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Infection and Immunity, September 2000, p. 4856-4864, Vol. 68, No. 9
Institut für Hygiene und Mikrobiologie
der Universität Würzburg, D-97080 Würzburg, Germany
Received 11 February 2000/Returned for modification 18 April
2000/Accepted 30 May 2000
The stx-flanking regions of 49 Shiga toxin-producing
Escherichia coli strains and nine Shigella
dysenteriae serotype 1 strains containing either stx,
stx1, stx2, or
stx2 variant genes, were examined. We analyzed
these regions by PCR using a set of primers with one primer
specific for the respective stx gene and a second primer
complementary to sequences of Stx phages H-19B and 933W. We further
characterized the amplification products by restriction endonuclease digestion and nucleotide sequencing. PCR products of
stx1-containing E. coli strains of
serogroups O157, O26, and 0103 showed the same lengths and similar
restriction patterns. However, we failed to amplify the 3'
stx-flanking region in
stx1-harboring E. coli
O111:H Shiga toxin (Stx)-producing
Escherichia coli (STEC) can cause a broad spectrum of human
diseases ranging from watery diarrhea to hemorrhagic colitis and the
hemolytic-uremic syndrome (5). Shiga toxins have been
generally accepted to be the main agent in the development of these
diseases (5, 16).
Two Stx subgroups have been described in E. coli: Stx1 and
Stx2. The structural genes for Stx1 and Stx2 are encoded in the genome
of temperate, lambdoid bacteriophages in E. coli O157 and O26 strains (9, 28-30). Such Stx-encoding phages of
E. coli O157 and O26 strains were reported to have similar
morphologies with regular hexagonal heads and short tails
(20). E. coli O157 phages are similar in
genome size and have highly related restriction fragment length
polymorphism (RFLP) patterns (17). stx RFLP pattern analysis of O157 and non-O157 STEC strains suggested a more
heterogeneous structure of phage DNA flanking the stx genes in E. coli O157:H7 than in non-O157 STEC strains
(23). Recently, the whole genome of the Stx2-encoding phage
933W and a 17.3-kb fragment of the Stx1-encoding phage H-19B were
determined by nucleotide sequencing (15, 19).
Sequence analysis of a part of the Stx1-encoding phage H-19B has shown
that the stxA1 and stxB1
genes are located in the late-phase region, downstream of and in the
same transcriptional orientation as a In bacteriophage The stx1 genes are separated from the lysis
genes by a 3-kb region (15), whereas the distance of the
stx2 genes to the lysis genes is 3.5 kb (9,
19). The region between stx genes and lysis genes
contains some open reading frames, the sequence of which show only
marginal homology with other genes in searches using sequence database
libraries. Only one open reading frame, L0105 (19), has been
described for the Stx2-encoding phage 933W in this region, which
encodes a protein with 50.8% identity to YjhS of E. coli
K-12. The function of this gene is yet unknown, but it is described as
a part of a fimbrial synthesis and iron transport gene cluster. A
similar 849-bp open reading frame whose putative gene product shows
20.5% identity to YjhS is found in the stx-flanking region
of the Stx1-encoding phage H-19B.
In this study, we investigated the stx-flanking regions of a
large set of distinct Stx-producing E. coli and S. dysenteriae strains to study the heterogeneity in this region and
to look for stx-continuous phage sequences.
Bacterial strains and plasmids.
E. coli strains
6061/96, 2969/99, 2791/97, 6366/97, 4865/96, 893/98, 5951/97, and
6480/96 were isolated at the Institute of Hygiene and Microbiology,
University of Würzburg, by routine methods. The characteristics
of these strains and of all other E. coli isolates used in
this study are presented in Table 1. E. coli C600(H19J) and C600(933W) are laboratory strains
lysogenized with phage H19J (29) and phage 933W
(17). S. dysenteriae serovar 1 isolates
RIMD3101010, H2540-34/82, H1250-36/74, H2765-34/81, H3104-34/89,
H4435-34/89, 1971, SZ 340/95, and W206 were kindly provided by
Jochen Bockemühl, Nationales Referenzzentrum für Enteritiserreger, Hygiene Institut, Hamburg, Germany.
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Structural Analysis of Phage-Borne stx Genes and Their
Flanking Sequences in Shiga Toxin-Producing Escherichia
coli and Shigella dysenteriae Type 1 Strains
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
strains. Stx2-producing E. coli
strains revealed amplification products of different lengths and
restriction patterns, suggesting greater heterogeneity than in
stx1-positive strains. We also obtained specific PCR products for two Stx2c-producing and seven Stx2f-producing E. coli strains when they were subjected to PCR analysis.
In nine S. dysenteriae type 1 strains, H-19B- and
933W-specific primers amplified only the 3' stx-flanking
region. The results of our study demonstrate that the stx
genes of all strains investigated are continuous with phage sequences.
Whereas almost all strains except E. coli
O111:H strains were associated with a S-like gene,
association with Q could not be demonstrated in nine S. dysenteriae type 1 strains and three E. coli strains.
Furthermore, we showed that the organization of the
stx-flanking regions is similar in all strains
investigated, whereas fine-structure analysis showed subtle differences
among the sequences examined. Our results support the hypothesis that stx genes in E. coli and S. dysenteriae are generally phage-borne.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Q homologue and upstream of
the homologue genes S, R, and Rz, which are necessary for the
release of phage particles. A similar structure was found in the
Stx2-encoding phage 933W (19).
, the Q protein functions as a transcription
antiterminator by modifying transcription complexes initiating at the
late promoter PR'. Neely and Friedman could show a direct effect of the phage H-19B Q protein on the levels of Stx expression, as
well as expression of the downstream lysis genes, and concluded that
the H-19B antiterminator Q acts as a transcriptional activator by
supporting the readthrough of the transcription terminator (14,
15).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Characteristics of E. coli strains used in
this study
Standard DNA techniques. Plasmid DNA was prepared with Qiagen plasmid mini and midi kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Genomic DNA was prepared as described previously (7). DNA was digested with restriction endonucleases (New England Biolabs, Schwalbach, Germany; Gibco-BRL, Karlsruhe, Germany), and restriction fragments were analyzed by separation on 0.6 to 0.9% agarose gels in 0.5-fold-concentrated Tris-borate-EDTA buffer and stained with ethidium bromide. Purification of DNA fragments from agarose gels was performed using a Prep-a-Gene kit (Bio-Rad, Munich, Germany) or a QIAex kit (Qiagen). If not indicated otherwise, transformation of plasmid DNA was performed according to standard protocols (22). For Southern blot hybridization, DNA was prepared and separated electrophoretically and subsequently transferred from agarose gels to Zeta-Probe GT blotting membranes (Bio-Rad). Hybridization assays were performed with a digoxigenin-labeled PCR generated stx1B-specific DNA probe (24) under stringent conditions in accordance with the manufacturer's instructions (Boehringer GmbH, Mannheim, Germany). Specific washing steps were performed twice, each for 5 min at 60°C in 1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% (wt/vol) sodium dodecyl sulfate.
PCR. Amplification was carried out in a total volume of 100 µl containing each deoxynucleoside triphosphate at 200 µM, 30 pmol of each primer, 10 µl of 10-fold-concentrated Expand High Fidelity buffer (with 10-fold-concentrated MgCl2), and 2.6 U of Expand High Fidelity Polymerase (Boehringer GmbH) according to the manufacturer's instructions.
Template DNA (500 to 1,000 ng) was denatured at 94°C for 30 s, annealed at the appropriate temperature for 60 s, and then extended for 3 min at 68°C. This amplification step was carried out for 10 cycles. In the following 20 cycles the elongation time was 3 min plus a 20-s elongation step for each cycle. A final extension step of 7 min at 72°C was also conducted. The PCR products were separated on a 0.8% agarose gel, and gel slices with the appropriate DNA fragment were excised. DNA was eluted and purified with the QIAquick PCR purification kit (Qiagen). The purified DNA amplificates were digested either with PvuII or with EcoRI and separated again on a 0.8% agarose gel. Oligonucleotides were purchased from ARK Scientific GmbH (Darmstadt, Germany) and were designed with the Oligo 4.06 program (National Biosciences, Inc.). All primers used in this study are listed in Table 2.
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Cloning of PCR products.
PCR products were ligated into pCR
2.1 vector (Original TA Cloning Kit, pCR II, pCR 2.1; Invitrogen) and
transformed into E. coli InvV
F' competent cells
according to the manufacturer's instructions.
Nucleotide sequencing. Nucleotide sequencing was carried out with universal and reverse primers for pUC/M13 vectors and customized primers. Separation of sequencing products was performed on 7% denaturing polyacrylamide gels in a 377 automatic sequencer (Perkin-Elmer/Applied Biosystems GmbH, Weiterstadt, Germany). Nucleotide sequence analysis of PCR products was performed with two clones obtained independently. DNA sequences were analyzed with the DNASIS program, version 2.0, from Hitachi Software (San Bruno, Calif.). Searches for homologies of DNA sequences were performed using the EMBL-GenBank database library.
Nucleotide sequence accession numbers.
The nucleotide
sequences determined and analyzed during this study were submitted to
the EMBL data library. Characteristics and accession numbers of these
sequences are presented in Table 3.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Analysis of the stx-flanking region of
Stx1-positive STEC isolates.
Twenty Stx1-producing E. coli (STEC) isolates representing the major non-O157 STEC
serotypes were selected to analyze their stx1-flanking sequences (Tables 1 and
4). We designed a set of primer
pairs, with one primer specific for stx1 and the
second primer specific for the stx1-flanking
sequences (Fig. 1A). These primers were
derived from the sequence of the Stx1-encoding phage H-19B
(15). We analyzed the 3'-stx-flanking region of
the published sequence of phage H-19B for open reading frames and found
four open reading frames, designated as orf85a, orf85b, orf282, and orf102. Primers specific for the 3'-stx-flanking regions
were chosen from the open reading frames orf85a and orf282 (Table 2, Fig. 2A). To verify the linkage between
stx1 and a Q-like gene, PCR amplification was
performed using primer pair Q-stx-f and 285 (see Fig. 1). We
further characterized the amplification products by subsequent
restriction endonuclease digestion with PvuII.
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strains, we revealed only
PCR products with primers Q-stx-f and 285 in the 5' region
and no products from the 3'-stx region (Table 4).
Nucleotide sequence analysis was performed from the
stx-flanking region spanning the stx1
gene and the S gene using E. coli O103:H2 1858/96. Two
PCR products of this strain revealed with primer pair
stxB1-f/s-stx-b1 were independently
cloned and sequenced. The fragments obtained were 3,199 bp in length. A
scheme of this sequence is shown in Fig. 2A. The sequence starts with
the stxB1 subunit gene. Downstream of
stxB1, two open reading frames of 228 bp (orf75)
and 1,851 bp (orf616) could be identified, and the sequence ended with
the S gene. The overall identity of this sequence to H-19B phage is
96%.
Since no PCR products were revealed from the investigated E. coli O111 strains, we determined the nucleotide sequence of
the 3'-stx-flanking region. We prepared genomic DNA of
E. coli O111:H strain 1639/77, digested
it with restriction endonuclease SphI, and separated the
fragments on an agarose gel. Hybridization with a
stxB1-specific probe revealed a signal band of
ca. 5 kb. DNA bands of this size were then excised from an agarose gel,
ligated in a SphI-digested pBR322 vector, and transformed
into E. coli DH5. Up to 600 transformants were spotted
onto a nylon membrane and were hybridized with an
stxB1-specific probe. Two of the transformants reacted with this probe, and one of these was investigated further. Using universal and customized primers we could determine the whole
sequence of the fragment of 5,467 bp. The 5' end of 1,849 bp was
identical to the respective sequence of Stx1 phage H-19B. This sequence
comprises a part of the Q gene and the whole
stx1 gene (Fig. 2A).
The sequence downstream of stx1 of 3,618 bp
shows only 45% identity to the corresponding sequences of H-19B and
933W. Located 400 bp downstream of the stxB1
subunit was a region with 92% identity to the essential recombination
function (erf) gene of phage H-19B; in that phage the
erf gene is located upstream of the kil and cIII genes in
the early regulatory phase region. A short stretch of 200 bp was 82.9%
identical to the lambda Rz1 gene; in the lambda genome this stretch is
part of the lysis cassette. We also detected a sequence of 1,042 bp
with 73.2% identity to the sequence of the Salmonella
enterica serovar Typhimurium LT2 (strain 1196-T3) phage fels-1
(Fig. 2A).
Primers O111fl-1, O111fl-2, and O111fl-3 (Table 2) were designed from
the 3' stx1 region of strain 1639/77 and used
for PCR with strains ED-31, 78/92, and SK003. PCR products of
approximately 1,000, 2,000, and 3,600 bp were revealed for all strains
tested. This finding demonstrates that E. coli
O111:H strains carry a similar
stx1-flanking region which is distinct from that
of phages H-19B and 933W.
Analysis of the stx-flanking region of Stx2-positive
STEC isolates.
In order to perform similar investigations on
stx2 genes, we selected 20 Stx2-positive STEC
strains (Tables 1 and 5). The primers
used were derived from the Stx2-encoding phage 933W (9) and
are depicted in Table 2 and Fig. 1B.
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strain 3985/96.
PCR products spanning the stx2 gene and the S
gene were cloned and sequenced from each strain. The PCR product of
E. coli strain O26:H11 strain 1448/97 was 3,919 bp in
length. The sequence starts with the stxB2
subunit gene (Fig. 3A). Downstream of
the stxB2 gene, we could identify five
open reading frames of 325 bp (orf107), 1,938 bp (orf645), 180 bp
(orf59), 255 bp (orf84), and 150 bp (orf49) (Fig. 3A). Searches for
homologous sequences revealed that orf645 had 98% nucleotide sequence
identity to L0105 of the Stx2-encoding phage 933. L0105 may encode a
protein with 50.8% identity to E. coli K-12 YjhS
(19). All other open reading frames failed to match a
sequence of the database library.
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strain
3985/96 was 3,920 bp. The sequence also starts with the
stxB2 subunit gene (Fig. 3A). Downstream of the
stxB2 gene, we identified four open reading frames of 325 bp (orf107), 1,938 bp (orf645), 180 bp (orf59), and 273 bp (orf90). orf645 showed 99.3% identity to orf645 of E. coli O26:H11 strain 1448/97 and 92.1% identity to L0105 of phage 933W (19). The other three open reading frames did not show significant identities to published sequences.
The PCR product obtained with E. coli O26:H11 strain ED-147
was 3,896 bp long. The sequence also starts with the
stxB2 subunit gene (Fig. 3A). Between the
stxB2 gene and the S gene we found three open
reading frames of 325 bp (orf107), 1,938 bp (orf645), and 246 bp
(orf81). orf645 also showed 92.5% identity to L0105 of phage 933W. All
other open reading frames failed to match any database library sequences.
The sequence identity between the 3.9-kb fragments obtained with both
E. coli O26:H11 strains was 94.8%. Whereas the identity between E. coli O26:H11 strain 1448/97 and E. coli O145:H strain 3985/96 was 99.6%, the
similarity between E. coli O111:H ED-147
and E. coli O145:H strain 3985/96 was only
94.7%.
PCR and nucleotide sequence analysis of the
stx-flanking region of stx2
variants.
We extended our studies on some variants of
stx2 and carried out PCR and nucleotide sequence
analysis on stx2c and its flanking regions
harbored by E. coli O157:H strain E32511 and
E. coli O145:H strain 4865/96 and on six
stx2f variant genes from E. coli
pigeon isolates (Table 1). PCR of the stx2c
variants was performed using oligonucleotide 545 (Table 2), which is
specific for the stxB2 subunit gene, and
oligonucleotide s-stx-b, which is specific for the S gene.
Amplification products were cloned and sequenced.
strain 4865/96 was 3,922 bp. Downstream of the
stxB2c subunit gene we could identify two open
reading frames of 1,938 bp (orf645) and 180 bp (orf59). orf645 of
E. coli O145:H strain 4865/96 showed 92.3%
identity to L0105 of the Stx2-encoding phage 933W. We found an overall
identity to the 3'-flanking region to E. coli
O157:H strain E32511 of 86% and an 86.5% overall
identity to the 3'-flanking region of phage 933W.
Furthermore, we investigated the stx-flanking regions of six
E. coli strains containing the stx2f
variant by PCR. The primers used were derived from E. coli
T4/97 (26). Analyses were performed using oligonucleotide
128-1 (Table 2), which is specific for the
stxA2f subunit gene in combination with
oligonucleotide stx2f-s-b, which is
complementary to the S-like gene of E. coli strain O128:H2 strain T4/97. Using Stx2f-positive E. coli strains T4/97,
E-D 365, E-D 371, E-D 373, 5598/97, and H.I.8 as templates, we could amplify PCR products of 2 kb from each of the strains (Fig. 3B).
Nucleotide sequence analysis was performed from the
stx2f-flanking regions of E. coli
O128:H2 isolate T4/97 and O128:B12 isolate H.I.8. PCR products from
both strains, spanning from stx2 to the S gene,
were cloned and completely sequenced. The sequence achieved from
E. coli O128:H2 isolate T4/97 was 2,411 bp in length. This sequence starts directly adjacent to the stxA2f
subunit gene (Fig. 3B). Downstream of the stxB2f
subunit we could identify one open reading frame, orf67, of 204 bp with
unknown function. We also found an S-like gene with 68.5% identity to
the S gene of phage 933W and 71% identity to the S gene of phage
H-19B. The overall identities of the stx-flanking region to
the corresponding regions of phage 933W and phage H-19B were 42 and
43%, respectively.
The PCR product of E. coli O128:B12 strain H.I.8 was 1,892 bp (Fig. 3B). Downstream of stxB2f we could
detect one open reading frame, designated orf60, with unknown function
and an S-like gene. The sequence showed an overall identity of
99% to the corresponding sequence of the E. coli
O128:H2 isolate T4/97, but a single base-pair exchange resulting in a
stop codon in orf60 revealed a putative truncated protein (Fig. 3B).
Analysis of the stx-flanking region of S. dysenteriae serovar 1 isolates. We have sequenced a 6,014-bp stretch of stx-flanking DNA of S. dysenteriae H2765-34/81. A map of this fragment and the comparison to the corresponding sequence of H-19B and 933W are shown in Fig. 2B. We did not find a Q-like gene close to the stxA subunit in the 5' direction. Instead of this there is an insertion element with high sequence homology to IS600 of S. sonnei. In the 3' direction of stx we found five open reading frames. Three of them, orf109, orf60, and orf87, did not show significant sequence identities to known genes. The first 1,140 bp of orf 536 were identical to those of L0105, as described by Plunkett et al. (19). However, homology ceased in the last third of the open reading frame, and it was shorter than that of L0105.
When we looked for overall homology of the stx-S region, we found 68.2% sequence identity with 933W and only 54% with H-19B. The S-like gene demonstrated 95.4% sequence identity with the S-gene of phage 933W and 91.8% with that of H-19B. We selected S. dysenteriae type 1 isolates RMD3101010, H2540-34/82, H1250-36/74, H3104, 34/89, H4435-34/89, B1971, HSZ340/95, and W206 and subjected them together with H2765-34/81 to PCR analyses. Amplification products of approximately 2,400 bp were obtained with primer pair stxB1-f/422 for each S. dysenteriae type 1 isolate, and amplification products of approximately 3000 bp length were obtained with primer pair stxB1-f/s-stx-b1. Restriction of the latter PCR product with restriction endonuclease SphI revealed two fragments of 1,000 and 2,000 bp for eight of the nine strains. The S. dysenteriae type 1 H2765-34/81 isolate showed a different restriction pattern, with two products of 1,300 and 2,000 bp. No amplification products were obtained with primer pair stxB1-f/457. Moreover, for all investigated S. dysenteriae type 1 isolates no amplification products were revealed with primer pairs stxB1-f/85a-stx1-b, stxB1-f/282-stx1-b, 545/457, 545/422, and 545/s-stx-b. To confirm a linkage between stx genes and an IS600 element, PCR amplification was performed using the primer pair of IS600-f and 285. An association with an IS600 element was revealed in seven of nine strains. In the cases of H2540-34/82 and B1971, we failed to generate a PCR product using the latter primer pair.| |
DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Although it was demonstrated earlier that particular stx genes are phage encoded or at least are adjacent to phage sequences (3, 9, 15, 19), we could show with our present experiments that in addition to stx1 and stx2, the stx2 variants stx2c, stx2f, and stx of S. dysenteriae are also flanked by phage sequences. Moreover, fine structure analysis of the stx-flanking regions yielded similarities as well as crucial differences in different Stx-converting bacteriophages.
The results of our study indicate that the physical linkage of stx and phage sequences may be a general feature of Stx-producing bacteria, regardless of the serotype or the species. In most strains, stx is located between a Q-like antiterminator gene and a holin S gene. These genes represent the central region of the late-phase region of phage lambda or other lambdoid bacteriophages. Restriction analysis of PCR products suggested that this regulatory region contained a similar structure in most of the Stx1-positive E. coli strains except E. coli O111.
Different amplification product lengths and different restriction patterns in Stx2-positive E. coli strains indicate a greater sequence divergence than was observed in Stx1-positive strains. However, these different patterns could not be correlated to a specific STEC serotype. Divergence in Stx2 phages isolated from different STEC serotypes was also observed in a study performed by Wagner et al. (32). They demonstrated that Stx phages isolated from seven STEC strains could be placed into five distinct groups by phage genomic RFLP. In addition, when E. coli K-12 was lysogenized with these phages, toxin and phage production differed markedly compared to the RFLP groups.
Comparison of the nucleotide sequence of the 3'-stx-flanking region of three Stx2-positive strains with the corresponding region of phage 933W showed an overall identity of 86% (Fig. 3A). For each of the E. coli strains we could identify the same open reading frame, orf645 (Fig. 3A), with high homology to L0105, which is described as being in the region between the stx genes and the lysis genes for phage 933W (19). This open reading frame may encode a protein with 50.8% identity to E. coli K-12 YjhS (19).
PCR analysis of Stx2c-positive and Stx2f-positive E. coli strains demonstrated for the first time a linkage of stx2 variants to the holin S gene. Sequence comparisons of two Stx2c-positive E. coli strains revealed a high overall homology in the 3'-flanking region and in the presence of orf645. A similar result was obtained with two Stx2f-positive strains. The stx-flanking region of a human and a pigeon isolate differed in only one nucleotide. Although both PCR products were nearly identical, exchange of one nucleotide caused a truncated open reading frame in E. coli O128:B12 strain H.I.8. The distance between the A-subunit gene and the S gene is shorter in stx2 variants than in classical stx2-positive strains.
E. coli H.I.8 was isolated from a patient with infant diarrhea in the United States and was described in 1977 (4, 11), whereas E. coli T4/97 was isolated from a feral pigeon in Würzburg, Germany, in 1999 (26). Our analysis of the stx-flanking regions demonstrated high similarity in that region, indicating close relationship. Since the Stx2f variant was found as an exclusive toxin type in pigeons (26), one could conclude that either horizontal gene transfer may have occurred or that STEC isolates have been transferred between pigeons and humans. One should also keep in mind that pigeons may serve as a reservoir to enlarge the gene pool which may be used by pathogenic enterobacteria.
Analysis of the similar region of S. dysenteriae type 1 which was performed by our group and also independently published by McDonough and Butterton (13) also revealed a linkage of stx with phage sequences. However, Q-like genes were not obtained. Instead of this, an IS600 element was located close to the stxA subunit gene.
With the investigations described here, we could clearly show that stx of S. dysenteriae is linked with phage sequences. However, phages could not be induced in such strains. McDonough and Butterton (13) have analyzed the location of the stx gene in S. dysenteriae type 1 3818T and found genes with high sequence identity to genes of lambdoid phages that were close in proximity to stx. Compared with the gene order found in phage 933W, attL, int, and xis genes seem to be present in functional form, but the right side containing the head and tail genes is not present in this strain. Those authors suggested that recombination events between insertion sequences led to deletions and gene rearrangements. This investigation is in agreement with our study. However, in some isolates we did not find an IS600 element upstream of stx.
From our results, we conclude that a specific genetic order in the vicinity of stx genes is generally present in Stx phages. This could be necessary for controlled expression and release of the toxins together with infectious phage particles. This assumption is in agreement with results published by Neely and Friedman, who have shown the direct interaction between the antiterminator Q and the transcription of the Shiga toxins for phage H-19B (14, 15).
Regarding the region between the stx genes and the S gene, we found a typical mosaic structure, which is common for lambdoid, double-stranded DNA phages (6). Although phages share a similar morphology, a similar mode of replication, and a similar genomic structure they can be unrelated at the nucleotide level. It has been proposed that double-stranded tailed phages share common ancestries and that they undergo a profuse exchange of their functional genetic elements drawn from a large common genetic pool. This is also supported by our finding that the E. coli O111 strains analyzed contained sequences which originally stem from S. enterica serovar Typhimurium phage fels-1, thus providing evidence for exchange of genetic material between S. enterica serovar Typhimurium and E. coli.
The results of our study clearly show that in all analyzed Stx-producing bacteria the stx genes are linked with phage sequences, regardless of the serotype, geographical distribution, animal or human origin, stx genotype, or the species. We suggest that stx genes can be generally considered to be phage-borne. However, location of stx adjacent to phage genes does not necessarily mean that Stx is encoded by intact, functional phages, or that it can be transduced via release of maturated phage particles.
The analysis of the stx-flanking regions is a convenient method that gives us important information on the genetic background in which stx genes are located. However, to more precisely learn about the infection characteristics such as receptor proteins and integration sites, complete phage sequences need to be determined.
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ACKNOWLEDGMENTS |
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We thank Beatrix Henkel for excellent technical assistance. We further thank Michael McClelland, San Diego, Calif., for his interest and helpful discussions. We also thank Lisa Durso, Lincoln, Nebr., for critical reading of the manuscript.
This work was supported by grants from the Deutsche Forschungsgemeinschaft.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Mikrobiologie und Hygiene der Universität Würzburg, Josef-Schneider-Str.2, D-97080 Würzburg, Germany. Phone: 49-931-2013905. Fax: 49-931-2013445. 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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