Previous Article | Next Article 
Journal of Bacteriology, September 1998, p. 4516-4522, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Vibrio cholerae O139 Bengal: Combined Physical and
Genetic Map and Comparative Analysis with the Genome of V. cholerae O1
Gopal
Khetawat,
Rupak K.
Bhadra,
Sujata
Kar,
and
Jyotirmoy
Das*
Biophysics Division, Indian Institute of
Chemical Biology, Calcutta 700 032, India
Received 26 January 1998/Accepted 25 June 1998
 |
ABSTRACT |
A combined physical and genetic map of the genome of strain SG24 of
Vibrio cholerae O139 Bengal, a novel non-O1 strain having epidemic potential, has been constructed by using the enzymes NotI, SfiI, and CeuI. The genome of
SG24 is circular, and the genome size is about 3.57 Mb. The linkages
between 47 NotI and 32 SfiI fragments of
V. cholerae SG24 genomic DNA were determined by combining
two approaches: (i) identification of fragments produced by enzyme I in
fragments produced by enzyme II by the method of fragment excision,
redigestion, and end labeling and (ii) use of the linking clone
libraries generated from the genome of classical O1 strain 569B. The
linkages between nine CeuI fragments were determined
primarily by analyses of partial fragments of the
CeuI-digested genome. More than 80 cloned homologous and
heterologous genes, including several operons, have been positioned on
the physical map. The map of the SG24 genome represents the second map
of a V. cholerae genome, and a comparison of this map with
that of classical O1 strain 569B revealed considerable
diversity in DNA restriction sites and allowed identification of
hypervariable regions. Several genetic markers, including virulence
determinant genes, are in different positions in the SG24 and 569B
genomes.
| |
INTRODUCTION |
Vibrio cholerae, a
noninvasive, gram-negative bacterium, is the causative agent of the
diarrheal disease cholera. The specificity of the somatic O antigen of
V. cholerae resides in the polysaccharide moiety of the
lipopolysaccharide present in the outer membrane, which forms the basis
of the serological classification of this organism (42). The
V. cholerae strains causing epidemic cholera have, until
recently, been confined to serogroup O1, which consists of two
biotypes, classical and El Tor. The classical biotype was responsible
for cholera epidemics till 1961, when the El Tor biotype displaced it.
V. cholerae strains other than O1, which are collectively called non-O1 vibrios, can cause only sporadic infections and are
believed to lack the potential to cause epidemics (30). One
of the two events, the more alarming one, has dominated the global
cholera scenario in the present decade; this was the unprecedented emergence in late 1992 in India of a novel strain of V. cholerae which does not agglutinate with O1 polyvalent antiserum
but has epidemic and endemic potential, a phenomenon that has never
occurred in the recorded history of cholera (1, 13, 36).
Strains isolated from different parts of India and Bangladesh during
the epidemic were found to be of clonal origin (5, 6) and
were classified as new serovar O139, synonym Bengal. The other event was the dramatic and unexpected reappearance of epidemic cholera caused
by V. cholerae O1 El Tor in South America in January 1991, after a 100-year absence on that continent (21). These two
events have necessitated a renewed look into all aspects of the
organism that are related to pathogenesis. The epidemic caused by
V. cholerae O139 persisted for about a year (31,
32) and was again displaced by El Tor. Several lines of evidence
have, however, suggested that O139 originated from the El Tor biotype
(4, 6, 10, 13, 43) by the acquisition of a 35-kb DNA segment
which replaced most of the O1 antigen-encoding rfb gene
cluster of the recipient strain (8, 14). Thus, serogroup
O139 combines the virulent properties of epidemic strains with the
outer appearance of nonepidemic strains.
By using restriction enzymes which have a single site in either the
core region or the direct repeat sequence (RS) of the CTX genetic
element (27), it was shown that the genomes of most of the
O139 strains have two copies of the CTX genetic element in tandem
connected by two RSs (6). The chromosomal location of the
CTX genetic element in an O139 strain is the same as that reported for
El Tor vibrios. The organization of the virulence gene cassettes in
different O139 strains showed genetic heterogeneity in the population.
While most of the epidemic O139 strains have two copies of the CTX
genetic element, in some strains the number of elements has been
amplified and in at least one strain a copy of the element has been
deleted (6).
The genomes of El Tor strains isolated immediately before and after an
O139 outbreak showed extensive restriction fragment length polymorphism
(RFLP) among themselves and with the genome of O139 (33,
46). In late 1996, the appearance of a V. cholerae O139 strain having altered antibiotic sensitivity compared to that of
the O139 previously seen (29) has complicated the
epidemiological scenario of V. cholerae and has necessitated
an examination of possible rearrangements in the genome underlying such
rapid changes in phenotypic traits, which are unexpected in
well-characterized clonal strains within such a short period. In view
of the fact that the genetic basis of V. cholerae tropism
and pathogenesis is still mostly unknown, comparative genome mapping
studies to appraise the extent of genome diversity will be of interest,
particularly since the emergence of new variants of this organism
having epidemic potential with altered genotypes or phenotypes is
turning out to be widespread rather than exceptional (20).
The physical map of a classical O1 strain has been constructed
(12, 25), and there was previously no second map for
comparison of the genomes of V. cholerae strains in more
detail. It is in this context that the present report describes the
construction of a macrorestriction map of the genome of O139 by use of
the enzymes NotI, SfiI, and CeuI.
About 80 homologous and heterologous genes and operons have been
positioned on the physical map. A comparison of the V. cholerae O139 genome with that of classical O1 revealed several
gross differences.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
V.
cholerae classical O1 strain 569B, El Tor O1 strain CO457, and
O139 Bengal strain SG24, which were used in this study, were obtained
from the National Institute of Cholera and Enteric Diseases, Calcutta,
India. V. cholerae cells were grown in a gyratory shaker at
37°C in nutrient broth containing 0.1 M NaCl (pH 8.0) and maintained
as described previously (38). Escherichia coli cells were grown in a gyratory shaker at 37°C in Luria-Bertani broth
with appropriate antibiotics whenever required.
Preparation of high-molecular-weight genomic DNA and enzyme
digestion.
Agarose plugs of cells in the logarithmic phase of
growth were prepared and digested with restriction enzyme
NotI, SfiI, or CeuI as described
previously (25, 39). For CeuI digestion, five or
six sliced agarose pieces were incubated in 2× buffer supplied by the
manufacturer. After 15 min, the buffer was replaced with 100 µl of
fresh 1× buffer containing 10 µg of bovine serum albumin and 4 U of
the enzyme, and incubation was continued at 37°C for 3 h. For
partial digestion with CeuI, six agarose pieces were
digested with 1 U of the enzyme in 100 µl of the reaction mixture for
2 h at 37°C.
PFGE and hybridization experiments.
Pulsed-field gel
electrophoresis (PFGE) of enzyme-digested DNA was carried
out in a Pulsaphor Plus System with a hexagonal electrode array
(Pharmacia, Uppsala, Sweden) as described previously (25, 33,
39). Phage 
multimeric DNA and yeast chromosomal DNA were used
as molecular mass markers. Preparation of 32P-labeled DNA
probes and Southern blot hybridization conditions were as described
previously (25, 39). End labeling of DNA fragments following
NotI, SfiI, or CeuI digestion was done
by incubating the agarose blocks in a buffer containing Klenow enzyme and [
-32P]dCTP and subjecting them to PFGE
followed by autoradiography.
Isolation, redigestion, and radiolabeling of DNA fragments.
After PFGE, restriction fragments of genomic DNA digests were excised
from low-melting-point agarose gels under long-wavelength UV light, and
the agarose slice containing the DNA fragment was digested with a
second enzyme and labeled as described previously (25).
| |
RESULTS AND DISCUSSION |
Restriction fragment analysis and genome size.
Previous
studies on the V. cholerae genome (6, 25, 39)
showed that the enzymes NotI and SfiI digest the
genomic DNA into a small number of large fragments that can be resolved
by PFGE. To construct the physical map of the genome of V. cholerae O139, intact genomic DNA of strain SG24 in agarose blocks
was digested with NotI, SfiI, and CeuI
and the resulting fragments were separated by PFGE. The
endonuclease CeuI, which cleaves the genomic
DNAs of all of the organisms examined so far at the 23S rRNA of the
rrn operon and nowhere else in the genome (22,
26), has nine sites in the genome of V. cholerae O1
and O139 (33).
Ethidium bromide-stained gels of PFGE-separated genomic DNA
fragments of strain SG24 showed that NotI, SfiI,
and CeuI digestion yielded 35, 28, and 8 fragments,
respectively (Fig. 1). For comparison, the NotI, SfiI, and CeuI digestion
profiles of the genomes of El Tor O1 strain CO457, which displaced
O139, and classical O1 strain 569B were also examined. To identify the
smaller fragments that are not resolved in stained gels, the
restriction fragments were end labeled with [-32P]dCTP
in agarose slices before PFGE and autoradiograms were subsequently examined. In addition to those identified in stained gels, 11 NotI fragments, 4 SfiI fragments, and 1 CeuI fragment were detected in the autoradiograms (Fig.
2). The NotI and
SfiI digestion profiles of the genomes of SG24, CO457, and
569B exhibited distinct RFLP. The polymorphism is more pronounced
between SG24 and 569B. The CeuI digestion profiles of SG24
and El Tor strain CO457 were identical (Fig. 1), which was expected
since O139 originated from El Tor. Parenthetically, the CeuI
digestion profiles of El Tor strain VC44, a strain isolated immediately
before the O139 epidemic, and SG24 exhibited RFLP (33).
View larger version (101K):
[in this window]
[in a new window]
|
FIG. 1.
PFGE separation of NotI-, SfiI-,
and CeuI-digested genomic DNAs of V. cholerae CO457 (lanes a), SG24 (lanes b), and 569B (lanes c). The
enzyme-digested DNA was separated on 1% FastLane Agarose (FMC) with
pulse time ramping between 5 and 25 s for 22 h at 10 V/cm and
3°C for NotI and SfiI. For the separation of
CeuI fragments, electrophoresis was carried out by using
pulse times of 5 (4 h), 10 (4 h), 25 (4 h), and 100 (12 h) s at 10 V/cm
and 3°C. The gels were stained with ethidium bromide. Marker
molecular sizes, in kilobases, are shown in the margins.
|
|
View larger version (60K):
[in this window]
[in a new window]
|
FIG. 2.
Autoradiogram of PFGE-separated NotI (lane
a), SfiI (lane b), and CeuI (lane c) fragments of
genomic DNA of V. cholerae O139 strain SG24.
DNA in an agarose block was digested with the enzyme, end labeled with
[-32P]dCTP, and electrophoresed as described in the
legend to Fig. 1. The gels were dried and exposed to X-ray film.
Restriction fragments were named on the basis of the enzymes that
generated the fragments (N, NotI; S, SfiI; C,
CeuI) and were numbered on the basis of size in descending
order.
|
|
Restriction fragments were named on the basis of the enzymes that
generated the fragments (N,
NotI; S,
SfiI; C,
CeuI) and
were numbered on the basis of size in descending
order (Fig.
2).
NotI fragments N2 and N3, fragments N6 and
N7, fragments N8, N9,
and N10, fragments N13 and N14, fragments N17 and
N18, fragments
N21 and N22, fragments N25 and N26 and
SfiI
fragments S2 and S3
and fragments S18 and S19 are the same in size. The
genome size
of strain SG24 was estimated to be about 3.57 Mb (Table
1), as
opposed to the 2.2 Mb reported
previously (
6). Better resolution
of DNA fragments following
enzyme digestion and refinement of
PFGE methods by altering various
parameters allowed us to resolve
several previously reported
NotI fragments as doublets and triplets
(Table
1). Thus, the
genome size of O139 is close to that of
classical O1 strains
(
25).
CeuI cleavage map of the V. cholerae O139
genome.
CeuI digestion of the V. cholerae
SG24 genome produced nine fragments ranging from 1,400 to 6 kb (Fig. 2;
Table 1). It has been reported that, as in other organisms,
CeuI has a site only in the rrn operon of
V. cholerae (33). Thus, there are nine rrn operons in the V. cholerae O139 genome. The
CeuI map of the SG24 genome was constructed primarily from
the analyses of 10 partial fragments of the CeuI-digested
genome (Table 2). The linkage between the
CeuI fragments of the genome of V. cholerae SG24
is thus C2-C1-C3-C8-C5-C7-C6-C9-C4-C2, and the genome is circular (see
Fig. 5). The CeuI linkage map of the genome of O139 is
different from that of classical O1 strain 569B (see Fig. 6).
NotI and SfiI cleavage maps of the V. cholerae O139 genome.
The linkages between 47 NotI and 32 SfiI fragments of V. cholerae SG24 genomic DNA were determined by combining
three approaches. These included (i) identification of fragments
produced by enzyme I in fragments produced by enzyme II by the method
of fragment excision, redigestion, and end labeling, (ii) use of the
linking clone libraries generated from the genome of classical O1
strain 569B (26), and (iii) analysis of partial digestion
products.
The
CeuI linkage map was used as the skeleton to determine
the linkages between the
NotI and
SfiI fragments
of the genome
of
V. cholerae SG24. To identify linkages
between different
NotI
or
SfiI fragments,
individual PFGE-separated
CeuI fragments were
excised from
the gel, digested with
NotI or
SfiI, end labeled,
and subjected to PFGE (Fig.
3A).
Alternatively, the gel-excised
CeuI fragments were used as
probes for Southern blot hybridization
of the
NotI- or
SfiI-digested genome of
V. cholerae SG24
(Fig.
3B). Both of these approaches allowed clubbing of
NotI
and
SfiI
fragments that are linked. For example, when
gel-excised
CeuI
fragment C3 was digested with the enzyme
NotI, end labeled, resolved
by PFGE, and autoradiographed,
nine
NotI fragments (N17, N45,
N21, N16, N32, N40, N39, and
two flanking fragments arising from
N4 and N19; Fig.
3A, lane a), and
seven
SfiI fragments (S6, S24,
S30, S22, S31, and two
flanking fragments, S4 and S18; Fig.
3A,
lane c) lit up, showing that
these fragments are linked. The
NotI
or
SfiI
fragments overlapping the two ends of C3 were determined
by hybridizing
fragment C3 with the PFGE-resolved
NotI- or
SfiI-digested
genomic DNA (Fig.
3B, part a). The
order in which these fragments
are present in the genome was determined
by using position-specific
probes, such as linking clones (Table
3),
NotI or
SfiI
fragments
that are included in the
CeuI fragment, and
cloned-gene probes
(Table
4). Similar
analyses were performed with gel-excised
CeuI,
NotI, and
SfiI fragments.
View larger version (48K):
[in this window]
[in a new window]
|
FIG. 3.
(A) Identification of NotI or SfiI
fragments of the V. cholerae SG24 genome in isolated
CeuI fragments. CeuI fragments C3 (lanes a and c)
and C6 (lanes b and d) were completely digested with NotI or
SfiI, end labeled, and separated by PFGE. Identification of
NotI fragments in isolated SfiI fragments.
SfiI fragments S6 (lane e) and S9 (lane f) were completely
digested with the enzyme NotI, end labeled, and separated by
PFGE. NotI (NM)- or SfiI (SM)-digested,
end-labeled SG24 genomic DNA was used as a marker for
identification of linked fragments. Asterisks denote the flanking DNA
fragments. (B) Assignment of the flanking NotI or
SfiI fragments (shown in panel A) of V. cholerae SG24 genomic DNA in isolated CeuI
fragments. Radiolabeled CeuI fragment C3 (a) or C6 (b) was
used as the probe for Southern hybridization with the NotI-
or SfiI-digested genome of V. cholerae SG24.
For identification of the flanking NotI fragments in
isolated SfiI fragments of the genomic DNA of strain
SG24, radiolabeled SfiI fragment S6 (c) or S9 (d) was used
as the probe for Southern hybridization with the NotI- or
SfiI-digested SG24 genome. Hybridizations were carried out
at 60°C. The filters were washed under stringent conditions as
described in Materials and Methods.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Linkages between NotI and SfiI
fragments of the genome of strain SG24 of V. cholerae
O139 determined by using linking clones or restriction fragments
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Homologous and heterologus genes positioned on the
physical map of the genome of strain SG24
of V. cholerae O139
|
|
The
NotI linking clone library generated from the genome of
classical O1 strain 569B (
25) has been extensively used to
determine
the linkages between
NotI fragments. Some
representative examples
of hybridization experiments using linking
clones as probes are
shown in Fig.
4. The linkages between the
NotI fragments of the
569B genome determined by using
linking clones NLH44 and NLB29
(
25) are different from those
of the
NotI-digested genome of
SG24 (data not shown).
Linking clone NLH19 linked
NotI fragments
N15 and N26 of the
569B genome (
25), but it hybridized with
only
NotI fragment N15 of the SG24 genome, indicating deletion
of
the linked fragment (Fig.
4). Besides
NotI linking clones,
several
SfiI and
CeuI fragments of the O139 genome having a single
NotI site served as
NotI linking clones, and
NotI and
CeuI fragments
having a single
SfiI site were used as
SfiI linking clones (Fig.
4; Table
3).
View larger version (70K):
[in this window]
[in a new window]
|
FIG. 4.
Determination of linkages between NotI
fragments of SG24 genomic DNA by using a NotI
linking clone library of the 569B genome and isolated SfiI
fragments of the SG24 genome. Nick-translated linking clones NLB2,
NLB51, NLB92, NLB130, NLH19, NLH35, and NLH44 and SfiI
fragment S20 were hybridized with PFGE-separated NotI
fragments of the SG24 genome. NotI-digested and end-labeled
genomic DNA was used as molecular size markers (lane M) for
identification of linked fragments. The NotI fragments that
are linked to each other are marked.
|
|
Mapping of similar-size fragments.
To identify and position
similar-size fragments on the map, a combination of the following
approaches was adopted: (i) analysis of PFGE-separated genomic
DNA digested with any two of the three enzymes NotI,
SfiI, and CeuI, (ii) hybridization of comigrating fragments generated by one enzyme with the genomic DNA digested with a second enzyme, and (iii) use of suitable linking clones or
cloned genes. For example, to position NotI fragments N2 and N3, N6 and N7, and N13 and N14, which appeared as doublets on the map,
genomic DNA was digested with NotI and
CeuI and double digestion products were separated by PFGE.
The digestion profile revealed that one of the two fragments N2 and N3,
N6 and N7, or N13 and N14 has a CeuI site(s) and the other
does not (data not shown). One of the fragments in the N2-N3 doublet is
located in fragment C2 (which will be referred to as N2), and the other
is located in fragment C6 (which will be referred to as N3). This was
confirmed by Southern blot hybridization of the
NotI-digested O139 genome by using fragment C6 (Fig. 3B,
part b) or C2 as the probe and by end-labeling experiments. Linking
clone NLH44 hybridized with fragment N8 and the doublet N28-N29 (data
not shown). When the same probe was used for the Southern hybridization
of NotI-SfiI-double-digested genomic DNA,
it hybridized with fragment N8 and another smaller fragment designated
X (generated from either N28 or N29), indicating the presence of an
SfiI site(s). The fragment in the N28-N29 doublet hybridizing with linking clone NLH44 will be referred to as N29. Linking clone NLB51 hybridized with N1 and N28 and with S3. Besides, dnaK also hybridized with N28 and S3. Linking clone NLH8
hybridized with C3 and S6 and links fragment N16 and either N21 or N22
(which will be referred to as N21). Linking clone NLB29 links fragments N5 and N22 (Table 3) and also hybridized with C2 and S7. While positioning comigrating NotI fragments on the map, it was
revealed that N8-N9-N10 is a triplet (Table 1). This was confirmed by digesting the gel-excised triplet with the enzyme AscI or by
hybridization using fragment C1, C2, or C4 as the probe (data not
shown). Doublets N2-N3 and N6-N7 were also digested with
AscI, which confirmed that they are doublets (data not
shown). The other comigrating fragments were similarly positioned on
the map. By combining all of the approaches, a multienzyme
macrorestriction map of the genome of V. cholerae O139
strain SG24 has been constructed (Fig.
5).
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 5.
Combined physical and genetic map of the genome of
V. cholerae O139 strain SG24 determined by using
enzymes NotI, SfiI, and CeuI. The
restriction fragments are numbered on the basis of size (Table 1). An
asterisk denotes tentative assignment. The genetic markers are
described in Table 4. The positioning of the genetic markers in a
particular fragment is arbitrary, and the positions of the markers in a
single fragment do not reflect their true order in the chromosome.
CeuI sites were taken as the positions of the rrn
operons.
|
|
Positioning of genetic markers on the physical map.
More than
80 cloned genes (Table 4) and nine rrn operons have been
positioned on the physical map of the V. cholerae O139 genome by hybridization using homologous and heterologous probes (Fig.
5). The gene probes used comprised O139 antigen-specific genes, the
hemolysin gene, DNA repair genes, heat shock protein genes, cholera
toxin genes, trimethoprim-sulfamethoxazole resistance genes, and some
other virulence determinant genes (Table 4). In the V. cholerae genome, the CeuI sites are located only in the
rrn operons (33) similar to those reported for
several other bacteria (22). The genes have been positioned
on the physical map on fragments they hybridized with and do not
represent the exact locations and orientations on the fragment. Genes
hybridizing with the same fragment have been positioned on the fragment
arbitrarily, and the positioning does not reflect the true order of the
genes in the chromosome.
Comparison of the physical maps of O139 and classical O1 strain
569B.
A comparison of the physical map of the V. cholerae O139 genome constructed in the present study with that of
classical O1 strain 569B (25) revealed conservation in
certain regions but gross rearrangements in the genome of O139 with
respect to the classical O1 genome (Fig.
6). Restriction site variability between the two genomes is another feature emerging from the comparison. A
fraction of sites is conserved between the two strains. The genome of
V. cholerae O139 has gained a number of NotI
sites. The variable regions in the genomes of both strains might be due to exchange of genetic elements. Extensive RFLP was recorded between the genomes of classical O1, El Tor, and O139 following digestion with
NotI, SfiI, or CeuI (Fig. 1) and is
more predominant between the O139 and classical O1 genomes. The genome
of SG24 has more NotI (total, 47) and SfiI
(total, 32) sites (Table 1) than does the genome of classical O1 strain
569B (25). Although the chromosomal locations of several
genes, like rpoH, the tcp gene cluster, etc., are
conserved (Fig. 6), they show RFLP because of loss or creation of
restriction sites.
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 6.
Comparison of the chromosome maps of V. cholerae O139 strain SG24 and V. cholerae
classical O1 strain 569B. For a better comparison, the circular maps
were linearized. A triangle on a line connecting the two genomes
indicates a conserved region, and regions in the chromosomes that have
undergone rearrangement are indicated by an X on a connecting line. C
and N represent CeuI and NotI maps,
respectively.
|
|
NotI linking clones of the genome of the classical O1 strain
allowed identification of regions of the O139 genome that might
have
undergone rearrangements. Several linking clones of the genome
of
classical O1 strain 569B hybridized with only one fragment
of the
NotI-digested O139 genome (Fig.
4), indicating that either
the
NotI site was lost or one of the linked
NotI
fragments underwent
deletion in the O139 genome. While
NotI
linking clone NLH44 linked
fragments N6 (175 kb) and N22 (42 kb) in the
NotI-digested 569B
genome, it showed linkages between
fragments N8 (136 kb) and N29
(38 kb) of the
NotI-digested
SG24 genome. This clone hybridized
with the
SfiI-digested
genomes of strains 569B and O139 in fragments
S3 (280 kb) and S1 (380 kb), respectively (data not shown). Linking
clone NLB29 hybridized with
one similar-size
NotI fragment of
both the 569B and SG24
genomes, and the other fragment showed
RFLP.
The copy number of direct RS IS
1004 is reduced to four in
the genome of O139 from eight in that of classical O1 (
9).
Most
of the genetic markers examined exhibited RFLP in the
NotI- or
SfiI-digested genomic DNAs of
O139 and classical O1. In classical
O1 strains, the CTX genetic element
is present in two copies that
are separated by about 1.5 Mb in the
genome (
12). The genome
of
V. cholerae O139,
which originated from El Tor, has two copies
of the element in tandem,
as expected (
5,
6). One copy of
the CTX genetic element in
classical O1 strain 569B is closely
linked to several other virulence
determinant genes like the
tcp operon and
toxT
and is located in
NotI fragment N14 (
12). The
two
copies of the CTX genetic element present in tandem in the
O139 genome
are located about 1 Mb apart from the
tcp operon and
the
toxT gene (Fig.
5). In the genome of
V. cholerae O139, the
position of
toxR, the positive
transcriptional activator gene
regulating the expression of several
major virulence genes, including
cholera toxin gene
ctxAB,
has changed from that in strain 569B
and is no longer linked to the
groEL,
dam, and
secY genes (Fig.
5 and
6). Another variable region stretches the CTX genetic element
along
with mutator genes
mutU and
mutS. The locations
of the
mutK,
cspA, and RNA methyltransferase
genes in the genome of O139 are
different from those in the classical
O1 genome (
12). The presence
of such hypervariable regions
in the genome has been reported
in several bacterial pathogens
(
11,
15,
17,
34,
35,
37,
41). However, the
rfb
and
hlyA loci in the genomes of
SG24 and 569B are
conserved.
The number and location of
rrn operons in the genomes of
enteric bacteria appear to be highly conserved. In contrast to seven
rrn operons in the genomes of the different gram-negative
bacteria
examined so far (
22), the genomes of
V. cholerae O1 and O139
have nine (
33). While the number
of
rrn operons in strains 569B
and SG24 remained the same,
their positions in the chromosome
have altered, suggesting major
genomic rearrangements between
O1 and O139 (Fig.
6). A similar
observation has been reported
in
Salmonella typhi, an
enteric pathogen causing typhoid fever.
It was suggested that the
genetic events responsible for rearrangement
in the genome of
S. typhi are presumably due to recombination
between the
rrn genes and not at other sites (
23,
24). The
role of
rrn operons in imparting genome instability in
V. cholerae has yet to be investigated. However, in
contrast to
S. typhi,
where the lengths of the
CeuI fragments remain unaltered in almost
all of the strains
studied, the sizes of the
CeuI fragments of
the genomes of
O1 and O139 strains are different (Fig.
1). It
has been reported that
V. cholerae strains belonging to non-O1-non-O139
serogroups have 10
rrn operons in their genomes
(
33). Intraspecies
variation in
rrn operons has
not been reported in other organisms.
Genomic rearrangements at high frequency might play a positive role in
the developmental processes of bacteria. To survive
in the environment
while maintaining epidemic potential and to
elude all of the associated
onslaughts following infection, pathogenic
organisms might prefer
to store their virulence factors in relatively
plastic regions of the
chromosome, which would give them the kind
of flexibility necessary
to sustain their pathogenic potential.
| |
ACKNOWLEDGMENTS |
We thank M. K. Waldor, New England Medical Center, Boston,
Mass., for providing plasmid pSXT1 and K. Yamamoto, Osaka University, Osaka, Japan, for plasmid pKY017.
This work was supported by research grant BT/R&D/PRO-109/15/8/96 from
the Department of Biotechnology, Government of India. S.K. is the
recipient of a Research Associateship from the Council of Scientific
and Industrial Research, Government of India.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biophysics
Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Rd., Calcutta 700 032, India. Phone: 91-33-473 0350/5197/5368.
Fax: 91-33-473 0350/5197/0284. E-mail:
biophy{at}cal.vsnl.net.in.
Present address: Department of Medicine, Hematology Division, The
Johns Hopkins University Medical Institutes, Baltimore, MD 21205.
Present address: Department of Epidemiology and Public Health,
Yale University School of Medicine, New Haven, CT 06520-8034.
| |
REFERENCES |
| 1.
|
Albert, M. J.,
A. K. Siddique,
M. S. Islam,
A. S. J. Faruque,
M. Ansaruzzaman,
S. M. Faruque, and R. B. Sack.
1993.
Large outbreak of clinical cholera due to Vibrio cholerae non-O1 in Bangladesh.
Lancet
341:704[Medline].
|
| 2.
|
Bandyopadhyay, R., and J. Das.
1994.
DNA adenine methyl transferase encoding gene (dam) of Vibrio cholerae.
Gene
140:67-71[Medline].
|
| 3.
|
Bera, T. K.,
S. K. Ghosh, and J. Das.
1989.
Cloning and characterization of the mutL and mutS genes of Vibrio cholerae: nucleotide sequence of the mutL gene.
Nucleic Acids Res.
17:6241-6251[Abstract/Free Full Text].
|
| 4.
|
Berche, P.,
C. Poyart,
E. Abachin,
H. Lelivre,
J. Vandepitte,
A. Dodin, and J.-M. Fournier.
1994.
The novel epidemic strain O139 is closely related to the pandemic strain O1 of Vibrio cholerae.
J. Infect. Dis.
170:701-704[Medline].
|
| 5.
|
Bhadra, R. K.,
S. Roychowdhury, and J. Das.
1994.
Vibrio cholerae O139 El Tor biotype.
Lancet
343:728[Medline].
|
| 6.
|
Bhadra, R. K.,
S. Roychoudhury,
R. K. Banerjee,
S. Kar,
R. Majumdar,
S. Sengupta,
S. Chatterjee,
G. Khetawat, and J. Das.
1995.
Cholera toxin (CTX) genetic element in Vibrio cholerae O139.
Microbiology
141:1977-1983[Abstract/Free Full Text].
|
| 7.
|
Bhattacharyya, D., and J. Das.
1997.
V. cholerae analogue of E. coli secY gene: identification, cloning and characterization.
Gene
196:261-266[Medline].
|
| 8.
|
Bik, E. M.,
A. E. Bunschoten,
R. D. Gouw, and F. R. Mooi.
1995.
Genesis of the novel epidemic Vibrio cholerae O139 strain: evidence for horizontal transfer of genes involved in polysaccharide synthesis.
EMBO J.
14:209-216[Medline].
|
| 9.
|
Bik, E. M.,
R. D. Gouw, and F. R. Mooi.
1996.
DNA fingerprinting of Vibrio cholerae strains with a novel insertion sequence element: tool to identify epidemic strains.
J. Clin. Microbiol.
34:1453-1461[Abstract].
|
| 10.
|
Calia, K. E.,
M. Muratagh,
M. J. Ferraro, and S. B. Calderwood.
1994.
Comparison of Vibrio cholerae O139 with V. cholerae O1 classical and El Tor biotypes.
Infect. Immun.
62:1504-1506[Abstract/Free Full Text].
|
| 11.
|
Canard, R.,
B. Saint-Joanis, and S. T. Cole.
1992.
Genomic diversity and organization of virulence genes in the pathogenic anaerobe Clostridium perfringens.
Mol. Microbiol.
6:1421-1429[Medline].
|
| 12.
|
Chatterjee, S.,
A. K. Mondal,
N. A. Begum,
S. Roychoudhury, and J. Das.
1998.
Ordered cloned DNA map of the genome of Vibrio cholerae 569B and localization of genetic markers.
J. Bacteriol.
180:901-908[Abstract/Free Full Text].
|
| 13.
|
Cholera Working Group.
1993.
Large epidemic of cholera-like disease in Bangladesh caused by Vibrio cholerae O139 synonym Bengal.
Lancet
342:387-390[Medline].
|
| 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 polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1.
Mol. Microbiol.
19:815-826[Medline].
|
| 15.
|
Dempsey, J. A. F.,
A. B. Wallace, and J. G. Cannon.
1995.
The physical map of the chromosome of a serogroup A strain of Neisseria meningitidis shows complex rearrangements relative to the chromosomes of the two mapped strains of the closely related species N. gonorrhoeae.
J. Bacteriol.
177:6390-6400[Abstract/Free Full Text].
|
| 16.
|
Fayet, O.,
T. Ziegelhoffer, and C. Georgopoulos.
1989.
The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures.
J. Bacteriol.
171:1379-1385[Abstract/Free Full Text].
|
| 17.
|
Jiang, Q.,
K. Hiratsuka, and D. E. Taylor.
1996.
Variability of gene order in different Helicobacter pylori strains contributes to genomic diversity.
Mol. Microbiol.
20:833-842[Medline].
|
| 18.
|
Kaper, J. B.,
H. Lockman,
M. M. Baldini, and M. M. Levine.
1984.
A recombinant live oral cholera vaccine.
Bio/Technology
2:345-349.
|
| 19.
|
Keasler, S. P., and R. H. Hall.
1993.
Detecting and biotyping Vibrio cholerae O1 with multiplex polymerase chain reaction.
Lancet
341:1661[Medline].
|
| 20.
| Khetawat, G., R. K. Bhadra, S. Nandi, and J. Das. Resurgent Vibrio cholerae O139: rearrangement of
CTX genetic elements and amplification of rrn operons.
Submitted for publication.
|
| 21.
|
Levine, M. M.
1991.
South America: the nature of cholera.
Lancet
338:45-46.
|
| 22.
|
Liu, S.-L.,
A. Hessel, and K. E. Sanderson.
1993.
Genomic mapping with I-CeuI, an intron-encoded endonuclease specific for genes for ribosomal RNA, in Salmonella spp., Escherichia coli and other bacteria.
Proc. Natl. Acad. Sci. USA
90:6874-6878[Abstract/Free Full Text].
|
| 23.
|
Liu, S.-L., and K. E. Sanderson.
1995.
Rearrangements in the genome of the bacterium Salmonella typhi.
J. Bacteriol.
177:3355-3357[Abstract/Free Full Text].
|
| 24.
|
Liu, S.-L., and K. E. Sanderson.
1996.
Highly plastic chromosomal organization in Salmonella typhi.
Proc. Natl. Acad. Sci. USA
93:10303-10308[Abstract/Free Full Text].
|
| 25.
|
Majumder, R.,
S. Sengupta,
G. Khetawat,
R. K. Bhadra,
S. Roychoudhury, and J. Das.
1996.
Physical map of the genome of Vibrio cholerae 569B and localization of genetic markers.
J. Bacteriol.
178:1105-1112[Abstract/Free Full Text].
|
| 26.
|
Marshall, P., and C. Lemieux.
1991.
Cleavage pattern of the homing endonuclease encoded by the fifth intron in the chloroplast large subunit rRNA-encoding gene of Chlamydomonas eugametos.
Gene
104:241-245[Medline].
|
| 27.
|
Mekalanos, J. J.
1983.
Duplication and amplification of toxin genes in Vibrio cholerae.
Cell
35:253-263[Medline].
|
| 28.
|
Miller, V. L., and J. J. Mekalanos.
1984.
Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR.
Proc. Natl. Acad. Sci. USA
81:3471-3475[Abstract/Free Full Text].
|
| 29.
|
Mitra, R.,
A. Basu,
D. Dutta,
G. B. Nair, and Y. Takeda.
1996.
Resurgence of Vibrio cholerae O139 Bengal with altered antibiogram in Calcutta, India.
Lancet
348:1181[Medline].
|
| 30.
|
Morris, J. G.
1990.
Non-O group 1 Vibrio cholerae: a look at the epidemiology of an occasional pathogen.
Epidemiol. Rev.
12:179-191[Free Full Text].
|
| 31.
|
Mukhopadhyay, A. K.,
S. Garg,
G. B. Nair,
S. Kar,
R. K. Ghosh,
S. Pajni,
A. Ghosh,
T. Shimada,
T. Takeda, and Y. Takeda.
1995.
Biotype traits and antibiotic susceptibility of Vibrio cholerae serogroup O1 before, during and after the emergence of the O139 serogroup.
Epidemiol. Infect.
115:427-434[Medline].
|
| 32.
|
Mukhopadhyay, A. K.,
S. Garg,
R. Mitra,
A. Basu,
K. Rajendran,
D. Dutta,
S. K. Bhattacharya,
T. Shimada,
T. Takeda,
Y. Takeda, and G. B. Nair.
1996.
Temporal shifts in traits of Vibrio cholerae strains isolated from hospitalized patients in Calcutta: a 3-year (1993 to 1995) analysis.
J. Clin. Microbiol.
34:2537-2543[Abstract].
|
| 33.
|
Nandi, S.,
G. Khetawat,
S. Sengupta,
R. Majumder,
S. Kar,
R. K. Bhadra,
S. Roychoudhury, and J. Das.
1997.
Rearrangements in the genomes of Vibrio cholerae strains belonging to different serovars and biovars.
Int. J. Syst. Bacteriol.
47:858-862[Abstract/Free Full Text].
|
| 34.
|
Philipp, W. J.,
S. Nair,
G. Guglielmi,
M. Lagranderie,
B. Gicquel, and S. T. Cole.
1996.
Physical mapping of Mycobacterium bovis BCG Pasteur reveals differences from the genome map of Mycobacterium tuberculosis H37Rv and from M. bovis.
Microbiology
142:3135-3145[Abstract/Free Full Text].
|
| 35.
|
Pyle, L. E.,
T. Taylor, and L. R. Flinch.
1990.
Genomic maps of some strains within the Mycoplasma mycoides cluster.
J. Bacteriol.
172:7265-7268[Abstract/Free Full Text].
|
| 36.
|
Ramamurthy, T.,
S. Garg,
R. Sharma,
S. K. Bhattacharya,
G. B. Nair,
T. Shimada,
T. Takeda,
T. Karasawa,
H. Kurazono,
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[Medline].
|
| 37.
|
Romling, U.,
J. Greipel, and B. Tummler.
1995.
Gradient of genomic diversity in the Pseudomonas aeruginosa chromosome.
Mol. Microbiol.
17:323-332[Medline].
|
| 38.
|
Roy, N. K.,
G. Das,
T. S. Balganesh,
S. N. Dey,
R. K. Ghosh, and J. Das.
1982.
Enterotoxin production, DNA repair and alkaline phosphatase of Vibrio cholerae before and after animal passage.
J. Gen. Microbiol.
128:1927-1932[Abstract/Free Full Text].
|
| 39.
|
Roychoudhury, S.,
R. K. Bhadra, and J. Das.
1994.
Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes.
FEMS Microbiol. Lett.
115:329-334[Medline].
|
| 40.
|
Sahu, G. K.,
R. Chowdhury, and J. Das.
1997.
The rpoH gene encoding  32 homology of Vibrio cholerae.
Gene
189:203-207[Medline].
|
| 41.
|
Schmidt, K. D.,
B. Tummler, and U. Romling.
1996.
Comparative genome mapping of Pseudomonas aeruginosa PAO with P. aeruginosa C, which belongs to a major clone in cystic fibrosis patients and aquatic habitats.
J. Bacteriol.
178:85-93[Abstract/Free Full Text].
|
| 42.
|
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.
|
| 43.
|
Waldor, M. K., and J. J. Mekalanos.
1994.
ToxR regulates virulence gene expression in non-O1 strains of Vibrio cholerae that cause epidemic cholera.
Infect. Immun.
62:72-78[Abstract/Free Full Text].
|
| 44.
|
Waldor, M. K.,
H. Tschape, and J. J. Mekalanos.
1996.
A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139.
J. Bacteriol.
178:4157-4165[Abstract/Free Full Text].
|
| 45.
|
Yamamoto, K.,
Y. Ichinose,
H. Shinagawa,
K. Makino,
A. Nakata,
M. Iwanaga,
T. Honda, and T. Miwatani.
1990.
Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products.
Infect. Immun.
58:4106-4116[Abstract/Free Full Text].
|
| 46.
|
Yamasaki, S.,
G. B. Nair,
S. K. Bhattacharya,
S. Yamamoto,
H. Kurazono, and Y. Takeda.
1997.
Cryptic appearance of a new clone of Vibrio cholerae serogroup O1 biotype El Tor in Calcutta, India.
Microbiol. Immunol.
41:1-6[Medline].
|
Journal of Bacteriology, September 1998, p. 4516-4522, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Oswald, W., Konine, D. V., Rohde, J., Gerlach, G.-F.
(1999). First Chromosomal Restriction Map of Actinobacillus pleuropneumoniae and Localization of Putative Virulence-Associated Genes. J. Bacteriol.
181: 4161-4169
[Abstract]
[Full Text]
Top
Abstract
Introduction
