated by homologous recombination-mediated exchange of
wb* gene clusters.
Identification of non-O1 and non-O139 strains containing
virulence genes. Based on the data described above, we rea-
soned that a homologous recombination event flanking the
gmhD and rjg genes may result in non-O1 and non-O139 patho-
gens similar to O139 strains. In order to identify O139 Bengal-
like strains that originated by exchange of wb* clusters, 300 V.
cholerae strains were screened for the presence of the ctxAB
and tcpA genes, which are known to be present in epidemic and
pandemic strains. DNA dot blot analysis was performed by
using the ctxAB and tcpA genes as the probes. Fifteen non-O1
and non-O139 strains were found to be tcpA
⫹
. All of these
strains also carried three other genes (aldA,toxT, and int)of
the VPI cassette, which indicated that the entire VPI may be
present in these strains. Thirteen strains carried the rstR and
rstA genes; nine of these strains also carried the ctxAB genes;
and two strains did not carry any of these three genes. Of the
four ctxAB mutant strains, one carried just the rstR and rstA
genes, while the three other strains carried the rstR and rstA
genes and the genes of the core region except the ctxAB genes
(data not shown). Further characterization of 4 of the 15 tcp
⫹
strains (serogroups O27, O37, O53, and O65) indicated that
they contained the entire VPI and a pre-CTX␾(CTX␾with-
out the ctx genes) or a CTX␾. These four strains had genetic
backgrounds very similar to those of the epidemic O1 strains
(see below), thus indicating that there was an O-antigen shift in
an O1 strain.
Genetic relatedness of the non-O1 and non-O139 strains to
the epidemic strains as determined by IS1004 fingerprinting
and RFLP analysis. In order to determine the genetic relat-
edness among the various strains and to determine which of
these strains resulted from an O-antigen shift in an epidemic
strain, IS1004 fingerprinting and RFLP analyses were per-
formed.
IS1004 is an IS element present in multiple copies in O1
classical and El Tor strains, and it has been used previously (8)
for typing V. cholerae strains. In agreement with a previous
report (8), O1 classical and El Tor strains exhibited unique
fingerprints; i.e., the classical and El Tor strains had six and
four IS1004 copies, respectively (Fig. 4a, lanes O1 cla and O1
El Tor). In the present analysis, the O27 and O139 strains were
identical to the El Tor strains (Fig. 4a, lanes O139 and O27),
and the O37 strain was similar to the classical strains (Fig. 4a,
lane O37). The O53 and O65 strains exhibited some similarity
to both O1 El Tor and O1 classical strains and thus appeared
to have diverged early from the progenitor of the O1 strains
(Fig. 4a, lanes O53 and O65).
Further evidence of genetic similarity among the strains was
obtained by RFLP analyses of two regions of chromosomes I
and II. For this analysis, the vps (vibrio polysaccharide synthe-
sis) (22) operon from chromosome I and a region on chromo-
some II that contains a site-specific methyl transferase gene
(smt-VCA 0198) were chosen. Restriction enzyme SphI-di-
gested genomic DNAs were hybridized with various probes
(described in Materials and Methods). The RFLP patterns of
the vps regions of the O1 classical and El Tor strains and the
four non-O1 and non-O139 strains (serogroups O27, O37,
O53, and O65) were identical, whereas many other non-O1
and non-O139 strains had extensive variations (data not
shown).
The smt region in the classical and El Tor strains exhibited
unique RFLP patterns (Fig. 4b). The RFLP patterns of the smt
region of the O139 and O27 strains were identical to those of
the El Tor strains (Fig. 4b, compare lanes O1 El Tor to lanes
O139 and O27), and the RFLP patterns of the O37, O53, and
O65 strains were similar but not identical to the pattern of the
smt region of either the classical or El Tor strains. Further-
more, the smt gene was found only in strains that have an O1
backbone (Fig. 4c). None of the non-O1 and non-O139 strains
used in this analysis (O9, O31, and O144) have an O1-like
backbone, as determined either by IS1004 fingerprinting (Fig.
4a, lanes O144, O9, and O31) or by RFLP analyses (Fig. 4b,
lanes O144, O9, and O31), and they lacked the smt gene.
Taken together, IS1004 fingerprinting and RFLP analysis re-
sults indicated that the four non-O1 and non-O139 strains
by orf-18 in O37. The stop codon of wbeV (indicated by the arrowhead pointing upward) overlaps with the start codon of orf-18 in O37. The stop
codon of wbeV of O1 is indicated by the arrowhead pointing downward. (C) Right junctions of the O1 and O139 sequences published previously
(14, 45). The homology breakpoint is at the start of the rjg ORF. The rjg genes of O1 and O139 strains have different N terminus and start codons.
In all these cases, the actual recombination crossover sites could be anywhere within homologous segments far away from the homology
breakpoints and not necessarily at the junctions.
VOL. 70, 2002 HORIZONTAL TRANSFER OF O-ANTIGEN CLUSTER 2447

were derived from an epidemic strain by wb* cluster exchange
and subsequently diverged. A DNA sequence analysis of one
of the wb* regions (serogroup O37) determined in this study
(see above) supports this conclusion.
Genetic organization of the CTX prophage and VPI regions
of the non-O1 and non-O139 strains. We reasoned that ex-
change of the wb* region in an O1 strain would result in a
non-O1 and non-O139 strain containing either the classical or
El Tor ctx and VPI regions. In order to determine the struc-
tural organization of the ctx region in the non-O1 and non-
O139 strains examined in this study, chromosomal DNAs were
digested with EcoRI (an enzyme that does not cut within the
CTX␾genome), and the fragments were hybridized with rstA,
rstR,ctx core, and ctxAB gene probes. As expected, there was
diversity in the arrangement and location of the CTX prophage
genomes in various strains. The sizes of the fragments of the
second CTX prophage copy (chromosome II) varied in the
three classical strains analyzed [Fig. 5a, upper panel, lanes 395
(O1 Cla), NIH35-A3 (O1 Cla), and 5011 (O1 Cla)]. Like O1 El
Tor and O139 strains, the O37 strain had a chromosome I
copy(ies) of CTX␾[Fig. 5a, upper panel, lanes N16961 (O1 El
Tor), E7946 (O1 El Tor), O37, and O139], and the O27 strain
also contained a single copy (Fig. 5a, lane O27). The same
fragment was detected when the blot was hybridized with rstA
or rstR or ctx core probes in all of the strains except the O53
and O65 strains (Fig. 5a, lower panel). These two strains pro-
duced a single band when they were hybridized with rstA or
rstR or ctx core probes (Fig. 5a, lower panel, lanes O53 and
O65) but did not hybridize with any band when the ctxAB
probe was used.
To further elucidate the genetic organization, the ctx region
on chromosome I was analyzed by RFLP. The region analyzed
encompassed about 54,715 bp, which included the RTX cas-
sette, CTX␾, and the sequences upstream and downstream of
them. Figure 5b shows that the O37 and O139 strains have an
El Tor-like organization (compare lane O1 El Tor to lanes O37
and O139). The O27, O53, and O65 strains resembled El Tor
strains in the RTX cassette segment (bands a and b were
produced only by El Tor strains and not by a classical strain
[Fig. 5b]) but differed from epidemic strains by lacking the
pTLC element (Fig. 5b, compare lanes O1 Cla, O1 El Tor,
O37, and O139 to lanes O27, O53, and O65, bands f/g, h, and i).
We analyzed the VPI region in the non-O1 and non-O139
strains examined in this study. Digestion of the genomic DNAs
with XmnI, followed by hybridization of the blot with five
different probes spanning the entire VPI region, resulted in
identification of seven and eight fragments in the O1 classical
and El Tor strains, respectively (Fig. 5c, lanes O1 Cla and O1
El Tor). Among the other strains, the O37 strain had a classical
VPI cluster (Fig. 5c, compare lanes O1 Cla and O37), the O53,
FIG. 4. IS1004 fingerprinting and RFLP analyses of the various serogroup strains. (a) IS1004 fingerprints of HpaII-digested chromosomal
DNAs of various V. cholerae strains. Since HpaII does not cut within IS1004, each band represents one copy of the element. (b) RFLP analysis
of the smt region of chromosome II of various V. cholerae strains. The genomic DNAs were digested with SphI and hybridized simultaneously with
multiple probes (smtrgn 1 to 5). The identities of the bands were deduced from their predicted sizes, based on the published V. cholerae genome
sequence (22), and they were confirmed by separately hybridizing the same blot with individual probes. The sizes of the molecular weight markers
(in kilobases) are indicated on the left. (c) Blot used in panel b rehybridized with an smt probe (primers M 668 and M 669). Lanes O144, O9, and
O27 were omitted since they did not show any hybridizing band with this probe.
2448 LI ET AL. INFECT.IMMUN.

O65, and O139 strains had an El Tor VPI cluster (compare
lane O1 El Tor to lanes O139, O53, and O65), and the O27
strain appeared to contain a novel VPI cluster (Fig. 5c, lane
O27). Further hybridization analyses with additional probes
indicated that the O27 strain had the entire VPI cluster with
two additional XmnI sites (data not shown).
DNA sequence of the rstR,ctxAB,tcpA, and aldA genes of the
non-O1 and non-O139 strains. In order to understand further
the origin of the ctx and VPI regions, the rstR gene (which
encodes the repressor of CTX␾and determines the phage
immune specificity) and the ctxAB genes of CTX␾and the tcpA
and aldA genes of the VPI were sequenced. These genes are
known to be different in classical and El Tor biotypes (15, 30,
31, 32). The results are summarized in Table 1.
The rstR genes were PCR amplified, cloned into the pCR2.1
vector, and sequenced. Strains of serogroups O37 and O139
had an rstR allele with El Tor specificity, the O53 and O65
strains had classical immune specificity and the O27 strain had
novel specificity. The rstR
O27
allele, whose specificity differs
from that of the classical, El Tor, and Calcutta CTX␾s, is
identical to the recently reported rstR-4** gene (38) encoding
an RstR repressor protein that is 86 amino acids long.
We sequenced the ctxAB genes of the two non-O1 and non-
O139 (O27 and O37) strains in order to identify the variations
in the ctx genes in various serogroups. The O53 and O65
strains did not possess ctx genes. The O27 and O37 strains
possessed highly conserved ctxAB genes; i.e., the 1,152 bp of
their ctxAB genes differed by less than three or four nucleo-
tides, and their proteins differed by two or three amino acids
(CtxA,
cla/ET
S46N
O37
; CtxB,
cla/ET
Q24H
O27
,
cla/ET
D28A
O27
,
FIG. 5. RFLP analysis of the virulence regions of the various strains. (a) EcoRI-digested genomic DNAs were probed with ctxAB gene probes
(upper panel) and the ctx core probe (lower panel). EcoRI does not cut within the CTX␾genome, and each hybridizing band therefore represents
one copy of CTX present in a strain. (b) SphI-digested genomic DNAs of the strains were probed with five probes spanning a 45-kb region
surrounding the CTX␾genome integrated on chromosome I. The probes and the sizes of the fragments and the corresponding bands are as
follows: ctxrgn 1, 1,106 and 13,818 bp (bands a and j); ctxrgn 2, 2,862 and 13,387 bp (bands e and b); ctxrgn 3, 3,255 and 1,365 bp (bands d and i);
ctxrgn 4, 2,651 and 2,180 bp (bands f and h); and ctxrgn 5, 2,651 and 11,400 bp (bands g and c). (c) Genomic DNAs digested with XmnI and probed
simultaneously with five different probes from the VPI region. The identities of the bands were confirmed by separate hybridization with individual
probes. The probes and the sizes of the fragments and the corresponding bands are as follows: ald, 11,064 bp (band b); tagD, 3,994 bp (band e)
and 837 bp (band h); tcpAdn, 3,273 bp (band f) and 1,070 bp (band g); toxT, 12,332 bp (band a); and vpi0845, 3,976 bp (band d) and 7,685 bp
(band c).
VOL. 70, 2002 HORIZONTAL TRANSFER OF O-ANTIGEN CLUSTER 2449

cla/ET
F46L
O37
, and
cla/ET
K55N
O37
, where cla indicates classical
and ET indicates El Tor). In addition to these changes, the
CtxB
O27,O37
alleles were identical to the classical alleles at two
other positions,
ET
Y39H
cla/O27/O37
and
ET
I68T
cla/O27/O37
.
A 1.4-kb fragment encompassing the tcpA gene was PCR
amplified, and the tcpA gene was sequenced. The tcpA
O37
allele was identical to the tcpA
cla
allele, the tcpA
O53,O65
alleles
were identical to the tcpA
ET
allele, and the O27 strain had a
novel allele. The tcpA
O27
allele is identical to the recently
reported tcpA-env allele (38) except for a single amino acid
substitution (
cla/ET
V9D
tcpA-env
). In contrast to the tcpA alleles,
the aldA genes of the strains exhibited very few variations (data
not shown).
DISCUSSION
Antigenic shift and emergence of novel pathogens. Exchange
of polysaccharide biosynthesis clusters resulting in novel
pathogens has been well documented in many bacterial patho-
gens, such as Streptococcus pneumoniae and Neisseria meningi-
tidis (16, 24, 51). Previous phylogenetic studies of V. cholerae
(8, 46) indicated that exchange of wb* regions may be common
in this species. Moreover, the O-antigen diversity and conser-
vation of the structural architecture of the wb* regions seen in
V. cholerae provide ample opportunity for O-antigen shifts
resulting from exchange of wb* regions. Serogroup changes in
nonpathogenic V. cholerae may not have serious consequences
and may go unnoticed, whereas in pathogenic backgrounds,
such as O1, antigen shifting may result in novel pathogens (e.g.,
V. cholerae O139 Bengal, which causes cholera epidemics even
in populations immune to V. cholerae O1).
The non-O1 and non-O139 strains identified in this study
further expand the repertoire of V. cholerae strains with epi-
demic potential and underscore the idea that the emergence of
the O139 serogroup was not a unique event. The genetic re-
latedness of the four non-O1 strains, based on IS1004 finger-
printing and RFLP analyses, was supported by an extensive
multilocus sequence typing analysis in which these four strains
always clustered with the epidemic strains (data not shown).
Interestingly, the four strains diverged further by acquiring
different virulence cassettes or parts of cassettes. For example,
the O37 serogroup strain with a classical backbone and a clas-
sical VPI acquired an El Tor CTX␾, the O53 and O65 strains
with an El Tor backbone acquired a preclassical CTX␾(with-
out the ctx genes), and the O27 strain with an El Tor backbone
acquired a novel CTX␾and a VPI cluster. Thus, it appears
that the genetic backbone, wb* cluster, VPI, and CTX regions
have evolved as independent units.
The O37 wb* region DNA sequence determined in our study
revealed that the O37 strain resulted from an exchange of the
O1 wbe cluster with a wb* O37 cluster, similar to the event that
occurred in the O139 serogroup. However, the homology
breakpoints in O37 are different from the breakpoints of the
O139 junctions. Unlike O139, in which the entire wbe region
has been replaced by the wbf region, in O37 some of the O1
wbe genes closer to the right junction have been retained,
which provides further support for the idea that the O37 strain
emerged from an O1 strain by O-antigen shifting. Although the
precise crossover points in this recombination event cannot be
predicted from the sequence, phage-mediated transfer of the
donor wb* cluster would place the crossover sites closer to the
wb* junctions, due to the large size of the wb* clusters (25 to
45 kb) and the general packaging limits of transducing phages.
On the other hand, conjugational transfer does not impose
such constraints, and the actual crossover could occur any-
where, even kilobases away from the junctions in the homol-
ogous segments flanking the divergent wb* clusters. It is also
possible that the wb* junctions are hyperrecombinogenic. Such
a proposal has been advanced before (50), and chi-like se-
quences have been found at the junctions of wb* clusters in
Escherichia coli and Klebsiella spp.
The sequence of the O37 wb* region has also revealed the
putative genes responsible for synthesis of the receptor of an
O1-specific generalized transducing phage, CP-T1. The O1
O-antigen has been postulated to be the receptor for the phage
(21), and CP-T1 has recently been reported (10) to be able to
infect O37 serogroup strains as well. Our sequence analysis
suggests that the three genes (wbeV,galE, and wbeW) shared
by the O1 and O37 wb* regions are directly involved in a step
in the synthesis of the phage receptor component of the O-
antigen.
Based on the XL-PCR and hybridization data (Fig. 1), we
predict that the O27, O53, O65, O77, O80, and O139 strains
probably have similar left junctions. The left and right junc-
tions in the O53 and O65 strains may be similar since the sizes
of the EcoRI junction fragments are the same (Fig. 1c) and the
sizes of the wb* regions of these two strains are also similar,
indicating that the strains may have minor variations in their
wb* regions. These hybridization data further suggest that the
three other strains (serogroups O27, O53, and O65) which
have backbones very similar to those of O1 strains probably
arose by exchange of wb* clusters.
Heterogeneity in the genetic organization of CTX prophage.
Heterogeneity in the genetic organization of the CTX pro-
phage region in various strains of V. cholerae has been docu-
mented previously. For example, O1 classical strains have two
copies of the CTX␾, one located on each of the two chromo-
somes. Also, some El Tor strains contain a single copy, while
many others have two or more copies arranged in tandem on
chromosome I (5, 12, 15, 31, 32, 34). V. cholerae O139 strains
are similar to O1 El Tor strains in that they have two copies of
the CTX␾arranged in tandem on chromosome I (5, 56). There
was remarkable diversity in the CTX␾s of the four strains
identified in this study with respect to the arrangement of the
copies and their gene sequences.
VPI is not unique to epidemic serogroups. Unlike the ctx
region, the VPI has not been analyzed in great detail in any
strain other than O1 classical and El Tor strains (30) and,
recently, several environmental strains (38). A few studies have
examined the tcpA gene from non-O1 and non-O139 sero-
group strains, and they have identified several tcpA variants
(13, 20, 39, 40). These studies, together with our results, clearly
demonstrate that the VPI is not unique to the epidemic and
pandemic O1 and O139 Bengal strains, as was originally re-
ported (28). While some of the non-O1 and non-O139 patho-
gens (O27, O37, and O139) have been derived from O1 strains,
independent acquisition of these virulence factors via phage-
mediated transfer in multiple O serogroup strains has also
been observed, and many of these non-O1 and non-O139
2450 LI ET AL. INFECT.IMMUN.

strains have a full complement of the VPI cluster (unpublished
data).
Model for the emergence of virulent V. cholerae strains.
Since TCP has been demonstrated to be the receptor for
CTX␾, a two-step model for the origin of virulent V. cholerae
strains has emerged (10, 19, 35). In the first step, VPI is hor-
izontally acquired by a nontoxigenic strain, and the TCP pro-
duced by the VPI-containing strain serves as the receptor for
CTX␾, which leads to the second step, in which the CTX␾
carrying ctxAB genes is acquired. This model is supported by
the findings that (i) CTX␾and VPI are associated with a
majority of the O1 and O139 strains and (ii) CTX␾
⫺
VPI
⫹
non-O1 and non-O139 strains have been occasionally found,
whereas CTX␾
⫹
VPI
⫺
non-O1 and non-O139 strains are rare
(19). However, to account for these rare strains, a TCP-inde-
pendent mechanism of acquisition of CTX␾has been pro-
posed, which involves a generalized transducing phage, CP-T1
(10). Data obtained during our studies not only support the
two-step model (none of the 300 non-O1 and non-O139 strains
screened by us was CTX␾
⫹
VPI
⫺
) but also delineate some of
the additional steps involving O-antigen exchange (Fig. 6). As
proposed before (11), classical and El Tor biotype strains orig-
inated, diverged, and subsequently acquired the classical and
El Tor VPI and CTX␾s from a nonpathogenic V. cholerae
progenitor (O1 serogroup) strain that did not possess either
VPI or CTX␾. The pre-CTX phages evolved independently, as
revealed by their distinct rstR alleles (11; unpublished data),
and ctxAB genes were subsequently acquired by a horizontal
transfer event, as revealed by the different ctx alleles in O27
and O37 strains.
Based on our data, the O37 strain seems to have arisen from
the classical strain progenitor (classical backbone and VPI) by
changing its O-antigen, diverging, and subsequently acquiring
an El Tor CTX␾. The O53 and O65 strains seem to have
originated from an O1 progenitor (backbone showing similar-
ity to both O1 classical and El Tor) by changing O-antigens
(sequentially or independently) and then diverging and acquir-
ing an El Tor VPI and a classical pre-CTX␾. The O27 strain
originated from an El Tor progenitor (El Tor backbone) and
acquired a novel VPI (novel tcpA allele) and a novel CTX␾.
The tcpA gene and presumably the rstR gene evolved by re-
combination from a common VPI and CTX␾rather than as
different phages. The idea of a mosaic pattern of similar and
divergent genes within VPI was proposed recently based on
analyses of the VPI of several environmental V. cholerae strains
(38). A comparison of the complete VPI sequences of a clas-
sical strain and an El Tor strain also supports the role of
recombination in the evolution of VPI (30). Thus, a change in
the O-antigen of the O27 strain may have occurred in the
progenitor prior to acquisition of the VPI and CTX phages, or
the O27 strain may have originated directly from an El Tor
strain and its rstR and tcpA genes may have subsequently been
altered by allelic exchange.
Our data demonstrate that genetic switching of O-antigen
biosynthesis regions resulted in the emergence of at least some
non-O1 and non-O139 V. cholerae strains having pathogenic
potential (i.e., containing the known V. cholerae virulence re-
gions VPI and CTX prophage). The nonrandom distribution of
these virulence markers in various V. cholerae serogroups (not
all 200 serogroups have these virulence factors) suggests that
horizontal transmission of virulence genes may not be similarly
effective or frequent in various V. cholerae strains. The under-
lying mechanisms for this phenomenon are not clear at this
time. However, the data presented in this paper improve our
understanding of the evolution of the species and provide
insight into the possible mechanisms for emergence of epi-
demic V. cholerae strains and serogroups from nonepidemic V.
cholerae strains and serogroups. From a public health stand-
point, our data raise the possibility that existing V. cholerae
vaccines may provide little or no protection against the newly
identified pathogenic strains, and they suggest that there is a
need for novel strategies for developing vaccines against V.
cholerae.
ACKNOWLEDGMENTS
We thank Nick Ambulos and Lisa Sadzewicz (UMAB Bioploymer
Laboratory) for DNA sequencing and primer synthesis. Thanks are
also due to Rick Blank, Arnold Kreger, and an anonymous reviewer
for helpful comments on the manuscript and to Judy Johnson for
providing access to the Smith strain collection.
Funding for this study was provided by a grant from the Department
of Veterans Affairs (to J.G.M.), by grant RO1 GM60791 from the
National Institutes of Health (to J.G.M.), by a University of Maryland
intramural grant (to S.S.), and by BREF intramural support from the
Department of Veterans Affairs (to S.S.).
REFERENCES
1. Albert, M. J., M. Ansaruzzaman, P. K. Bardhan, A. S. G. Faruque, S. M.
Faruque, M. S. Islam, D. Mahalanabis, R. B. Sack, M. A. Salam, A. K.
Siddique, M. D. Yunus, and K. Zaman. 1993. Large epidemic of cholera-like
disease in Bangladesh caused by Vibrio cholerae O139 synonym Bengal.
Lancet 342:387–390.
2. Aldova, E., K. Laznickova, E. Stepankova, and J. Lietava. 1968. Isolation of
nonagglutinable vibrios from an enteritis outbreak in Czechoslovakia. J. In-
fect. Dis. 118:25–31.
FIG. 6. Model for emergence of the non-O1 and non-O139 strains
with epidemic potential by O-antigen switching. A dendrogram of the
genetic relatedness of the various serogroups, based on IS1004 finger-
printing analysis, is shown on the left. The O37 strain appears to have
been derived from an O1 preclassical strain (classical VPI cluster) by
O-antigen shifting, divergence, and subsequent acquisition of an El
Tor CTX␾. The El Tor strains diverged from classical strains, acquired
a distinct CTX␾, and probably acquired a tcpA allele by recombination
(30). The O139 strain appears to have been derived from an El Tor
strain by O-antigen switching, and the O27 strain appears to have been
derived from an El Tor progenitor and to have acquired a distinct
CTX␾and a distinct VPI. It is also probable that O27 emerged from
an El Tor strain by O-antigen switching and subsequently changed its
rstR and tcpA genes by allelic exchange. The O53 and O65 strains have
an El Tor VPI cluster and are identical to each other. They have
genetic backbones resembling those of both classical and El Tor
strains, and hence, they probably emerged from an O1 progenitor,
diverged, and subsequently acquired a classical pre-CTX␾and and an
El Tor VPI. These two strains probably underwent O-antigen switch-
ing events independently or sequentially, from O1 to O53 and O65. Cl,
classical; ET, El Tor; NT, novel type (38).
VOL. 70, 2002 HORIZONTAL TRANSFER OF O-ANTIGEN CLUSTER 2451

3. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,
and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation
of protein database search programs. Nucleic Acids Res. 25:3389–3402.
4. Bailey, M. J. A., C. Hughes, and V. Koronakis. 1997. RfaH and the ops
element, components of a novel system controlling bacterial transcription
elongation. Mol. Microbiol. 26:845–851.
5. Basu, A., A. K. Mukhopadhyay, C. Sharma, J. Jyot, N. Gupta, A. Ghosh,
S. K. Bhattacharya, Y. Takeda, A. S. Faruque, M. J. Albert, and G. B. Nair.
1998. Heterogeneity in the organization of the CTX genetic element in
strains of Vibrio cholerae O139 Bengal isolated from Calcutta, India and
Dhaka, Bangladesh and its possible link to the dissimilar incidence of O139
cholera in the two locales. Microb. Pathog. 24:175–183.
6. Berche, P., C. Poyart, E. Abachin, H. Lelievre, J. Vandepitte, A. Dodin, and
J.-M. Fournier. 1994. The novel epidemic strain O139 is closely related to
pandemic strain O1 of Vibrio cholerae. J. Infect. Dis. 170:701–704.
7. Bhattacharya, S. K., M. K. Bhattacharya, G. B. Nair, D. Dutta, A. Deb, T.
Ramamurthy, S. Garg, P. K. Saha, P. Dutta, A. Moitra, B. K. Mandel, T.
Shimada, Y. Takeda, and B. C. Deb. 1993. Clinical profile of acute diarrhoea
cases infected with the new epidemic strain of V. cholerae O139: designation
of disease as cholera. J. Infect. Dis. 27:11–15.
8. Bik, E. M., R. D. Gouw, and F. R. Mooi. 1996. DNA fingerprinting of Vibrio
cholerae strains with a novel insertion sequence element: a tool to identify
epidemic strains. J. Clin. Microbiol. 34:1453–1461.
9. Bik, E. M., A. E. Bunschoten, R. J. L. Willems, A. C. Y. Chang, and F. R.
Mooi. 1996. Genetic organization and functional analysis of the otn DNA
essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139. Mol.
Microbiol. 20:799–811.
10. Boyd, E. F., and M. K. Waldor. 1999. Alternative mechanism of cholera toxin
acquisition by Vibrio cholerae: generalized transduction of CTX␾by bacte-
riophage CP-T1. Infect. Immun. 67:5898–5905.
11. Boyd, E. F., A. J. Heilpern, and M. K. Waldor. 2000. Molecular analyses of
a putative CTX␾precursor and evidence for independent acquisition of
distinct CTX␾s by toxigenic Vibrio cholerae. J. Bacteriol. 182:5530–5538.
12. Boyd, E. F., K. E. Moyer, L. Shi, and M. K. Waldor. 2000. Infectious CTX␾
and the vibrio pathogenicity island prophage in Vibrio mimicus: evidence for
recent horizontal transfer between V. mimicus and V. cholerae. Infect. Im-
mun. 68:1507–1513.
13. Chakraborty, S., A. K. Mukhopadhyay, R. K. Bhadra, A. N. Ghosh, R. Mitra,
T. Shimada, S. Yamasaki, S. M. Faruque, Y. Takeda, R. R. Colwell, and G. B.
Nair. 2000. Virulence genes in environmental strains of Vibrio cholerae.
Appl. Environ. Microbiol. 66:4022–4028.
14. Comstock, L. E., J. A. Johnson, J. M. Michalski, J. G. Morris, Jr., and J. B.
Kaper. 1996. Cloning and sequence of a region encoding a surface polysac-
charide of Vibrio cholerae O139 and characterization of the insertion site in
the chromosome of Vibrio cholerae O1. Mol. Microbiol. 19:815–826.
15. Davis, B. M., H. H. Kimsey, W. Chang, and M. K. Waldor. 1999. The Vibrio
cholerae O139 Calcutta bacteriophage CTX␾is infectious and encodes a
novel repressor. J. Bacteriol. 181:6779–6787.
16. Dillard, J. P., M. Caimano, T. Kelly, and J. Yother. 1995. Capsules and
cassettes: genetic organization of the capsule locus of Streptococcus pneu-
moniae. J. Dev. Biol. Stand. 85:261–265.
17. Donnenberg, M. S., and J. B. Kaper. 1991. Construction of an eae deletion
mutant of enteropathogenic Escherichia coli by using a positive selection
suicide vector. Infect. Immun. 59:4310–4317.
18. Fallarino, A., C. Mavrangelos, U. H. Stroeher, and P. A. Manning. 1997.
Identification of additional genes required for O-antigen biosynthesis in
Vibrio cholerae O1. J. Bacteriol. 179:2147–2153.
19. Faruque, S. M., M. J. Albert, and J. J. Mekalanos. 1998. Epidemiology,
genetics, and ecology of toxigenic Vibrio cholerae. Microbiol. Mol. Biol. Rev.
62:1301–1314.
20. Ghosh, C., R. K. Nandy, S. K. Dasgupta, G. B. Nair, R. H. Hall, and A. C.
Ghose. 1997. A search for cholera toxin (CT), toxin coregulated pilus (TCP),
the regulatory element ToxR and other virulence factors in non-O1/non-
O139 Vibrio cholerae. Microb. Pathog. 22:199–208.
21. Guidolin, A., and P. A. Manning. 1985. Bacteriophage CP-T1 of Vibrio
cholerae. Identification of the cell surface receptor. Eur. J. Biochem. 153:
89–94.
22. Heidelberg, J. F., J. A. Eisen, W. C. Nelson, R. A. Clayton, M. L. Gwinn, R. J.
Dodson, D. H. Haft, E. K. Hickey, J. D. Peterson, L. Umayam, S. R. Gill,
K. E. Nelson, T. D. Read, H. Tettelin, D. Richardson, M. D. Ermolaeva, J.
Vamathevan, S. Bass, H. Qin, I. Dragoi, P. Sellers, L. McDonald, T. Utter-
back, R. D. Fleishmann, W. C. Nierman, O. White, S. L. Salzberg, H. O.
Smith, R. R. Colwell, J. J. Mekalanos, J. C. Venter, and C. M. Fraser. 2000.
DNA sequence of both chromosomes of the cholera pathogen Vibrio chol-
erae. Nature 406:477–483.
23. Hobbs, M., and P. R. Reeves. 1994. The JUMPstart sequence: a 39 bp
element common to several polysaccharide gene clusters. Mol. Microbiol.
12:855–856.
24. Jiang, S. M., L. Wang, and P. R. Reeves. 2001. Molecular characterization of
Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide
gene clusters. Infect. Immun. 69:1244–1255.
25. Johnson, J. A., C. A. Salles, P. Panigrahi, M. J. Albert, A. C. Wright, R. J.
Johnson, and J. G. Morris, Jr. 1994. Vibrio cholerae O139 synonym Bengal
is closely related to Vibrio cholerae El Tor but has important differences.
Infect. Immun. 62:2108–2110.
26. Kamal, A. M. 1971. Outbreak of gastro-enteritis by non-agglutinable (NAG)
vibrios in the republic of the Sudan. J. Egypt. Public Health Assoc. XLVI:
125–159.
27. Kaper, J. B., J. G. Morris, Jr., and M. M. Levine. 1995. Cholera. Clin.
Microbiol. Rev. 8:48–86.
28. Karaolis, D. K., J. A. Johnson, C. C. Bailey, E. C. Boedeker, J. B. Kaper, and
P. R. Reeves. 1998. A Vibrio cholerae pathogenicity island associated with
epidemic and pandemic strains. Proc. Natl. Acad. Sci. USA 95:3134–3139.
29. Karaolis, D. K., S. Somara, D. R. Maneval, Jr., J. A. Johnson, and J. B.
Kaper. 1999. A bacteriophage encoding a pathogenicity island, a type-IV
pilus and a phage receptor in cholera bacteria. Nature 399:375–379.
30. Karaolis, D. K., R. Lan, J. B. Kaper, and P. R. Reeves. 2001. Comparison of
Vibrio cholerae pathogenicity islands in sixth and seventh pandemic strains.
Infect. Immun. 69:1947–1952.
31. Kimsey, H. H., and M. K. Waldor. 1998. CTX ␾immunity: application in the
development of cholera vaccines. Proc. Natl. Acad. Sci. USA 95:7035–7039.
32. Kimsey, H. H., G. B. Nair, A. Ghosh, and M. K. Waldor. 1998. Diverse CTX
␾s and evolution of new pathogenic Vibrio cholerae. Lancet 352:457–458.
33. Manning, P. A., U. H. Stroeher, and R. Morona. 1994. Molecular basis for
O-antigen biosyntheis in Vibrio cholerae O1: Ogawa-Inaba switching, p. 77–
94. In I. K. Wachsmuth, P. A. Blake, and O. Olsvik (ed.), Vibrio cholerae and
cholera: molecular to global perspectives. American Society for Microbiol-
ogy, Washington, D.C.
34. Mekalanos, J. J. 1983. Duplication and amplification of toxin genes in Vibrio
cholerae. Cell 35:253–263.
35. Mekalanos, J. J., E. J. Rubin, and M. K. Waldor. 1997. Cholera: molecular
basis for emergence and pathogenesis. FEMS Immunol. Med. Microbiol.
18:241–248.
36. Mooi, F. R., and E. M. Bik. 1997. The evolution of epidemic Vibrio cholerae
strains. Trends Microbiol. 4:161–165.
37. Morris, J. G., Jr., G. E. Losonsky, J. A. Johnson, C. O. Tacket, J. P. Nataro,
P. Panigrahi, and M. M. Levine. 1995. Clinical and immunological charac-
teristics of Vibrio cholerae O139 Bengal infection in North American volun-
teers. J. Infect. Dis. 171:903–908.
38. Mukhopadhyay, A. K., S. Chakraborty, Y. Takeda, G. B. Nair, and D. E.
Berg. 2001. Characterization of VPI pathogenicity island and CTX␾pro-
phage in environmental strains of Vibrio cholerae. J. Bacteriol. 183:4737–
4746.
39. Nandi, B., R. K. Nandy, A. C. Vicente, and A. C. Ghose. 2000. Molecular
characterization of a new variant of toxin-coregulated pilus protein (TcpA)
in a toxigenic non-O1/non-O139 strain of Vibrio cholerae. Infect. Immun.
68:948–952.
40. Novais, R. C., A. Coelho, C. A. Salles, and A. C. Vicente. 1999. Toxin-
coregulated pilus cluster in non-O1, non-toxigenic Vibrio cholerae: evidence
of a third allele of pilin gene. FEMS Microbiol. Lett. 171:49–55.
41. Ramamurthy, T., S. Garg, R. Sharma, S. K. Bhattacharya, G. B. Nair, T.
Shimada, T. Takeda, T. Karasawa, H. Kurazano, A. Pal, and Y. Takeda.
1993. Emergence of novel strain of Vibrio cholerae with epidemic potential in
southern and eastern India. Lancet 341:703–704.
42. Rhine, J. A., and R. K. Taylor. 1994. TcpA pilin sequences and colonization
requirements for O1 and O139 Vibrio cholerae. Mol. Microbiol. 13:1013–
1020.
43. Shimada, T., E. Arakawa, K. Itoh, T. Okitsu, A. Matsushima, Y. Asai, S.
Yamai, T. Nakazato, G. B. Nair, M. J. Albert, and Y. Takeda. 1994. Extended
serotyping scheme for Vibrio cholerae. Curr. Microbiol. 28:175–178.
44. Smith, H. L. 1979. Serotyping of non-cholera vibrios. J. Clin. Microbiol.
10:85–90.
45. Sozhamannan, S., Y. K. Deng, M. Li, A. Sulakvelidze, J. B. Kaper, J. A.
Johnson, G. B. Nair, and J. G. Morris, Jr. 1999. Cloning and sequencing of
the genes downstream of the wbf gene cluster of Vibrio cholerae serogroup
O139 and analysis of the junction genes in other serogroups. Infect. Immun.
67:5033–5040
46. Stine, O. C., S. Sozhamannan, Q. Gou, S. Zheng, J. G. Morris, Jr., and J. A.
Johnson. 2000. Phylogeny of Vibrio cholerae based on recA sequence. Infect.
Immun. 68:7180–7185.
47. Stroeher, U. H., and P. A. Manning. 1997. Vibrio cholerae serotype O139:
swapping genes for surface polysaccharide biosynthesis. Trends Microbiol.
5:178–180.
48. Stroeher, U. H., G. Parasivam, B. K. Dredge, and P. A. Manning. 1997.
Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthe-
sis. J. Bacteriol. 179:2740–2747.
49. Stroeher, U. H., K. E. Jedani, and P. A. Manning. 1998. Genetic organization
of the regions associated with surface polysaccharide synthesis in Vibrio
cholerae O1, O139 and Vibrio anguillarum O1 and O2: a review. Gene
223:269–282.
50. Sugiyama, T., N. Kido, Y. Kato, N. Koide, T. Yoshida, and T. Yokochi. 1997.
Evolutionary relationship among rfb gene clusters synthesizing mannose
homopolymer as O-specific polysaccharides in Esherichia coli and Klebsiella.
Gene 198:111–113.
2452 LI ET AL. INFECT.IMMUN.

51. Swartley, J. S., A. A. Marfin, S. Edupuganti, L.-J. Liu, P. Cieslak, B. Perkins,
J. D. Wenger, and D. S. Stephens. 1997. Capsule switching of Neisseria
meningitidis. Proc. Natl. Acad. Sci. USA 94:271–276.
52. Tacket, C. O., R. K. Taylor, G. Losonsky, Y. Lim, J. P. Nataro, J. B. Kaper,
and M. M. Levine. 1998. Investigation of the roles of toxin-coregulated pili
and mannose-sensitive hemagglutinin pili in the pathogenesis of Vibrio chol-
erae O139 infection. Infect. Immun. 66:692–695.
53. Taylor, R. K., V. L. Miller, D. B. Furlong, and J. J. Mekalanos. 1987. The use
of phoA gene fusions to identify a pilus colonization factor coordinately
regulated with cholera toxin. Proc. Natl. Acad. Sci. USA 84:2833–2837.
54. Taylor, R. K. 1999. Virus on virus infects bacterium. Nature 399:312–313.
55. Terai, A., K. Baba, H. Shirai, O. Yoshida, Y. Takeda, and M. Nishibuchi.
1991. Evidence for insertion sequence-mediated spread of the thermo-
stable direct hemolysin gene among Vibrio species. J. Bacteriol. 173:5036–
5046.
56. Waldor, M. K., and J. J. Mekalanos. 1994. Emergence of a new cholera
pandemic: molecular analysis of virulence determinants in Vibrio cholerae
O139 and development of a live vaccine prototype. J. Infect. Dis. 170:278–
283.
57. Waldor, M. K., and J. J. Mekalanos. 1996. Lysogenic conversion by a fila-
mentous phage encoding cholera toxin. Science 272:1910–1914.
58. World Health Organization. 2001. Cholera vaccines. Wkly. Epidemiol. Rec.
W. H. O. 76:117–124.
59. Yamasaki, S., T. Shimizu, K. Hoshino, S. T. Ho, T. Shimada, G. B. Nair, and
Y. Takeda. 1999. The genes responsible for O-antigen synthesis of Vibrio
cholerae O139 are closely related to those of Vibrio cholerae O22. Gene
237:321–332.
Editor: D. L. Burns
VOL. 70, 2002 HORIZONTAL TRANSFER OF O-ANTIGEN CLUSTER 2453