Microbiology (2005), 151, 311–322
DOI 10.1099/mic.0.27621-0
Molecular evolution of Vibrio pathogenicity island-2
(VPI-2): mosaic structure among Vibrio cholerae
and Vibrio mimicus natural isolates
William S. Jermyn and E. Fidelma Boyd
Department of Microbiology, UCC, National University of Ireland-Cork, Cork, Ireland
Correspondence
E. Fidelma Boyd
[email protected]
Received 13 September 2004
Revised
30 September 2004
Accepted 30 September 2004
Vibrio cholerae is a Gram-negative rod that inhabits the aquatic environment and is the
aetiological agent of cholera, a disease that is endemic in much of Southern Asia. The 57?3 kb
Vibrio pathogenicity island-2 (VPI-2) is confined predominantly to toxigenic V. cholerae O1
and O139 serogroup isolates and encodes 52 ORFs (VC1758 to VC1809), which include
homologues of an integrase (VC1758), a restriction modification system, a sialic acid metabolism
gene cluster (VC1773–VC1783), a neuraminidase (VC1784) and a gene cluster that shows
homology to Mu phage. In this study, a 14?1 kb region of VPI-2 comprising ORFs VC1773 to
VC1787 was identified by PCR and Southern blot analyses in all 17 Vibrio mimicus isolates
examined. The VPI-2 region in V. mimicus was inserted adjacent to a serine tRNA similar to VPI-2
in V. cholerae. In 11 of the 17 V. mimicus isolates examined, an additional 5?3 kb region encoding
VC1758 and VC1804 to VC1809 was present adjacent to VC1787. The evolutionary history
of VPI-2 was reconstructed by comparative analysis of the nanH (VC1784) gene tree with
the species gene tree, deduced from the housekeeping gene malate dehydrogenase (mdh),
among V. cholerae and V. mimicus isolates. Both gene trees showed an overall congruence; on
both gene trees V. cholerae O1 and O139 serogroup isolates clustered together, whereas
non-O1/non-O139 serogroup isolates formed separate divergent branches with similar clustering
of strains within the branches. One exception was noted: on the mdh gene tree, V. mimicus
sequences formed a distinct divergent lineage from V. cholerae sequences; however, on the
nanH gene tree, V. mimicus clustered with V. cholerae non-O1/non-O139 isolates, suggesting
horizontal transfer of this region between these species.
INTRODUCTION
Vibrio cholerae, the causative agent of cholera, is a major
cause of morbidity and mortality in many parts of the
world where clean water is not available; in some areas of
Southern Asia, cholera is endemic. V. cholerae is a natural
inhabitant of the aquatic ecosystem, and most V. cholerae
isolates do not possess the ability to cause cholera. Among
V. cholerae natural isolates, only O1 and O139 serogroups
are associated with epidemic and pandemic cholera. The V.
cholerae O139 serogroup first emerged in 1992 as a cause
of epidemic cholera (Albert et al., 1993; Cholera Working
Group, 1993). It is now believed that O139 serogroup
isolates arose from an O1 strain that acquired the O139
antigen (Berche et al., 1994; Bik et al., 1995; Stroeher et al.,
1997; Waldor & Mekalanos, 1994). All V. cholerae O1 and
O139 serogroup isolates encode two main virulence
factors: toxin co-regulated pilus (TCP) and cholera toxin
(CT). TCP is encoded on a pathogenicity island, the Vibrio
Abbreviations: CT, cholera toxin; TCP, toxin co-regulated pilus; VPI-2,
Vibrio pathogenicity island-2.
0002-7621 G 2005 SGM
pathogenicity island (named VPI-1 in this paper), and CT
is encoded by a filamentous phage, CTXw (Karaolis et al.,
1998; Kovach et al., 1996; Waldor & Mekalanos, 1996).
Acquisition of TCP and CT by V. cholerae is sequential: first
V. cholerae isolates acquired TCP, an essential intestinal
colonization factor and the receptor for CTXw (Taylor
et al., 1987; Waldor & Mekalanos, 1996), and then they
acquired CT via CTXw (Waldor & Mekalanos, 1996). Several
additional genomic regions have also been identified,
predominantly among epidemic O1 and O139 serogroup
isolates; these include RS1w, Vibrio seventh pandemic
island-I (VSP-I), VSP-II and VPI-2 (Faruque et al., 2002;
Davis et al., 2002; Dziejman et al., 2002; Jermyn & Boyd,
2002; O’Shea et al., 2004a). RS1w is associated with the CTX
prophage in V. cholerae El Tor isolates and is required for
the production of CTXw (Davis & Waldor, 2000). VSP-I
and VSP-II are genomic islands identified by microarray
analysis among V. cholerae El Tor isolates (Dziejman et al.,
2002). It was suggested by Dziejman et al. (2002) that the
genes encoded on the VSP-I and VSP-II islands are likely
to be responsible for the unique characteristics of the
seventh pandemic (El Tor) strains.
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311
W. S. Jermyn and E. F. Boyd
VPI-2 is a 57?3 kb chromosomal region encoding ORFs
VC1758 to VC1809 which has all the characteristics of a
pathogenicity island (Jermyn & Boyd, 2002). All toxigenic
V. cholerae O1 and O139 serogroup isolates contained VPI2, whereas non-O1/nonO139 non-toxigenic isolates lacked
the region. VPI-2 encodes several gene clusters: a type-1
restriction modification system, which may protect the
bacteria from viral infection (Bickle & Kruger, 1993); a nan–
nag gene cluster homologous to genes involved in sialic
acid metabolism, which may act as a carbon and nitrogen
source (Vimr et al., 2004); neuraminidase, which acts on
higher-order gangliosides in the intestine converting them
to GM1 gangliosides, with the subsequent release of sialic
acids (Galen et al., 1992); and a region with homology to
Mu phage. Among most V. cholerae O139 serogroup isolates, only a 20 kb region of VPI-2 was present, as ORFs
VC1761 to VC1788 had been deleted, indicating the
instability of the region (Jermyn & Boyd, 2002).
The species V. mimicus is closely related to V. cholerae;
however, V. mimicus is phenotypically and genotypically
distinct from V. cholerae and can be readily differentiated
from V. cholerae (Boyd et al., 2000a; Byun et al., 1999;
Davis et al., 1981; O’Shea et al., 2004a; Reen & Boyd, 2004).
V. mimicus, unlike V. cholerae, is negative in sucrose,
Voges–Proskauer, corn oil and Jordan tartrate reactions
(Davis et al., 1981). Comparative sequence analysis of the
housekeeping gene malate dehydrogenase (mdh) from V.
mimicus isolates showed the mean pairwise divergence
between V. cholerae and V. mimicus was approximately
10 %, which is equivalent to the divergence between Salmonella enterica LT2 and E. coli K-12 (Boyd et al., 1994, 2000a;
Byun et al., 1999; O’Shea et al., 2004a). In addition, analysis
of groE-I and groEL-II on chromosomes 1 and 2, respectively, of V. cholerae and V. mimicus also demonstrated the
divergence of both species from one another (Reen & Boyd,
2004). The natural habitat of V. mimicus, similar to V.
cholerae, is the aquatic ecosystem, where it has been found
both as a free-living bacterium and in association with
phytoplankton and crustaceans (Acuna et al., 1999; Campos
et al., 1996). Consumption of V. mimicus-contaminated
shellfish has been linked to the development of gastroenteritis (Acuna et al., 1999). The virulence determinants of
V. mimicus have not been well characterized. Recently, it was
demonstrated that some V. mimicus isolates also harbour
VPI-1 (TCP) and CTXw (CT) (Boyd et al., 2000b). Several V.
mimicus strains isolated from clinical and environmental
sources were shown to produce multiple toxins, including
a haemolysin, zonula occludens toxin, a heat-stable enterotoxin, as well as a CT-like toxin (Campos et al., 1996;
Chowdhury et al., 1994; Ramamurthy et al., 1994; Shi et al.,
1998; Spira & Fedorka-Cray, 1983, 1984).
In this paper, we examined 17 V. mimicus clinical and
environmental isolates of various serogroups for the
presence of the 57?3 kb VPI-2 region. ORFs VC1773 to
VC1787 were found to be present in all 17 V. mimicus stains
examined. In addition, 11 V. mimicus isolates contained
312
ORFs VC1758 (integrase) and VC1804 to VC1809. We
reconstructed the phylogenetic relationship of the nanH
(VC1784) gene among a range of V. cholerae and V. mimicus
isolates and compared it to the species tree, as deduced
from the housekeeping gene mdh. On the mdh gene tree, V.
mimicus isolates formed a separate divergent lineage from
V. cholerae. On the nanH gene tree, however, V. mimicus
isolates clustered with V. cholerae non-O1/non-O139 isolates, indicating that the nanH gene from these species is
closely related. Analysis of additional genes spanning VPI-2
among a subset of V. cholerae and V. mimicus were in
agreement with the nanH data, suggesting that VPI-2 was
horizontally transferred between these species.
METHODS
Bacterial strains. A total of 22 V. cholerae and 17 V. mimicus
isolates were used in this study. These isolates were derived from
both clinical and environmental sources with a wide geographic distribution (Table 1). V. cholerae and V. mimicus strains were grown
in Luria–Bertani (LB) broth. All strains were stored at 270 uC in
broth containing 20 % (v/v) glycerol.
DNA isolation. Chromosomal DNA was extracted from each isolate
using the G-nome DNA isolation kit (BIO 101). A single colony was
inoculated into 3 ml LB broth and incubated overnight, with shaking, at 37 uC. The cells were pelleted at 3000 r.p.m. for 5 min and
resuspended in 1?85 ml suspension solution. The cells were lysed
and treated with RNase and protease solutions. Following incubation at 37 uC for 2 h, any remaining proteins were precipitated out
and the sample was centrifuged. Supernatants were transferred to a
clean Eppendorf tube (1?5 ml) and treated with both 100 % ethanol
and TE buffer. The DNA was spooled out and air-dried to remove
excess alcohol. Following drying, the DNA was dissolved in TE
buffer, pH 8, and stored at 220 uC.
Molecular analysis. To assay for the presence of VPI-2 among
V. mimicus isolates, 17 primer pairs that span the entire 57?3 kb
VPI-2 region and two primer pairs that flank the island were used
for PCR analysis (Jermyn & Boyd, 2002). PCR was performed in
volumes of 20 ml containing 100 ng genomic DNA, 200 mM primer,
1?25 mM dNTPs, 50 mM MgCl2, and 1U Taq DNA polymerase
(Bioline). The amplification conditions were pre-incubation at
96 uC for 60 s, followed by 30 cycles of 94 uC for 30 s, 48–55 uC
(depending on the primer pair) for 30 s and 72 uC for a time chosen
based on the size of the expected fragment (60 s kb21). All PCRs
were carried out in a PTC-200 Peltier Thermal Cycler (MJ Research).
PCR products were resolved by electrophoresis on a 0?8 % agarose
gel made with 16 TAE (consisting of 40 mM Tris/acetate and
1 mM EDTA) and containing ethidium bromide. PCR amplification
products were visualized by UV transillumination.
Southern hybridization was carried out with DNA probes generated
from PCR products from the reference V. cholerae strain N16961 as a
template (Heidelberg et al., 2000). PCR products were purified using
the ConceRT-PCR purification kit (Gibco, BRL). For Southern
hybridization analysis, DNA from each strain was digested with the
restriction enzyme EcoRI (Roche Molecular Biochemicals), and the
fragments were separated by electrophoresis in 0?6 % TAE agarose.
The gel was depurinated with 0?25 M HCl, denatured with a solution of 5 M NaCl and 10 N NaOH, and neutralized with 1 M Tris
and 206 SSC. The fragments were transferred to nitrocellulose
membranes by a posiblotter (Stratagene). DNA was fixed to the
membrane by UV cross-linking. Approximately 100 ng probe DNA
was conjugated to horseradish peroxidase using the ECL direct
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Microbiology 151
Evolution of Vibrio pathogenicity island-2
Table 1. V. cholerae and V. mimicus strains used in PCR and Southern hybridization analysis
Strain
V. cholerae
569B
O395
SM115
N16961
GP33
GP155
F1939
3038
2740-80
1811-98
MO2
E714
V45
V46
V47
V51
V52
V54
151
SG14
9581
9582
V. mimicus
ES6
ES13
ES24
ES26
ES28
CS6
CS11
CS16
CS17
CS18
CS45
CS61
PT5
PT48
9583
523-80
531-90
Serogroup (biotype)
Source
Location
Year
O1 (classical)
O1 (classical)
O1 (El Tor)
O1 (El Tor)
O1 (El Tor)
O1 (El Tor)
O1 (El Tor)
O1 (El Tor)
O1 (El Tor)
O1 (El Tor)
O139
Unknown
Rough
O141
O141
O141
O37
O8
O37
O54
O41
Rough
Clinical
Clinical
Clinical
Clinical
Clinical
Unknown
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Environmental
Clinical
Clinical
Clinical
India
India
Bahrain
Bangladesh
USSR
Australia
Rwanda
Vietnam
USA
Bangladesh
Madras
USA
Bangladesh
USA
USA
USA
Sudan
Thailand
Mexico
India
India
India
1947
1964
1978
1975
1971
1979
1994
Unknown
1980
1998
1992
Unknown
1978
1978
1984
1987
1968
Unknown
1998
1992/93
1990
1990
O26
O32
O137
O23
O8
O34
O137
Rough
O131
O41
O36
O128
O115
O115
O115
O115
O115
Environmental
Environmental
Environmental
Environmental
Environmental
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Bangladesh
Bangladesh
USA
USA
Japan
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
1985
1985
1980
1980
1990
nucleic acid labelling system (Amersham Pharmacia Biotech), and
after hybridization overnight, was detected by the ECL chemiluminescent substrate. Hybridization membranes were washed according
to manufacturer’s instructions.
Pulsed-field gel electrophoresis. To determine whether the ORF
VC1758 encoding an integrase was located in the proximity of
VC1773–VC1787, we carried out pulsed-field gel electrophoresis
(PFGE). Genomic DNA was prepared from the V. mimicus strains
PT5 and 523-80 as follows. The bacteria were grown in LB broth
overnight at 37 uC. The cells were centrifuged and resuspended in SE
buffer (consisting of 75 mM NaCl and 25mM EDTA) to an OD620
of 1?0. An equal volume of the cell suspension and 2 % agarose was
mixed and cast in a mould. The individual agarose inserts were
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treated with PEN buffer (0?5 M EDTA, 1 %, w/v, N-lauroylsarcosine,
1 mg Pronase ml21) and washed with TE buffer (100 mM Tris/Cl,
10 mM EDTA). After processing, the inserts were incubated for at
least 6 h with SfiI at 50 uC. Electrophoresis was performed with a
CHEF-DR II electrophoresis system (Bio-Rad) in 16 TBE buffer
(45 mM Tris, 45 mM boric acid, 1 mM EDTA) at 13 uC. Pulse
ramps were 3 to 80 s for 21 h at 160 V in 1?0 % agarose. After
electrophoresis, the gel was stained with ethidium bromide, washed
and treated for Southern hybridization analysis.
DNA sequencing. The primer pairs used for PCR amplification
and subsequent sequencing are listed in Table 2. The nucleotide
sequence of an internal fragment of six genes spanning the VPI-2
region was obtained: VC1764, encoding a methyl accepting subunit;
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W. S. Jermyn and E. F. Boyd
Table 2. Oligonucleotide primer sequences used for PCR analysis and Southern hybridization probe generation
Primer
Sequence (5§ to 3§)
Product size
NF2
1806R
mdh1
mdh2
1764F
1764R
1781F
1781R
1783F
1783R
nanH3
nanH4
1799F
1799R
1806F
1806R
GATTGTCATTGTTGAAACGGA
TAGGATCTCGCCTGGTTTTCCG
ATGAAAGTCGCTGTTATT
GTATCTAACATGCCATCC
GCTCTAACTGCTGTATTCATGC
GGCTGCTTTCGTCAATAAG
CCAAACTGTCGTTTCCATAC
GGGTTGCGTTATTGAACC
ATGCGGCAATGGTTTGG
CGGTATTCAATGCTGTACC
TGACAGTCCAGCCAAACAGG
CAAGTTATACCGCCATCTC
GACTTGTAGTTCATTGTCGGTC
ATATCGCTCACCACCAGGTC
CATTCTTCGCAACTCGTTAG
TAGGATCTCGCCTGGTTTTCCG
2?9 kb
This study
0?9 kb
Boyd et al. (2000a)
1?0 kb
This study
0?6 kb
This study
0?6 kb
This study
0?7 kb
This study
0?9 kb
This study
0?9 kb
This study
nanE (VC1781), encoding N-acetylmannosamine-6-phosphate
2-epimerase; nagC (VC1783), encoding N-acetylglucosamine-6phosphate deacetylase; nanH (VC1784), encoding neuraminidase;
VC1799, encoding an integrase; and VC1806, encoding a replicase.
For the nanH gene, fragments were amplified from the chromosomal DNA of 22 V. cholerae strains and three V. mimicus strains,
whereas for the remaining five genes, five V. cholerae strains were
examined, which included a classical strain O395, an O139 serogroup strain MO2, and three non-O1/non-O139 strains V46, V47
and V52, and a V. mimicus strain PT5. PCR was performed in a
20 ml reaction mixture using conditions as described earlier. PCR
amplicons were purified using the ConceRT-PCR purification kit
(Gibco-BRL) in accordance with manufacturer’s instructions.
Following purification, an aliquot of 10 ml was used as a sequencing
template. All sequencing was carried out by MWG-Biotech.
Bioinformatic and phylogenetic analysis. The VC1764, nanE,
nagC, nanH, VC1799 and VC1806 nucleotide sequences were aligned
with the CLUSTAL W multiple sequence alignment program (http://
www.ebi.ac.uk/clustalw/) (Higgins et al., 1996). The housekeeping
locus mdh was also included in the analysis for the same set of
strains (O’Shea et al., 2004a). Phylogenetic analysis was performed
on the nanH and the mdh sequences with the Molecular Evolutionary Genetics Analysis (MEGA) version 2.1 (Kumar et al., 2001).
Phylogenetic trees were constructed using the neighbour-joining
method based on synonymous nucleotide sites, with corrections for
multiple substitutions by the Jukes–Cantor method (Jukes & Cantor,
1969; Saitou & Nei, 1987; Kumar et al., 2001). Rates (per sites) of
synonymous (kS) and non-synonymous (kN) substitutions were
calculated by the method of Nei and Gojobori (1986). We also
calculated the kN/kS ratio, which is a measure of the selective constraints on a gene. The VPI-2 region was analysed using the Basic
Local Alignment Search Tool (BLAST) at the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/COG/).
RESULTS
Distribution of VPI-2 (ORFs VC1758–VC1809)
among V. mimicus
Previously, we identified the 57?3 kb VPI-2 region among
toxigenic V. cholerae isolates (Fig. 1). To determine whether
314
Reference
the VPI-2 region was present in V. mimicus, PCR assays
were carried out on 12 clinical and five environmental V.
mimicus isolates using 17 primer pairs spanning the VPI-2
region (Tables 1 and 2). In all 17 V. mimicus isolates
examined, positive PCR products were obtained with four
of the 17 primer pairs: 148F/146B (encompassing VC1773–
VC1776), 147F/146A (encompassing VC1777–VC1779),
nagC1/nagA2 (encompassing VC1782–VC1783) and nanH3/
nanH4 (encompassing VC1784). Thus, with primer pair
148F/146B, a 4?2 kb PCR band was obtained, and similarly
with primer pair 147F/146A, a 2?3 kb PCR product was
obtained from all strains examined (Fig. 2). The primer
pair nagC1/nagA2 gave a 2?1 kb PCR product band and
primer pair nanH3/nanH4 gave a 0?7 kb PCR product
from all V. mimicus strains examined (Fig. 2). In addition,
10 clinical V. mimicus isolates and one environmental V.
mimicus isolate gave positive PCR products with three additional primer pairs: nint3/nint4 (encompassing VC1758),
VC3A/VC4A (encompassing VC1804–VC1807) and VC3B/
VC4B (encompassing VC1806–VC1809). With the primer
pair nint3/nint4, a 1?0 kb product was obtained; with the
primer pairs VC3A/VC4A and VC3B/VC4B, 2?5 kb and
2?3 kb products were obtained, respectively (Fig. 2). To
verify the PCR results, we carried out Southern hybridization analysis (Fig. 3). Using probes 148F, 147F, nagC and
nanH, positive hybridization fragments were obtained
from all 17 isolates, as expected, which span the region
of interest. The probe RadC, spanning VC1786–VC1789,
however, also gave a positive hybridization fragment in all
isolates, even though PCR analysis failed to amplify this
region. Using DNA probe nint spanning VC1758, and
DNA probes VC3A and VC3B spanning VC1804–VC1809,
Southern hybridization confirmed the presence of these
regions in 11 V. mimicus isolates only. No hybridization
fragments were obtained using probes specific for regions
VC1759–VC1772 and VC1789–VC1803, demonstrating
their absence in all V. mimicus isolates examined. In
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Microbiology 151
Evolution of Vibrio pathogenicity island-2
Fig. 1. Schematic representation and genetic organization of VPI-2 in V. cholerae O1 and O139 serogroups and V. mimicus
isolates. ORFs are represented by open arrows indicating the direction of transcription. The spotted filled arrows indicate
ORFs VC1773–VC1787. The dashed arrows indicate VC1758 (int gene) and VC1804–VC1809. Unfilled arrows represent
the remaining VPI-2 ORFs. A black arrow represents the tRNA serine.
Fig. 2. PCR analysis of the VPI-2 region among 16 V. mimicus strains. Lanes: 1 and 20, 1 kb molecular mass marker; lane 2,
V. cholerae strain N16961; lanes 3–18 V. mimicus isolates; lane 19, V. cholerae strain O395. (a) PCR amplification using the
primer pair 148F and 146B; (b) PCR amplification using the primer pair nanH3 and nanH4; (c) PCR amplification using the
primer pair VC3B and VC4B.
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315
W. S. Jermyn and E. F. Boyd
Fig. 3. Southern hybridization analysis of
EcoRI-digested chromosomal DNA to verify
the presence of the VPI-2 in clinical and
environmental V. mimicus isolates. Lanes:
1, N16961; 2, PT5; 3, 9583; 4, 523-80;
5, 531-90; 6, CS16; 7, CS18; 8, CS61;
9, ES28; 10, ES26; 11, ES24; 12, ES13;
13, ES6. Lane 1 contained the V. cholerae
strain N16961, whereas lanes 2–13 contained V. mimicus strains. (a) Hybridization
analysis with DNA probe 148F encompassing ORFs VC1773–VC1776; (b) hybridization analysis with DNA probe VC3A
encompassing ORFs VC1804–VC1807. No
hybridization band was detected in five V.
mimicus strains (lanes 6, 9–11 and 13),
indicating that the ORFs VC1804–VC1807
were absent in these strains.
summary, examination of the results of the PCR and
Southern hybridization analysis indicate that the VPI-2
in 11 V. mimicus isolates was 19?4 kb in size and consisted
of 22 ORFs (VC1758, VC1773–VC1787 and VC1804–
VC1809), whereas in six V. mimicus isolates the VPI-2
region consisted of a 14?1 kb region encompassing 15 ORFs
VC1773–VC1787.
We used PCR analysis and DNA sequencing to establish
whether the region VC1773–VC1787 was contiguous with
VC1804–VC1809. Using the PCR primer pair NF2 (located
within the ORF VC1784) and 1806R (located within the
ORF VC1806), we obtained a PCR product of 2?9 kb
from the 11 isolates containing the 19?4 kb VPI-2 region.
Nucleotide sequencing of the 2?9 kb PCR product from
V. mimicus strain PT5 showed the presence of the ORFs
VC1785, VC1786, VC1787, VC1804 and VC1805. Analysis
of the region VC1785–VC1805 in V. cholerae strain N16961
revealed a 7 bp sequence AACTATT in the 39 end of the
ORF VC1787 and an identical 7 bp sequence in the 59 end of
the ORF VC1804. However, examination of the sequence
in V. mimicus strain PT5 revealed that the last 5 bp of
VC1787 and the first 12 bp of VC1804 were deleted, so only
one copy of the 7 bp sequence AACTATT remained intact.
Genomic mapping of the VPI-2 in V. mimicus
To determine whether the 19?4 kb VPI-2 in V. mimicus was
inserted in the same insertion site as that in V. cholerae, we
carried out PCR analysis using primers designed from the
insertion site of the VPI-2 in V. cholerae. Using the primer
pair tRNAF (located within the serine tRNA) and int4
(located within ORF VC1758), a PCR product of 1?3 kb
was identified in 11 isolates containing the 19?4 kb VPI-2
region. Nucleotide sequencing of the 1?3 kb product from
V. mimicus strain PT5 showed that VPI-2 was located
adjacent to a serine tRNA. Sequencing revealed that there
Table 3. Sequence variation in the 611 bp nanH gene fragment of VPI-2 and in the 648 bp
fragment of the chromosomally encoded mdh gene derived from V. cholerae and V. mimicus
strains
Abbreviations: Total poly., total polymorphic sites; Syn., synonymous sites; Non-syn., non-synonymous
sites; kS, rate per site of synonymous substitution; kN, rate per site of non-synonymous substitution.
Gene and
species
nanH
V. cholerae
V. mimicus
All
mdh
V. cholerae
V. mimicus
All
316
Number
of strains
Number of sites
kS
kN
kN/kS
Total poly.
Syn.
Non-syn.
22
3
25
49
0
52
41
0
43
8
0
9
0?086
0
0?092
0?004
0
0?004
0?047
0
0?043
22
3
21
24
0
82
23
0
79
1
0
3
0?035
0
0?159
0?001
0
0?003
0?029
0
0?019
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Evolution of Vibrio pathogenicity island-2
was an intergenic region of 313 bp between the serine tRNA
and VC1758, the first ORF of VPI-2 in V. mimicus.
PFGE was used to determine if VC1758 was located in
close proximity to the ORFs VC1773–VC1787, since PCR
analysis failed to amplify any product using a primer pair
designed between these regions. Using the probes nint3/
nint4 (encompassing VC1758) and 148F/146B (encompassing VC1773–VC1776), an identical hybridization fragment
of approximately 200 kb was obtained from V. mimicus
strain PT5. Similarly for V. mimicus strain 523-80, an
identical hybridization fragment of approximately 120 kb
was obtained from each probe. This indicated that the
ORFs VC1758 and VC1773–VC1776 were located in
proximity to each other, on the same hybridization
fragment, on the V. mimicus chromosome.
Genetic variation at the nanH locus
To determine the evolutionary history of the VPI-2 region
among V. cholerae and V. mimicus, we sequenced a 611 bp
(nucleotides 177–788) fragment of the nanH gene from 22
V. cholerae strains and three V. mimicus isolates. Sequencing revealed variation at 49 nucleotide sites among the 22
V. cholerae nanH sequences examined, 41 of which were
synonymous substitutions (silent sites) and eight of which
were non-synonymous substitutions (amino acid replacement sites) (Table 3, Fig. 4). No nucleotide differences were
Fig. 4. (a) Distribution of the polymorphic
bases within the nanH gene of V. cholerae
and V. mimicus. The nanH sequence of V.
cholerae strain N16961 was used as the
consensus sequence and only polymorphic
sites in the nanH gene are shown.
Nucleotides identical to those of N16961
are represented by dots. The position of
each nucleotide in the gene is indicated by
the value at the top of the alignment. The 1,
2 or 3 at the bottom of the alignment
indicates the position of each base within a
codon. The asterisks indicate non-synonymous
substitutions resulting in amino acid changes.
Alleles numbered 1 to 8 indicate grouping
of identical sequences. (b) Distribution of
the polymorphic amino acid residues within
the NanH protein sequence of V. cholerae
and V. mimicus.
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W. S. Jermyn and E. F. Boyd
found between the epidemic V. cholerae O1 and O139
isolates examined. Of the 49 polymorphic sites identified,
there were 14 singleton sites found among V. cholerae nonO1/non-O139 serogroup isolates. For example, V. cholerae
O141 serogroup strain V46 contained seven singleton sites,
O54 serogroup strain SG14 contained three singleton sites
and O37 serogroup strain 151 contained two singleton
sites. The maximum nanH sequence difference between V.
cholerae strains was 5?23 % between strains 151 and V46.
All three V. mimicus strains exhibited identical nanH
nucleotide sequence to one another and contained three
unique polymorphic sites compared to the V. cholerae
nanH sequence (nucleotides 314, 549 and 609) (Table 3,
Fig. 4). The mean pairwise percentage difference among
the V. cholerae and V. mimicus nanH sequences was 2?00 %.
Of the 203 NanH amino acid sites examined, nine polymorphic amino acid sites were found among the V. cholerae
and V. mimicus strains (Fig. 4). Eight amino acid polymorphisms were found among V. cholerae isolates and three
among V. mimicus isolates, with one amino acid polymorphic site unique to V. mimicus. Six of the nine amino
acid replacement polymorphisms were conservative and
involved substitution of amino acids with similar properties. The remaining three amino acid polymorphisms
all involved substitution of a hydrophobic residue with a
hydrophilic one.
Sequence analysis of the 22 V. cholerae mdh sequences
revealed variation at 24 nucleotide sites, of which 23 were
synonymous substitutions and one was a non-synonymous
substitution (Table 3). All three V. mimicus strains exhibited
identical mdh sequences to one another, and contained 58
unique polymorphic sites compared to V. cholerae mdh
sequences, two of which resulted in amino acid changes
(Table 3). The mean pairwise percentage difference among
the V. cholerae and V. mimicus mdh sequences was 3?73 %
and the maximum difference was 12?03 %.
The variability observed at the nanH locus was greater than
that observed at the mdh locus for the same set of isolates.
To determine whether the selective constraints at these
loci are different, that is, whether nanH is under positive
selection or not, we examined the synonymous (kS) and
non-synonymous (kN) substitution rates and calculated
the kN/kS ratio for each gene (Table 3). Amino acid
replacements in a protein that incur an advantage to the
cell are fixed at a higher rate than silent site substitutions
resulting in a kN/kS ratio greater than 1. Amino acid
replacements in a protein that are deleterious to the cell
are fixed at a lower rate by purifying selection, with a kN/kS
ratio less than 1. Substitutions in a protein that are neither
selectively advantageous nor deleterious are neutral, with a
kN/kS ratio equal to 1. The nanH gene kN/kS ratio was not
high, indicating that this gene is not under positive selection
(Table 3).
Evolutionary genetic relationships of VPI-2
among V. cholerae and V. mimicus
To reconstruct the evolutionary history of the VPI-2 region
from V. cholerae and V. mimicus, a phylogenetic tree based
on the nanH gene was constructed. The nanH sequences
from 25 isolates were analysed using the Jukes–Cantor
distance method and a neighbour-joining gene tree was
constructed based on synonymous polymorphic sites only
(Fig. 5). From Fig. 5 it can be seen that all V. cholerae O1
and O139 serogroup strains clustered together on the
nanH gene tree. This cluster also included two V. cholerae
non-O1/non-O139 serogroup strains: V52, a toxigenic O37
serogroup strain; and V45, a rough isolate, which gave an
Fig. 5. Evolutionary relationships based on
synonymous site variation in the nanH and
mdh genes among V. cholerae and V. mimicus isolates. Phylogenetic trees were constructed by the neighbour-joining method
based on the Jukes–Cantor distance method
(Jukes & Cantor, 1969; Saitou & Nei, 1987).
Bootstrap values based on 1000 computergenerated trees are indicated at the branch
node.
318
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Evolution of Vibrio pathogenicity island-2
identical nanH sequence to the O1 and O139 serogroup
isolates. The other V. cholerae non-O1/non-O139 serogroup strains were grouped into two divergent clusters on
the nanH gene tree. The first cluster contained V. cholerae
strains V46, V47, V51 and SG14. The second cluster
contained the strains V54, 9581, 9582, 151 and E714 in
addition to V. mimicus strains PT5, PT48 and 9583.
A comparison of the nanH gene tree with the species
tree, based on the chromosomal housekeeping gene mdh,
revealed a similar clustering of strains in each tree, with the
exception of the V. mimicus isolates (Fig. 5). The V. cholerae
O1 and O139 serogroup isolates formed a cluster, which
included strains V45, V52 and V54 on the mdh gene tree.
Similarly, V. cholerae non-O1/non-O139 strains V46, V47,
V52 and SG14 clustered together, as did strains 151, 9581,
9582 and E714, on separate branches of the mdh tree.
However, a notable feature of the gene trees is the placement of V. mimicus isolates: unlike the nanH gene tree,
where V. mimicus isolates clustered with V. cholerae isolates,
on the mdh gene tree V. mimicus isolates formed a separate
distinct divergent lineage. The clustering of the nanH
sequence from V. mimicus with V. cholerae indicates that
horizontal transfer of nanH between V. cholerae and V.
mimicus has occurred. Interestingly, the branch lengths of
the mdh tree among V. cholerae isolates are short, which
suggests that V. cholerae isolates are closely related and
diverged from one another only very recently. The branch
lengths on the nanH tree are longer than the mdh gene tree,
which suggests that this region is evolving faster (Fig. 5).
To further determine the evolutionary history of the entire
VPI-2 region among V. cholerae and V. mimicus isolates,
we sequenced gene fragments from five additional ORFs,
VC1764, VC1781, VC1783, VC1799 and VC1806, which
spanned VPI-2. Nucleotide analysis of the five ORFs, for
six V. cholerae strains, N16961, O395, MO2, V46, V47 and
V52, provided similar findings to the nanH (VC1784)
gene sequence analysis (Table 4). Strains N16961, O395,
MO2 and V52 exhibited identical nucleotide sequences
for the five ORFs VC1764, VC1781, VC1783, VC1799 and
VC1806. Four ORFs, VC1781, VC1783, VC1799 and VC1806,
of strains V46 and V47 exhibited different nucleotide
sequences when compared to the consensus sequence in
strain N16961. In contrast, the nucleotide sequence of the
ORF VC1764 was identical between V46, V47 and N16961.
Comparison of the nucleotide sequence of three ORFs,
VC1781, VC1783 and VC1806, between V. mimicus strain
PT5 and V. cholerae strain N16961 revealed similar findings
to the nanH (VC1784) nucleotide comparison.
DISCUSSION
In this study, we identified regions homologous to VPI-2
from V. cholerae in V. mimicus isolates. Of the 17 V. mimicus
clinical and environmental isolates examined, all isolates
contained a 14?1 kb region of VPI-2, which encompassed
ORFs VC1773–VC1787, encoding homologues of neuraminidase and genes involved in sialic acid metabolism
(Fig. 1). This region in V. mimicus was inserted adjacent
to a serine tRNA similar to the VPI-2 region in V. cholerae.
In addition, 11 of the 17 V. mimicus isolates examined also
contained VPI-2 ORFs VC1758 (encoding an integrase)
and VC1804–VC1809. Further genomic mapping of these
regions in V. mimicus indicated that ORFs VC1773–VC1787
were located upstream of VC1804–VC1809; however, PCR
analysis failed to amplify any region between VC1758 and
VC1773–1787 or VC1804–1809. Nonetheless, by PFGE
analysis VC1758 was found located on the same genomic
fragment as VC1773–VC1787 and VC1804–VC1809, indicating that additional DNA may be present between
VC1758 and VC1773 which was outside the range of PCR
amplification. Previously, we showed that, among V.
cholerae O139 serogroup isolates, VPI-2 was truncated,
with only a 20 kb region (VC1758–1760 and VC1789–
VC1809) present, which included a number of Mu phagelike genes (Jermyn & Boyd, 2002). These data and the data
presented in this study suggest that the VPI-2 is highly
unstable. The size differences and instability of VPI-2 may
be associated with the presence of a Mu-like phage, since
Table 4. Number of variable sites in selected genes of VPI-2 between the consensus sequence of V. cholerae O1 strain
N16961 and the sequences of V. cholerae and V. mimicus strains
Gene
VPI-2
VC1764
VC1781
VC1783
VC1784
VC1799
VC1806
Chromosomal
VC0432
Function
Fragment
size
V. cholerae
O395
MO2
V52
V46
V47
V. mimicus
PT5
Methyl accepting subunit
N-acetylmannosamine-6-phosphate 2-epimerase
N-acetylglucosamine-6-phosphate deacetylase
Neuraminidase
Integrase
Replicase
818
559
481
611
643
657
bp
bp
bp
bp
bp
bp
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
19
22
7
17
0
7
12
18
5
17
9
13
19
7
Malate dehydrogenase
648 bp
1
0
0
8
13
71
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W. S. Jermyn and E. F. Boyd
Mu phage insertion and replication occurs by transposition
and thus can induce chromosomal rearrangements such
as deletion and insertion events.
The deletion of the type 1 restriction modification gene
cluster and the nan–nag gene cluster from the V. cholerae
O139 serogroup isolates may be the reason why this
serogroup is no longer a dominant cause of epidemic
cholera on the Indian subcontinent. The retention of VPI-2
in toxigenic V. cholerae O1 serogroup isolates suggests that
this region may provide a competitive advantage in vivo,
whereas the presence of the nan–nag region in all V. mimicus
isolates examined suggests that it may be an essential region
for survival in the environment. Interestingly, the nan–nag
gene cluster is also present in the genomes of Vibrio
vulnificus strains CMCP6 and YJ016 as well as Vibrio fischeri
strain ES114, but is absent from the genome of Vibrio
parahaemolyticus strain RIMD2210633 (W. S. Jermyn,
unpublished data). Among Vibrio species sequenced to
date, only V. cholerae and V. mimicus have been shown
to encode neuraminidase (nanH), which cleaves sialic
acid from sialoglycoconjugates (Corfield, 1990). V. vulnificus, V. parahaemolyticus and V. fischeri, however, encode
N-acetylneuraminic acid synthase, which can synthesize
sialic acid de novo, whereas V. cholerae and V. mimicus must
acquire sialic acid from an external source.
There were 49 polymorphic substitutions among the nanH
sequences examined in V. cholerae, which was approximately twice as many polymorphic sites compared to mdh
in the same set of strains (Table 3). This could indicate
that nanH is under some selective pressure for change or
that there is relax pressure at this locus. Analysis of the
nanH sequence demonstrated that most of the polymorphic
sites were silent site substitutions that do not result in
amino acid changes, and the kN/kS ratio at this locus was
much lower than 1, indicating that mutations in this gene
are probably deleterious to the host (Table 3). In fact, there
was a limited number of replacement polymorphisms at
this locus, indicating functional constraints on this protein.
Analysis of the sequenced nanH fragment revealed that
a number of key features and motifs, which are essential
for enzyme function, were conserved. For example, in
the active site of NanH, two arginines that stabilize the
carboxylic acid group of sialic acid were conserved. Also
the FRIP region, which is involved in substrate affinity,
was conserved. This indicated that the enzyme function was
maintained between Vibrio isolates.
In order to elucidate the evolutionary history of VPI-2
among V. cholerae and V. mimicus, comparative sequence
analysis of the nanH gene tree with the species tree, deduced
from the housekeeping gene mdh, was carried out. In a
number of studies, the mdh locus has been shown to be
an excellent indicator of the overall genetic diversity of
a species, since it is not under selective pressure and is
present in all isolates of a species (Boyd et al., 1994, 1996;
Byun et al., 1999; O’Shea et al., 2004a; Reen & Boyd,
2004). Clustering of V. cholerae isolates on the nanH gene
320
tree was similar to the clustering of isolates on the mdh
gene tree; however, the congruence between the trees did
not extend to nanH from V. mimicus. On the nanH
gene tree, V. mimicus did not form a separate divergent
branch from V. cholerae as it does on the mdh tree,
but instead clustered with V. cholerae non-O1/non-O139
isolates (Fig. 5). We examined five additional loci on the
VPI-2 among six V. cholerae isolates and one V. mimicus
isolate (Table 4). These five loci, which span the nan–nag
gene cluster and regions in the 59 and 39 of VPI-2, overall
gave a similar pattern of divergence and clustering as
nanH. This indicates that nanH, and by extension the
nan–nag genes, were horizontally transferred between V.
mimicus and V. cholerae. We favour an evolutionary
scenario in which VPI-2 was present in the most recent
common ancestor of V. mimicus and horizontally transferred to V. cholerae soon after these species diverged.
Horizontal transfer between V. cholerae and V. mimicus has
previously been documented (Boyd et al., 2000b; Faruque
et al., 1999). The presence of both CTXw and VPI in V.
mimicus natural isolates has been demonstrated (Boyd
et al., 2000b). Nucleotide sequence identity of genes from
both CTXw and VPI derived from V. mimicus and V.
cholerae has been reported, which strongly suggests that
contemporary horizontal transfer of DNA between these
species has occurred (Boyd et al., 2000b). Both V. cholerae
and V. mimicus occupy similar environmental niches and
it is possible that V. mimicus acts as an environmental
reservoir of novel DNA for V. cholerae. Horizontal gene
transfer between Vibrio species appears to be an emerging
theme in the evolution of this genus, since transfer of a
8?5 kb region encoded on the Vibrio seventh pandemic
island-II (VSP-II) between V. cholerae and V. vulnificus has
also recently been described (O’Shea et al., 2004b).
ACKNOWLEDGEMENTS
Research in E. F. B.’s laboratory is funded by a PRTLI-3 grant from the
Higher Education Authority Ireland and an Enterprise Ireland basic
research grant.
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