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Infection and Immunity, September 2000, p. 4850-4855, Vol. 68, No. 9
Institut für Hygiene und Mikrobiologie
der Universität Würzburg 97080 Würzburg, Germany
Received 18 January 2000/Returned for modification 28 February
2000/Accepted 8 June 2000
An infectious Shiga toxin (Stx) 2e-converting bacteriophage
( Shiga toxin (Stx)-producing
Escherichia coli (STEC) strains are a worldwide cause of
diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome (HUS)
(8). Stx identified in human STEC isolates comprise Stx1,
Stx2 and variants of Stx2, including Stx2c, Stx2d, and Stx2e (21,
31, 35). STEC strains associated with diarrhea and HUS usually
produce Stx1, Stx2, and Stx2c, either alone or in various combinations
(6, 24). In contrast, Stx2d was frequently identified in
STEC isolates from asymptomatic carriers (21). Stx2e is
typically produced by STEC strains that cause pig edema disease and
belong to serogroups O138, O139, and O141 (15). However,
Stx2e-producing STEC strains have also been isolated, albeit rarely,
from patients with diarrhea (20) and HUS (35). These human isolates belonged to serogroups O101 and O9 that have not
been reported in STEC strains associated with pig edema disease. Interestingly, Stx2e-producing STEC belonging to serogroup O101 have
been isolated from slaughtered healthy pigs (2), suggesting this animal species as a potential reservoir of human infections. Several stx2e genes have been cloned and
sequenced from STEC O101 isolates originating from a patient with
diarrhea and from healthy pigs, respectively (4), and were
demonstrated to be identical or almost identical to
stx2e present in an STEC O139 isolate from a pig
with edema disease (36).
Stx1 and Stx2 are encoded in the genome of temperate bacteriophages
(11, 32, 33). Phages that contain the structural genes for
Stx1 and Stx2 have been isolated from STEC O157 and O26 strains, and
their morphology, genome sizes, and restriction fragment length
polymorphism patterns have been characterized (23, 32, 38).
Moreover, the Stx1-converting phage H-19B isolated from STEC O26:H11
strain H19 and the Stx2-converting phage 933W originating from STEC
O157:H7 strain EDL 933 (19) have been characterized by
nucleotide sequencing and shown to have a genetic structure related to
that of bacteriophage In contrast to stx1 and
stx2, the stx2e genes in
STEC strains associated with pig edema disease have been reported to be
located in the chromosome because no Stx-converting phages could be
isolated from such strains (15, 37). No data have been
provided in the literature on the location of
stx2e genes in sporadic Stx2e-producing STEC
isolates of human origin described until now (20, 35). The
lack of information on the localization of the
stx2e gene prompted us to investigate the
presence of Stx2e-converting bacteriophages in
stx2e-harboring E. coli strains
isolated from patients in our laboratory. We were able to isolate and
characterize such a phage from the human STEC isolate 2771/97.
Bacterial strains, phages, plasmids, and growth media.
Eleven Stx2e-producing E. coli strains were included in this
study. Eight of them were isolated in 1997 and 1998 from patients with
diarrhea in Würzburg, Germany. These strains were found to be
positive by PCR with primers LP43 and LP44
(stxA2) and were then determined to contain
stx2e by PCR with primers FK9 and FK10 (4). Strain VUB-EH60 was isolated from a patient with
diarrhea in Belgium (20), strain E-D53 was isolated from a
healthy pig (2), and strain E57 was isolated from a pig with
diarrhea (12). E. coli laboratory strain DH5 PCR.
Primer pairs FK9-FK10 and FK1-FK2 were used to amplify
the stxA2e and stxB2e
subunit genes, respectively, as described by Franke et al. (3,
4). A 5-µl volume of each PCR product was separated on 1%
agarose gels, and the bands were visualized by staining with ethidium
bromide. The presence of stx1,
stx2, stx2c,
stx2d, eae, and enterohemorrhagic
E. coli hly genes was investigated using PCR primers and the
conditions described previously (21, 28, 30).
Preparation of phage particles.
Fresh-grown colonies of
stx2e-harboring E. coli strains were
suspended in LB broth and incubated with vigorous shaking until they
reached an optical density at 600 nm (OD600) of 0.1 to 0.3. After adjusting the cultures to a final mitomycin C (Sigma-Aldrich) concentration of 0.5 µg/ml, the bacterial suspensions were incubated overnight. The cultures were then centrifuged at 16,000 × g for 10 min, and the supernatants were filtered through membrane
filters with a pore size of 0.22 µm (Schleicher & Schuell, Dassel,
Germany). The filtrates were diluted 10-fold from 1:102 to
1:106. Portions (100 µl) of each dilution were mixed with
100 µl of 0.1 M CaCl2 and 300 µl of a log-phase culture
of E. coli DH5 Plaque hybridization.
Plaques were transferred to a nylon
membrane (Zeta-Probe GT; Bio-Rad, Munich, Germany) according to a
standard procedure (26) and hybridized with a
digoxigenin-labeled stxA2e probe as described below.
Southern blot hybridization.
Genomic DNA was isolated from
E. coli 2771/97 as described by Heuvelink et al.
(9) and digested with EcoRI and ClaI
(Gibco BRL, Eggenstein, Germany). Restriction fragments were separated on 0.6% agarose gels. Digested DNA was then transferred to a nylon membrane by capillary blotting (26) and fixed with a UV
cross-linker (Stratalinker; Stratagene, Heidelberg, Germany) using the
autocross-link mode (120 mJ/cm2). A 1,004-bp DNA fragment
of the stxA2e gene resulting from amplification with primers FK9 and FK10 (3, 4) was labeled with
digoxigenin and used as a probe. The probe labeling was performed by
incorporating digoxigenin-11-deoxyuridine-triphosphate (Boehringer
GmbH, Mannheim, Germany) during PCR as described previously
(29). Stringent hybridization was achieved with the DIG DNA
Labeling and Detection Kit (Boehringer GmbH) according to the
manufacturer's instructions.
Enzyme immunoassay.
Stx was detected with an enzyme
immunoassay (Premier EHEC; Meridian Diagnostics, Inc., Cincinnati,
Ohio, for the detection of Stx1 and Stx2). Briefly, fresh-grown
colonies of Stx2e-producing E. coli strains were suspended
in LB broth, supplemented with 5 mM CaCl2, and incubated
with vigorous shaking until they reached an OD600 of 0.1 to
0.3. The number of CFU was determined by plating 10-fold dilutions on
LB agar. The cultures were divided in two samples. One sample was
treated with mitomycin C as described above; the other was processed
without an inducing agent. After overnight incubation, the cultures
were centrifuged in a microcentrifuge for 10 min with 13,000 × g. Portions (100 µl) of the supernatants, either undiluted or
diluted 1:10, 1:50, 1:100, 1:500, 1:1,000, or 1:2,000 in LB medium,
were used in the enzyme immunoassay according to the manufacturer's
instructions. Duplicate samples were run in parallel, and the whole
experiment was performed twice. Absorbance measurements were performed
bichromatically at 450 and 620 nm with a BioFlow Multiskan MCC/340
ELISA Reader. Calculations were performed with OD values of between 0.1 and 1.0. Specific Stx2e concentrations were expressed as absorbance
values divided by the number of bacteria (in CFU/milliliters) present
in the suspensions at the time of induction.
Electron microscopy.
A Transduction of the stx2e gene.
To
determine the ability of phage
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of a Shiga Toxin 2e-Converting
Bacteriophage from an Escherichia coli Strain of Human
Origin
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
P27) was isolated from Stx2e-producing Escherichia coli
ONT:H
isolate 2771/97 originating from a patient with
diarrhea. The phage could be transduced to E. coli
laboratory strain DH5
, and we could show that lysogens were able to
produce biologically active toxin in a recA-dependent
manner. By DNA sequence analysis of a 6,388-bp HindIII
restriction fragment of P27, we demonstrated that the
stx2e gene was located directly downstream of
ileZ and argO tRNA genes. Although no analogue
of an antiterminator Q encoding gene was present on this fragment, a
lysis cassette comprising two holin genes which are related to the
holin genes of Pseudomonas aeruginosa phage CTX and a
gene homologous to the endolysin gene gp19 of phage PS3
were detected. The results of our study demonstrated for the first time
that Stx2e can be encoded in the genome of an infectious bacteriophage.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(18, 22). Analysis of a 17-kb
region of the genome of phage H-19B demonstrated that the
stx1 gene was located downstream of a gene encoding an analogue of the transcription activator Q of lambdoid phages and upstream of the analogues of genes encoding lysis functions (18). Phage 933W and phage VT2-Sakai had a related structure in this region (14, 22). Functional studies in
phage H-19B suggested a role for the Q protein and the lysis genes in the release of toxin from the bacterial cell (18).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(Gibco-BRL) was used as a host for bacteriophage P27 and recombinant
plasmids. Phage 933W isolated from E. coli O157:H7 strain
EDL 933 (19) and bacteriophage (ATCC 97537) were used as
controls in transduction experiments and plaque assays. Recombinant
plasmid pIM10 containing the recA gene of E. coli
(5) was a kind gift of G. Blum-Oehler and J. Hacker,
Würzburg, Germany. Bacteria were routinely grown in Luria-Bertani (LB) broth or on LB agar plates. When required, media were supplemented with 100 µg of ampicillin (Sigma-Aldrich, Deisenhofen, Germany) per ml.
and then investigated by a plaque assay
using a double-layer agar method (30). Plaques were counted
after overnight incubation at 37°C and subsequent plaque
hybridization with a stxA2e probe.
P27-containing suspension was
isolated from a stxA2e probe-positive plaque and
purified by cesium chloride centrifugation (26). A drop of
the phage suspension was deposited on copper grids with carbon-coated
Formvar films and stained with 2% KOH phosphotungstic acid (pH 7.2)
for 2.5 min. Samples were examined in a Phillips E.M. 301 electron
microscope operating at 80 kV.
P27 to transduce the
stx2e gene into E. coli DH5, a
plaque assay was performed. A soft agar layer displaying confluent
lysis was harvested into 1 ml of SM buffer (26). Tenfold
dilutions of such suspensions containing DH5 were plated onto LB
agar and incubated overnight at 37°C. Plates containing 100 to 150 colonies were analyzed by colony hybridization (25) using
the stxA2e probe prepared as described above.
E. coli DH5 colonies which hybridized with the
stx2e probe were subcultured three times on LB
agar plates by single colony streaking and tested for the presence of
stx2e after each subculture using PCR with
primer pairs FK9-FK10 and FK1-FK2.
recipients that remained
positive for the stx2e gene after the last
subculture, putative transductants were tested for immunity to
superinfection with phage P27 using a plaque assay. For this
purpose, plates with soft agar layers containing each of the suspected
lysogens as a host strain were prepared. A plate containing E. coli DH5 as a host strain was used as a control. Portions (15 µl) of suspensions of P27, phage 933W, and bacteriophage containing approximately 106 PFU were dropped onto each
plate. The presence of plaques was determined after incubation for
18 h at 37°C.
Vero cell assay. After incubation of LB broth cultures for 18 to 24 h at 37°C with agitation, bacteria were sedimented by centrifugation for 10 min at 4°C at 16,000 × g, and the supernatant was filter sterilized. After diluting the supernatants 1:25 with minimal essential medium (MEM) cell culture medium (Earle's MEM containing 1% of a solution containing 10,000 U of penicillin-10,000 U of streptomycin, plus 5% fetal calf serum, 1% nonessential amino acids, 1% MEM vitamins, and 1 mM Na-pyruvate; Biochrom, Berlin, Germany), 100-µl aliquots were transferred into the wells of a microtiter plate containing Vero cell monolayers (104 cells/well). Cytotoxic effects were determined after 24 to 48 h of incubation at 37°C in 5% CO2 by microscopic examination of the Vero cells and confirmed macroscopically by staining residual Vero cells with crystal violet (7).
Cloning and sequencing of the
stx2e-flanking regions.
Phage P27 DNA
was purified according to a standard procedure (26) and
digested by using different restriction enzymes. The digested DNA was
transferred to a nylon membrane and hybridized with an
stxA2e probe as described above. A 6.4-kb
HindIII fragment which hybridized with the probe was
chosen for cloning. It was excised from the gel, purified with a
QIAquick gel extraction kit (Qiagen GmbH), ligated into a
HindIII-digested pBluescript II KS(+) (Stratagene), and
transformed into E. coli DH5 according to standard
methods (26).
Nucleotide sequence accession number.
The nucleotide
sequence a 6,388-bp fragment of P27 DNA containing
stx2e and stx2e-flanking
regions was submitted to the EMBL database library and assigned
accession no. AJ249351.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Eight E. coli isolates from patients with diarrhea,
which were originally detected as
stx2e-harboring STEC by PCR with primers FK9 and
FK10 during routine diagnostic work in Würzburg, a patient isolate from Belgium, and two Stx2e-producing E. coli
strains isolated from pigs were investigated for the presence of
Stx-encoding phages. Liquid cultures of all strains were induced with
mitomycin C, and culture supernatants were tested for the presence of
infectious phage particles by a plaque assay using E. coli
DH5 as a host. Of 11 supernatants, 8 caused plaque formation on
DH5. Plaques were transferred to a nylon membrane and hybridized
with a stxA2e probe. Only the culture
supernatant of 1 of the 11 stx2e-containing isolates, E. coli ONT:H strain 2771/97, caused
formation of plaques on DH5 that hybridized with the
stxA2e probe. These plaques show a uniform and
turbid morphology and were substantially smaller than the plaques
formed by Stx2-converting phage 933W. Plaques were difficult to discern by visual inspection, and only the plaque hybridization approach allowed us to evaluate precisely the number of plaques formed by this
phage on a lawn of E. coli DH5.
We decided to further investigate this STEC strain and determined at first its virulence spectrum. Beside the stxA2e and stxB2e genes, which could be detected by PCR with primer pairs FK9-FK10 and FK1-FK2, other stx types, eae, or e-hly were not present.
Without inducing agent, we could prepare a phage lysate containing ca.
102 PFU/ml, indicating a low-level release of Stx2e phages.
After this, we prepared a high-titer phage lysate (4.7 × 108 PFU/ml) of P27 using mitomycin C induction. After
performing a plaque assay with the high-titer lysate and subsequent
plaque hybridization, we could show that all plaques hybridized with the stxA2e probe. This suggested the presence of
only one inducible phage in STEC strain 2771/97, which was designated
P27. Electron microscopic investigation revealed that the phage
particles were approximately 140 nm long and had regular hexagonal
heads ca. 45 to 50 nm in diameter and long, wide tails (Fig.
1).
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Lysogenization of E. coli DH5 with P27.
We
wanted to know whether it is possible to lysogenize E. coli
DH5 with P27 and thereby to convert it to the production of
Stx2e. Thus, 100-µl aliquots of a 1:10 dilution of the P27 phage
lysate were mixed with 300-µl aliquots of a E. coli DH5 log-phase culture and plated by the double-layer method. On such plates, confluent lysis was observed. The soft agar layers from two of
these plates were harvested and suspended in 1 ml of SM buffer each
(26). Tenfold dilutions of these suspensions were plated on
LB agar and incubated overnight at 37°C. We selected plates resulting
from a 106 dilution which contained 121 and 135 colonies,
respectively. These were in turn subjected to colony blot hybridization
(25) with an stxA2e probe. Of 121 colonies, 24 (19.8%) from the first plate and of 135 colonies, 13 (9.6%) from the second plate hybridized with the probe.
P27.
Lysogenization of T9 and T21 isolates with phage P27 was confirmed
by demonstrating their immunity to superinfection with this phage. None
of the lysogens supported plaque formation with phage P27 when used
as a host strain in the plaque assay. In contrast, both lysogens
remained sensitive to bacteriophage and to phage 933W.
Surprisingly, no phage particles were detected by the plaque assay, and
no phage DNA could be isolated from culture supernatants of the
lysogens after mitomycin C induction. Transformation with pIM10
restored the ability of the lysogens to release free phage particles
(see also below).
Production of Stx2e by E. coli 2771/97 and the lysogens
T9 and T21.
We have determined the ability to induce Stx2e
production by mitomycin C in wild-type strain 2771/97 and the lysogens
T9 and T21. Stx-enzyme immunoassay was performed with supernatants of induced and noninduced cultures. The results of this experiments are
shown in Fig. 2.
|
Mitomycin C, not induced with mitomycin; + mitomycin C, induced with 0.5 µg of mitomycin C per ml.
y axis, logarithmic scale of specific Stx2e concentration
expressed as absorbance values divided by the number of bacteria (in
CFU/milliliters) present in the suspensions at the time of induction.
, we transformed the lysogens with the
recA-positive plasmid pIM10. As can be seen in Fig. 2, the ability to produce Stx2e was restored in the lysogens containing a
functional recA gene. In lysogens T9 and T21, Stx2e
production increased 50- to 100-fold after induction by mitomycin C.
To investigate whether the lysogens T9 and T21 produce biologically
active toxin, we performed a Vero cell assay. All
recA-positive strains which were positive in the Stx-enzyme
immunoassay [2771/97, T9(pIM10), T21(pIM10)] were also cytotoxic in
the Vero cell assay, whereas control strains DH5(pIM10) and the
transductants without pIM10 did not show cytotoxicity to Vero cells
under the conditions described.
Analysis of stx2e-flanking regions of
P27.
Genomic DNA of P27 was digested with
HindIII, separated by agarose gel electrophoresis, and
hybridized with an stxA2e probe. A 6.4-kb
HindIII restriction fragment hybridized with the probe. This fragment was excised from the gel, purified, and cloned as described above. The cloned fragment was labeled with digoxigenin and
rehybridized with HindIII-restricted chromosomal DNA
from 2771/97. Only one 6.4-kb fragment hybridized with the probe,
suggesting that the cloned fragment is not a recombinant. Nucleotide
sequencing of the fragment revealed a length of 6,388 bp. We used the
GeneMark.hmm software for the search for ORFs and prediction of
appropriate translation start positions. ORFs proposed by GeneMark.hmm
were translated and compared with database libraries. For the final definition of ORFs, we used translation start codons in our sequence that were predicted by GeneMark.hmm with a high probability and which
fit best with the start positions of genes encoding homologues proteins
found in the database libraries. The results of these searches are
shown in Table 1, and a scheme of the
sequence features present on the fragment is shown in Fig.
3.
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P27 is 312 amino acids
in length, the N15 methylase comprises only 284 amino acids. We
detected downstream of the gene 52 homologue two tRNA genes with high
sequence identity to ileZ and argO of E. coli (see Table 1). The next two ORFs in 3' direction of
argO showed a high degree of homology with
stxA2e and stxB2e sequences.
The nucleotide sequence of stxA2e of P27
showed 99.8% identity to the stxA2e subunit
gene of Stx2e-producing strains ED-53 (accession no. X81416); 99.7% to
that of ED-68 (accession no. X81415), 412 (accession no. M36727), and
S1191 (accession no. M21534); 99.4% to that of ED-43 (accession no.
X81417); 99.3% to that of ED-42 (accession no. X81418); and 99.1% to stxA2e of R107 (accession no. U72191). The
nucleotide sequences of stxB2e of P27 were
identical to the corresponding sequences of S1191, 412, ED-43, ED-53,
and ED-68 and 99.2% identical to those of ED-42 and R107.
Moreover, the stxA2e and
stxB2e subunit genes carried by P27 shared
94.0 and 87.5% nucleotide sequence identity, respectively, with the A
and B subunits of the stx2 gene of phage 933W
(22).
Downstream of the stx2e genes and
orf6 we found two tandemly arrayed ORFs which could encode
two holins (Fig. 3). The amino acid sequences of both of the putative
proteins encoded by orf7 and orf8 demonstrated 27 and 30% amino acid identities, respectively, to the putative holins
(encoded by orf9 and orf10) of the
Pseudomonas aeruginosa phage CTX (17). Since
these low homologies did not give strong evidence for the presence of
holin genes, we performed a search for transmembrane domains according
to the suggestions proposed by Young et al. (39).
In each of the putative P27 holins we found three transmembrane
helices which spanned positions 28 to 51, 53 to 76, and 84 to 105 in
the first one (orf7) and positions 7 to 24, 33 to 55, and 60 to 77 in the second one (orf8). The structure predicted by
TMpred suggested a typical N-out-C-in structure which is
characteristic for class I holins of the lambda group (39).
The predicted protein of orf9 is 46% identical to Gp19
encoded by phage PS3 (accession no. AJ011579) and may constitute, together with the putative holin genes, a lysis cassette necessary for
the release of infectious P27 phage particles.
Comparison of the structure of the
stx2e-flanking region of P27 with the
corresponding region of phage 933W and the
stx2e-flanking region of STEC strain
S1191.
Comparison of homologues
stx2e-flanking sequences of P27 and phage
933W demonstrated similarities as well as crucial differences. In Fig.
3, a scheme of the 6,388-bp fragment of P27 (A) and a corresponding
segment of 9,760 bp of the stx2-flanking region 933W (B) is shown.
P27 is also preceded by tRNA genes.
We found two putative tRNA genes which are highly homologues to
ileZ and argO (Table 1). Moreover, we found
sequences related to a third one, argN, which is present in
phage 933W. Since a middle portion (ca. 30 bp) of the latter sequence
is substituted with heterologous nucleotides in P27, no tRNA
structure could be observed.
We found downstream of the stx2e operon a lysis
cassette which was different from the lysis cassettes of phage 933W or
phage H-19B. We could identify the holin genes by computer analysis of
the tertiary structure of their products but not by homology searches.
In the 5' direction of the tRNA genes, we found a methylase gene which
is not present in this area in 933W. Interestingly, a Q-like gene was
not present on our fragment.
On closer inspection of a 1,890-bp
stx2e-flanking sequence of STEC strain S1191,
which was available in the database (accession no. M21534), we found a
structure similar to that described for the P27. The fragment
started with an argO tRNA gene, followed by
stx2e. Downstream of
stxB2e, we found an ORF with high sequence identity to orf6 of P27. However, this ORF was shorter
than orf6 due to an internal 39 bp in-frame deletion. The
published sequence of STEC strain S1191 ended with 30 nucleotides which
are identical to the 5' end of the putative holin gene orf7.
All in all, the nucleotide sequence of this region is almost identical
to that of P27.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we demonstrated that Stx2e of STEC patient isolate
2771/97 is encoded in the genome of an infectious bacteriophage. The
morphology of this phage is distinct from that of phages 933W and
H-19B. The latter phages displayed either elongated hexagonal heads and
long, thin, flexible tails as phage H-19B (38) or regular
hexagonal heads and short, thin tails as phage 933W (22). In
contrast, phage P27 consists of a regular hexagonal head in combination with a long and wide tail (Fig. 1).
The presence of an infectious Stx2e-converting phage and the ability to convert a new host strain to the production of Stx supports the hypothesis that Stx phages play a role in horizontal gene transfer and the emergence of new STEC pathotypes. We could show in an earlier study that a derivative of a Stx2-converting phage from E. coli O157:H7 was able to lysogenize different enteropathogenic E. coli strains (27). Therefore, phages may be considered as highly mobile genetic elements with the capacity to spread stx genes among E. coli strains.
However, we were not able to detect infectious phage particles from the other 10 stx2e-harboring strains. The reasons for that phenomenon are not known. Either the stx2e genes of these strains could be encoded in the chromosome (37) or important sequences for maturation of the phage particles could be deleted, as is the case in Shigella dysenteriae type 1 (16), or else inactivated by insertion of insertion elements, as is speculated for the VT2-Sakai phage (14).
Interestingly, we could show that the published
stx2e-flanking sequence of STEC strain S1191
contains the same structural elements as P27. A tRNA gene as well as
a part of a putative holin gene could be detected on this fragment.
This finding raises the question of whether
stx2e of S1191 is located in the chromosome as
described previously (37) or in the genome of a
P27-related prophage.
The genetic organization of phage DNA flanking the stx genes
has been demonstrated to be conserved in Stx1- and Stx2-converting bacteriophages H-19B and 933W (18, 22). The structural genes for Stx1 and Stx2 are integrated in the late-phase regions of the
genomes of bacteriophages H-19B and 933W, respectively, both in
identical positions between functional analogues of the Q transcription activator gene and the holin S gene of the lysis cassette
(18, 22). Functional studies of bacteriophage H-19B demonstrated that the expression of stx1 and the
release of the toxin from the bacterial cell are dependent on this
late-phase region (18). Similarly, the expression of the
stx2 gene in phage 933W has been explained as a
part of the Q-dependent late transcript of the lysis genes
(22).
The sequence analysis of stx2e-flanking regions
of P27 DNA shows an organization that differs from that described
for phages H-19B and 933W. Although important structural elements of
the late regulatory region of lambdoid phages (i.e., lysis cassette) were detected on the sequenced fragment of P27, others, such as the
Q antiterminator gene, could not be found. A possible explanation for
the latter finding could be that the Q analogue is located in the
P27 genome more upstream of the stx2e gene,
in a region which was not analyzed in our study.
A gene whose product is related to an adenine-specific modification methylase encoded by gp52 of bacteriophage N15 was identified 385 bp upstream of the stx2e gene. Interestingly, a methylase gene (L0094) was also found 4,100 bp upstream of the stx2 gene in phage 933W (22).
The results presented here allow us to suggest that P27, although
belonging to the lambdoid group of phages, is not closely related to
H-19B or 933W. Phages may be considered important vehicles for the
spread of stx genes among E. coli strains living
in the same or different ecological niches. Since 10 Stx2e-producing strains included in our study could not be demonstrated to release infectious Stx-converting phage particles after induction, further studies are needed to clarify whether such stx2e
genes are located in a defective phage genome or whether they are not
associated with phage sequences.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft and, partially, by a grant from the Fundació Comptes de Barcelona, Barcelona, Spain. Maite Muniesa is a recipient of a scholarship from the Alexander von Humboldt Foundation (no. 15851).
We thank Barbara Plaschke, Olga Böhler, and Beatrix Henkel for excellent technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Hygiene und Mikrobiologie der Universität Würzburg, Josef Schneider-Str. 2, 97080 Würzburg, Germany. Phone: 49-931-201-3905. Fax: 49-931-201-3445. E-mail: hschmidt{at}hygiene.uni-wuerzburg.de.
Editor: A. D. O'Brien
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. |
Besemer, J., and M. Borodovsky.
1999.
Heuristic approach to deriving models for gene finding.
Nucleic Acids Res.
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