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Journal of Bacteriology, March 1999, p. 1767-1778, Vol. 181, No. 6
Laboratory of Genetics, University of
Wisconsin, Madison, Wisconsin 53706
Received 23 July 1998/Accepted 6 January 1999
Lysogenic bacteriophages are major vehicles for the transfer of
genetic information between bacteria, including pathogenicity and/or virulence determinants. In the enteric pathogen
Escherichia coli O157:H7, which causes hemorrhagic colitis
and hemolytic-uremic syndrome, Shiga toxins 1 and 2 (Stx1 and Stx2) are
phage encoded. The sequence and analysis of the Stx2 phage 933W is
presented here. We find evidence that the toxin genes are part of a
late-phage transcript, suggesting that toxin production may be coupled
with, if not dependent upon, phage release during lytic growth.
Another phage gene, stk, encodes a product resembling
eukaryotic serine/threonine protein kinases. Based on its position in
the sequence, Stk may be produced by the prophage in the lysogenic
state, and, like the YpkA protein of Yersinia species, it
may interfere with the signal transduction pathway of the mammalian
host. Three novel tRNA genes present in the phage genome may serve to
increase the availability of rare tRNA species associated with
efficient expression of pathogenicity determinants: both the Shiga
toxin and serine/threonine kinase genes contain rare isoleucine and
arginine codons. 933W also has homology to lom,
encoding a member of a family of outer membrane proteins associated
with virulence by conferring the ability to survive in macrophages, and
bor, implicated in serum resistance.
The production of one or more forms
of Shiga toxin (Stx) is a defining characteristic of enterohemorrhagic
Escherichia coli (EHEC), along with the capacity to evoke
attaching-and-effacing intestinal lesions and the presence of a
characteristic large plasmid (50). These strains,
particularly E. coli serotype O157:H7, have emerged as
an important public health concern worldwide as the causative agents of
a severe bloody diarrheal syndrome, hemorrhagic colitis, and an acute
renal disease, hemolytic-uremic syndrome. E. coli
O157:H7 is the subject of a recent text (44), as well as a
novel (21) and a nonfiction first-hand account of HUS
(40). The potent cytotoxins produced by these bacteria are
similar or nearly identical to those produced by Shigella
dysenteriae (59, 69). Although the terms "Shiga-like
toxins (SLT)" and "verotoxins (VT)" are still encountered, the
term "Shiga toxin (Stx)" refers to the entire family of related
toxins (16, 47); the Stx family contains two subgroups, Stx1
and Stx2, which are distinguishable serologically.
In the EHEC strains, as well as many other Stx-producing E. coli (STEC) strains, the toxins are encoded by lysogenic
bacteriophages (70, 71, 90, 94). The EHEC O157:H7
strain EDL933 produces both Stx variants Stx1 and Stx2: Stx2 is
encoded by the temperate bacteriophage 933W, while Stx1 is thought to
be encoded by a cryptic prophage (70, 71). The isolation of
Stx1-encoding phages from this strain has been reported, but the
phage called 933J was apparently a contaminant (70);
other isolates, less well characterized, seem to be 933W variants
that have exchanged the Stx1 structural genes for the Stx2 genes,
perhaps via a rare recombination event (79).
We have sequenced the entire Stx2 toxin-converting phage 933W, and we
describe our initial analysis below. This project is part of an ongoing
effort to sequence the entire genome of the EHEC O157:H7 strain EDL933;
the sequences of the large virulence plasmid pO157 and the chromosomal
pathogenicity island LEE have also been completed and are described
elsewhere (15, 73).
Strains and media.
EHEC EDL933 was obtained from C. W. Kaspar (Food Microbiology & Toxicology, University of
Wisconsin Electron microscopy.
CsCl-banded phage in 10 mM
MgCl2 were adsorbed to Pioloform-coated 400-mesh copper
grids and negatively stained with 1% (wt/vol) ammonium molybdate.
Negatively stained samples were viewed on a Philips CM120 STEM
instrument at 60 kV.
Preparation and sequencing of nucleic acids.
Viral DNA was
isolated from CsCl-banded phage, sheared by nebulization
(53), and shotgun cloned into M13 Janus (14). The "933 lysate" shotgun was prepared from polyethylene
glycol-precipitated cleared culture fluid of an EDL933 overnight
growth. The majority of the sequencing was carried out with Sequenase
and 35S label; additional data was collected with Prism
fluorescent dye terminators on ABI373 and ABI377 automated fluorescence
sequencers. Further sequence data was collected from a whole-genome
shotgun of the original-source lysogen EDL933 as part of an ongoing
effort to sequence-scan the entire genome of this pathogen. Sequence covering two final ambiguous areas was collected by PCR amplification from EDL933 genomic DNA. Sequence data was assembled and edited as
described previously (22) to yield a circular duplex
sequence of 61,663 bp.
Sequence analysis.
Open reading frame (ORF) identification,
homology searches, and other analyses were carried out as
described for the E. coli K-12 genome (11).
While a number of database search and sequence alignment tools were
used in the analysis, the percent identity values reported here are
from the implementation of the Clustal method in MegAlign (DNASTAR)
(24); unless explicitly stated otherwise, sequence
comparisons are for amino acid sequences. For some comparisons, E
values from BLAST 2.0 (2) are also noted. tRNAs were
initially found by visual inspection of the sequence and verified with
tRNAscan-SE (52). Control sequences for the tRNA search
included E. coli K-12 (86 tRNAs and 2 "pseudo-tRNAs") (11), bacteriophage T4 (8 tRNAs)
(49), and the non-tRNA-containing bacteriophage sequences of
lambda (85) and Nucleotide sequence accession number.
The 933W sequence has
been deposited in GenBank (accession no. AF125520) as a 61,670-bp
linear prophage sequence delimited by copies of the 7-bp att
core sequence.
Morphology of the 933W virion.
We examined the phages
spontaneously released from EDL933, and confirmed the shape
and dimensions reported for 933W and other Stx-converting phages
from serotype O157:H7 or O157:H
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Sequence of Shiga Toxin 2 Phage 933W from
Escherichia coli O157:H7: Shiga Toxin as a Phage
Late-Gene Product
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Madison), who obtained it from the American Type Culture
Collection (ATCC 43895). Phage 933W was routinely prepared from
overnight cultures of EDL933 as spontaneously released phage, separated
from cells by centrifugation and filtration. The initial phage titers
were ~105 PFU ml
1, but the titers fell more
than 20-fold after overnight storage at 4°C despite supplementation
with 10 mM CaCl2, 10 mM MgCl2, and/or 0.1%
(wt/vol) gelatin. Attempts to propagate the phage on E. coli K-12 strains in liquid culture were unsuccessful, but plate
lysates were prepared on lawns of E. coli K-12. Phage
were purified by precipitation with polyethylene glycol and/or by
equilibrium centrifugation in CsCl density gradients, using standard
techniques (3). Modified Luria-Bertani agar and broth were
supplemented with 10 mM CaCl2 (71); phage titers
were determined by using E. coli K-12 strain LE392 or K802.
80 (74). The sequence coverage
density in Fig. 3 was calculated and plotted by using S-PLUS (MathSoft,
Inc., Seattle, Wash.).
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
strains (70, 79, 98).
As shown in Fig. 1, the phage have
regular hexagonal heads, about 70 nm wide. They have been variously
reported as having no tails, very short tails, or short contractile
tails, apparently depending on how the virions land on the grid. In
many of our images, the phage particles exhibit clumping by some sort of tail-tail interaction. In such cases, the tails were more readily discerned as short contractile tails, about 27 nm long and 13 nm wide.
There are indications of a baseplate-like structure as well, but no
details could be made out.
View larger version (157K):
[in a new window]
FIG. 1.
Transmission electron micrograph of bacteriophage
933W. Bar, 200 nm.
Sequence of the 933W genome. The toxin genes of 933W were previously sequenced and are the basis of several diagnostic sequence probes (13, 36, 45, 46, 75). A few other sequences from 933W have been previously reported as well (23, 88).
Although we expected a strong overall similarity between phage 933W and lambda (70), this was not the case for the majority of the genome. Nonetheless, despite a virion morphology quite distinct from both the "classic" lambda phages and the P22 family, 933W has similarities to lambdoid phages at both the sequence and gene organization levels. Examination of the sequence reveals a divergent arrangement of ORFs and other features reminiscent of bacteriophage lambda and its relatives. Similarities to several different lambdoid phages can be noted at both the DNA and protein levels, so that 933W can be described as a mosaic of different phages. Within this backbone of common phage elements, several known or potential pathogenicity determinants are inserted into the so-called dispensable, nonessential, or accessory regions. A map based on the sequence of 933W is presented in Fig. 2, and Table 1 lists the annotated genes. The various features of the sequence are described below.
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Identification of the prophage attachment site. Like coliphage lambda, many temperate bacteriophages integrate into their host genomes via a site-specific recombination event between short common-core sequences within the phage (attP) and bacterial (attB) attachment sites, generating two composite core sequences (attL and attR) flanking the integrated linear prophage genome. Excision of the prophage involves a similar site-specific recombination event between attL and attR, to generate a circular phage genome.
The location of the 933W prophage in EDL933 was determined by examination of the data from a whole-genome shotgun library of that strain and confirmed by PCR across the attL and attR junctions. The prophage sequence is flanked by two copies of a 7-bp repeat (GTTTCAA) present only once in E. coli K-12 and only once in the circular phage sequence, which we conclude is the core of the 933W att sites. Integration of 933W disrupts the wrbA gene, which encodes the Trp repressor-binding protein, WrbA. During stationary phase, E. coli K-12 cells deficient in WrbA are less efficient than wild-type cells in their ability to repress the trp promoter (99). It was proposed that the WrbA protein functions as an accessory element in blocking TrpR-specific transcriptional processes that might be physiologically disadvantageous in the stationary phase of the bacterial life cycle. Of course, it is not known what the physiological situation might be in the intestinal tract. In a 933W lysogen, translation starting from the wild-type wrbA initiator codon could yield only a 20-residue peptide, containing the first 18 amino acids of WrbA. At the other end of the prophage, a phage-encoded start codon overlapping the stop codon of the integrase (ATGA) might allow the synthesis of a 192-amino-acid product retaining most of the WrbA sequence, although the first 13 residues of the wild-type protein would be replaced by a different 7-amino-acid sequence. This sequence alteration would truncate a conserved domain noted in a "WrbA family" of proteins (domain 4535 of ProDom; 38 residues), but the impact, if any, upon function is unknown.Identification of the virion DNA endpoints. Since the majority of our sequence data for 933W was determined by using a shotgun library of DNA derived from phage particles, if the packaged phage DNA had specific endpoints (i.e., like bacteriophage lambda), we should have generated a unique linear sequence upon its assembly. Moreover, based on our experience with nebulized shotgun libraries derived from other linear DNAs, the actual endpoints of the source DNA would be expected to be proportionally overrepresented, presumably because the end repair of enzymatically generated ends is much more efficient than the repair of ends generated by physical shearing. Instead, assembly of the sequence data yielded a partial concatamer with no unique ends or pileups. The identification of the integrated prophage endpoints indicated that the assembled sequence was a circular permutation of the prophage sequence. The 61,670-bp sequence is presented here in the prophage state, starting and ending with the 7-bp att core.
The lack of definite endpoints in the assembly of virion DNA sequences might indicate the absence of a defined cos-type end (although cohesive ends might have annealed to generate circles or linear concatamers prior to nebulization). The full data set used to complete the sequence is anything but random, since specific data was collected in a directed manner to deal with gaps and ambiguities. To more specifically address the question of the virion DNA endpoints, all of the sequence data collected from the "933 lysate" shotgun librarywhich does represent a "random" data setwas aligned with
the consensus prophage sequence. As shown in Fig.
3, our data supports a conclusion that
the virion endpoints of this phage are not fixed in the genome but
instead are distributed over a region of several kilobase pairs. We
suggest that this is the result of a headful packaging of sequences
longer than one full genome, as demonstrated for bacteriophages P22, P1, and T1 (8).
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Descriptions of selected ORFs. Genes with functional assignments are named, usually after the equivalent lambda genes when possible. ORFs with no functional assignment or only tentative assignments based on gene arrangement and location are designated only by ORF numbers. Genes (ORFs and RNAs) are labelled L0061 to L0142, from left to right as shown in Fig. 2; these labels are from a series of unique identifiers for the genes of EDL933. References to genes from E. coli K-12 include the corresponding identifier labels (b numbers) assigned to those genes in the complete genome sequence of that strain (11).
In all cases for which data is available, integrative recombination is mediated by a phage-encoded recombinase (integrase, or Int protein) which catalyzes the strand cleavage and rejoining, and excision usually requires the cooperative action of Int and a second phage-encoded excision protein (Xis). We have designated L0061, the ORF following attL in the prophage, as int; sequence comparisons suggest that it is a member of the integrase-recombinase family, although very distant from lambda, P22, and other lambdoid phages. The closest homologs are the putative integrases of the E. coli K-12 cryptic prophages Rac (40.1% identical to the product of ORF b1345, IntR) and Qin (52.0% identical to the product of ORF b1579, IntQ). Despite the sequence diversity exhibited by known Int proteins, all of these recombinases can be aligned in their C-terminal halves to reveal a conserved region implicated as the active site of this family (1, 4). The proposed 933W Int shows some similarity (10.5 to 15.3% identity; BLAST E values, 2 × 103 to 3 × 1040) to a great number of other Int sequences, and
examination of the alignments reveals that with a single exception, it
contains the conserved residues, including the tyrosine residue
identified as the active amino acid involved in a transient
phosphodiester linkage to the DNA during strand cleavage and rejoining.
The exception, a tyrosine residue instead of a histidine in the highly
conserved His-X-X-Arg motif, is also present in the Rac and Qin
integrases. Given the observation that in the P1 Cre integrase this
precise substitution reduced the recombination activity about sevenfold in vivo (1), the question arises of how efficient these
putative integrases are. However, this substitution is also present in the integrase of the Streptomyces lividans SLP1 element
(12), while the putative integrase of the Pseudomonas
aeruginosa phage CTX contains an N (asparagine) residue instead
of the H (39).
By analogy to lambda and other temperate phages, the 933W
ORF L0062, immediately upstream of int, may encode the phage
excisionase. A similar sequence is found in the Rac prophage
(25.3% identical to YdaQ, b1346), which is known to be excisable, but
not in Qin, where an IS2 insertion near intQ
is accompanied or followed by a deletion of flanking
sequences. With a few exceptions, Xis sequences show little
homology to one another, and even such generalizations as the
lambdoid Xis proteins being basic while those from gram-positive bacteria are often acidic (58) are rife with exceptions. The suggestion has been made that temperate phage excisionases have a
helix-turn-helix motif, as scored by the metric of Dodd and Egan
(26) for recognizing such motifs (83). This
metric generates SD scores, which are standard deviation units relative
to the appropriate mean; scores 
2.5 SD are indicative of a
helix-turn-helix motif. While most of the lambdoid Xis proteins do not
score well with the program (25), the best-scoring regions
of 933W Xis (score of 0.37 SD at position 33) and YdaQ (score of 0.34
SD at position 19) do align with the motif pointed out by Salmi et al.
(83), suggesting that some helix-turn-helix character may be
involved in these proteins as well. However, in the absence of any
experimental data, the possibility must be noted that excision of 933W
(and perhaps Rac) does not require an excisionase, as such. Excision of
the Staphylococcus aureus phages 13 and 42 requires no
phage-encoded product other than the integrase (17), and for
coliphage 186 the transcriptional repressor protein Apl also serves
as the phage-encoded excision factor (78).
ORF L0069 is homologous (89.7% identity) to "Ehly2," the
product of an ORF associated with an enterohemolysin 2 activity encoded by phage C3208 in E. coli O26:H11 (6). Both
of these hypothetical proteins are also similar to lambda Ea22 (L0069,
35.2% identity; Ehly2, 30.2% identity) and P22 EaD (L0069, 24.9%
identity; Ehly2, 25.3% identity), whose genes occur in analogous
positions within their respective genomes. In the absence of any
demonstrated hemolysin activity by the Ehly2 protein itself, this
similarity is best viewed as an indication that phage C3208 is also a
member of the lambdoid family. If this protein does have a cytotoxic
effect, it may contribute to the virulence of O157:H7.
The 933W sequence spanning ORFs L0073 to L0078 is similar to the
recombination region of lambda (97% identity over 3,698 bp), with both
the gene order and predicted amino acid sequences of individual genes
highly conserved; these ORFs are therefore designated exo
(97.3% identity), bet (99.6% identity), gam
(97.0% identity), kil (98.9% identity), cIII
(98.1% identity), and ssb (99.2% identity).
ORF L0080 was initially identified as a candidate for an analog of the
lambda regulatory gene N largely on the basis of its position within the sequence (its product shows 14.0% identity to
lambda N; 29.0% identity to P22 gene 24 protein). It was subsequently found to be very similar (96.9% identity) to the N gene of
H19-B.
The predicted product of ORF L0082 resembles the family of eukaryotic
serine/threonine protein kinases (12.6 to 20.1% identity to more
than 100 distinct serine/threonine kinases from a variety of
organisms; BLAST E values, 2 × 104 to 3 × 1020), and we have designated the gene
stk (for "serine/threonine kinase"). The sequence
similarities span the conserved regions in the catalytic domain of the
eukaryotic protein kinases, including both the ATP binding and active
sites. The Stk sequence is more similar to eukaryotic serine/threonine
protein kinases (e.g., 17.2% identity to STE20 of Saccharomyces
cerevisiae) than to other prokaryotic protein kinases, including
those of Mycobacterium tuberculosis (10.7% identity),
Streptomyces coelicolor (11.3% identity), Myxococcus
xanthus (12.6% identity), Bacillus subtilis (12.7%
identity), and Yersinia pseudotuberculosis and Y. enterocolitica (11.0% identity).
In bacteriophages lambda and 80, the NinR regions contain orf-221,
which encodes a phosphoprotein phosphatase resembling those of
mammalian origin (20). The function of this phage-encoded activity is unknown, although it presumably acts to modulate the signal
transduction pathways of the E. coli host in a manner
similar to the E. coli PrpA and PrpB phosphatases,
described by Missiakas and Raina (60). While 933W apparently
encodes a protein kinase, the phage does not encode a homolog
of this phosphatase. There is some suggestion that the
Yersinia protein kinase (YpkA) is involved in virulence by
interfering with the signal transduction pathway of the mammalian host
(38), and bacteriophage 933W may interfere with the host
systems in the same manner. The location of stk is analogous
to the position of the rexAB genes in lambda, suggesting
that it could be expressed in the lysogen.
ORF L0085 encodes the CI repressor of 933W, which appears to be a
hybrid of two species of repressors. The amino-terminal 89 amino acids
most closely resemble the repressor of phage HK022 (37.4% similarity),
while the rest of the sequence is almost identical to that of H19-B at
both the nucleotide (95.2% identity) and amino acid (96.6% identity)
levels. In the well-characterized lambda repressor, the amino-terminal
residues contain the DNA binding helix-turn-helix motif that interacts
with the operator sequence, while the carboxy-terminal domain of the
protein is involved in dimerization. A similar "hybrid"
repressor was recently noted in a comparison of the lysogeny modules
from two temperate Streptococcus thermophilus
bacteriophages (66).
ORF L0086 is the 933W cro homolog and encodes a
protein most similar to that of HK022 (43.4% identity). This is
consistent with the cI structure described above, given that
both CI and Cro must recognize the same operator sequences. ORF L0087
is the cII homolog, encoding a protein similar to
those of HK022 (91.9% identity) and H19-B (98.0% identity).
Confirming earlier hybridization and partial-sequence data
(23), the replication origin and replication genes of 933W
are nearly identical to those of lambda (94.0% nucleotide
sequence identity), as well as to H19-B (96.7% nucleotide sequence
identity). Sequence similarities allow assignment of L0088 and
L0089 as the replication genes O (98.0% identity to lambda;
98.7% identity to H19-B) and P (96.6% identity to lambda;
95.7% identity to H19-B), respectively, and L0090 as ren
(99.0% identity to lambda; 97.9% identity to H19-B). The replication
origins of 933W and H19-B have a 39-bp insert relative to that of
lambda, containing two additional iterons. This insert results in an
in-frame insertion of 13 amino acids in the replication protein O of
both Stx phages. An in vitro readthrough of the UAG termination
codon of the O gene has been found in bacteriophage
lambda (100); the conservation of the O sequences
includes this extended carboxy-terminal region.
The 933W sequence to the right of the replication origin contains
homologs of several lambda and P22 Nin region ORFs: L0093 (39.0% identity to the product of lambda orf-146; 39.3% identity to
P22 NinB), L0097 (91.6% identity to the product of lambda orf-204; 88.1% identity to P22 NinG), and L0098 (73.8% identity to the product
of lambda orf-68; 66.2% identity to P22 NinH), as well as HK022 Roi
(L0096, 78.9% identity). These ORFs occupy analogous positions in the
different phage genomes, and, except for L0093, there are also
homologs in H19-B.
ORF L0094 encodes a protein similar to a hypothetical
methylase of bacteriophage HP1 (30.1% identity) and the DNA
(N6-adenine) methyltransferase of bacteriophage T1
(22.3% identity). Although no functional characterization of this
protein is available, such a DNA modification might explain the
difficulties we experienced when attempting to digest 933W DNA with a
number of restriction enzymes.
ORF L0099 was identified as the homolog of the late regulatory
gene Q, most closely resembling the functional analog of
lambda Q from phage DLP12 (b0551, YbcQ; 77.2% identity); it
was subsequently found to be almost identical (96.5% identity) to the
Q gene product of H19-B.
The Stx2 subunits are encoded by stx2A and stx2B
(aka sltIIA and sltIIB) and were previously
characterized (43, 87, 89).
Downstream of the stx genes and seemingly part of the same
transcript, ORF L0105 encodes a protein similar (50.8% identity) to
E. coli K-12 YjhS (b4309). Examination of the H19-B
sequence reveals an unannotated 849-bp ORF downstream from the
stx1 genes (accession no. AF034975, bases 14651 to
15499), whose product is also similar to YjhS (20.5% identity). The
function of these genes is unknown, but yjhS is part of a
fimbrial synthesis and iron transport region which K-12 may have
acquired by horizontal transfer.
ORF L0107 is the 933W analog of the lysis (holin) gene S,
resembling those from the Qin prophage (79.2% identity) and H19-B (91.3% identity). Like a number of other lambdoid holin genes (9), this gene has two Met start codons separated by one
or two codons. However, in 933W neither of the codons between
the alternate starts is an arginine or lysine codon, and it is not clear whether a dual-start motif is involved in the regulation of 933W lysis.
L0108 is the R gene (endolysin) analog, also resembling that
of Qin (88.8% identity); although no R gene is annotated in
the H19-B sequence, the insertion of 4 bases near the end of that sequence (C between bases 16927 and 16928; AA between 17254 and 17255, and G between 17303 and 17304) would create an
R homolog running off the end of the entry, 91.4%
identical to L0108.
In other lambdoid phages, S and R are followed by
Rz, and 933W ORF L0110 is an Rz
homolog most like that from lambda (71.2% identity); a
homolog of the overlapping Rz1 reading frame
(48) is also present. 933W has an additional ORF,
L0109, inserted between R and Rz, whose product
resembles the P22 Ant antirepressor (34.2% identity). The function of
this protein in 933W is unknown, although its location suggests a
possible regulatory role in lysis of the host cell.
The end of the lysis region of 933W is similar to that of lambda
(91.0% identity over 940 bp). In addition to Rz (and
Rz1), this region contains a homolog (L0111; 96.9%
identity) of the lambda bor gene. In lambda lysogens,
bor expression has been implicated in serum resistance
(5), which may confer a selective advantage to cells
carrying the prophage.
Analogy to other phages was useful in the analysis of the first
"half" of the 933W genome. However, if the ORFs in the
remainder of this genome encode the virion structural and
morphogenic proteins, as continued analogies would argue, very
few can be even tentatively identified on the basis of
sequence comparisons. A number of complete or partial phage
sequences have been determined, and comparisons reveal
homologies between different phages for genes encoding enzymes, regulatory proteins, replication proteins, and various "accessory" products. However, the genes encoding the actual
structural proteins that comprise the virion seem to be drawn
from a much larger pool of potential sequencesas if the possible ways
to build morphologically similar phage particles are myriad and our sampling has merely skimmed the surface of the gene pool.
Based on the 933W virion DNA endpoint analysis, one might
expect the region starting with ORF L0112 to contain the genes involved in DNA packaging. Although the analogous genes from a number of phages
show little sequence homology, there is a conservation of gene
position and relative size (8, 29, 92). We have tentatively
assigned L0112 to L0114 as follows, with "informative" database
matches indicated: L0112, the terminase small subunit (bacteriophage P1
PacA, BLAST E value 0.78); L0113, the terminase large subunit
(bacteriophage T4 gp17; BLAST E value 0.005); and L0114, the prohead
portal protein (bacteriophage P22 gp1; BLAST E value 0.002). The
sequence matches have very poor scores, and these assignments must be
thought of as provisional.
One of the few structural-gene candidates that can be identified is
L0121, a putative tail fiber gene; its product shows 17.0% identity to
the Stf tail fiber of lambda and contains motifs described in various
phage tail fiber proteins (84). This ORF is also one of
several phage genes encoding structural components of bacteriophage virions in which the characteristic collagen-like repeats
(Gly-X-Y)n have been noted (91). The
predicted 933W tail fiber protein displays extensive homologies
to collagen (e.g., 26.8% identity to human alpha type I collagen),
with stretches of 40 and 38 repeats of the collagen motif. The repeats
have the bias toward proline at the second and third positions of the
motif that has long been known to occur in vertebrate collagens.
Furthermore, the collagen sequences in 933W tail fibers may well have
the triple-helix structure found in animal collagen: all
well-characterized phage tail fiber proteins are trimeric
(18), including the phage 
fibers that have sequence
similarity to the 933W protein. We note that the position of L0121 in
the genome of 933W is not analogous to that of the lambda
stf gene, but, given the difference in tail morphology between these phages, analysis by analogy is probably at its weakest for tail structural and morphogenic genes.
In addition to the bor homolog in the lysis region,
two other 933W ORFs may be involved in virulence. L0128 encodes a
homolog (28.6% identity) of the lambda Lom protein (77,
82), which encodes a member of a family of outer membrane
proteins associated with virulence in two species. Expressed
in lysogens, these proteins confer the ability to survive in macrophages.
L0137 encodes a member of the hok-gef-relF family of killer
proteins from "toxin-antitoxin" systems (90.4% identity to RelF; 78.4% identity to Gef). In these systems, the best characterized of
which are involved in plasmid maintenance, an unstable antisense RNA
prevents expression of the lethal protein by binding to the more stable
mRNA (33, 76). Examination of the sequence surrounding L0137
reveals that all of the sequence elements and potential secondary-structure features described for these systems (30, 35) are conserved in 933W. By analogy to the Hok system, L0137 is
designated hokW (for "host killing, 933W") and the
antisense RNA is designated sokW (for "suppression of
killing, 933W"). If these genes are expressed in the lysogenic state,
loss of the prophage would be selected against in a manner similar to
the selection against loss of plasmids. Interestingly, in the O157:H7 strain EDL933, at least four such systems are present: in addition to
phage 933W, the large virulence plasmid pO157 carries a Hok system
homolog (15), and data from the genomic sequence
indicates that Gef and RelF homologs are also present
(10). The target specificity of these systems resides in the
interactions between the antisense RNA and the mRNA, and for the
plasmid and phage sequences these seem to be different. Whether the
chromosomal loci interact with either of those systems to reinforce the
maintenance of the various pathogenicity determinants is unknown.
tRNAs. In the region between the late regulatory gene Q and the Shiga toxin genes, a tRNAIle-like sequence was previously found (88). Our reexamination of the sequence reveals genes for two additional tRNA sequences, and the proposed cloverleaf secondary structures of all three tRNAs are shown in Fig. 4. Most invariant or semi-invariant eubacterial tRNA residues are present in these sequences, and they may encode functional tRNAs although this has not been demonstrated.
|
although the E. coli
tRNACys has G15 · G48 (55), which is an
identity element for this tRNA (56).
tRNA3 (argO) has the anticodon UCU; with an unmodified U
as the first nucleotide of the anticodon, this could correspond to the codons AGA (Arg), AGG (Arg), AGU (Ser), and possibly AGC (Ser). As with tRNA2, this sequence does not closely resemble any tRNA sequences in the sequence databases. However, the tRNAArg
species encoded by bacteriophage T4, E. coli argU
(dnaY), and Salmonella argU (fimU) all
have UCU anticodons, and these species favor the arginine codon
AGA due to modification of U34 to 5-methoxycarbonylmethyluridine (mcm5U) (93). The argO sequence
contains the A20 and C35 determinants for E. coli
tRNAArg; A73 is replaced G73, but this has been observed in
some tRNAArg species (57).
It was suggested (88) that the tRNAIle sequence
was related to the integration site of the 933W prophage into the
E. coli chromosome, since a number of other
bacteriophages and pathogenicity-associated islands are inserted at or
near tRNA genes (19, 37). This is not the case for 933W,
although the similar E. coli K-12 chromosomal ileY locus is the insertion target of coliphage 186 (78). However, the proximity of the tRNA genes to the
stx genes might reflect the outcome of another recombination
event during which these sequences were initially acquired by the
phage. If this is the case, are the tRNAs functional or are they just
along for the ride? Examination of codon usage (Table
2) suggests that the phage-encoded tRNAs
could serve to supplement the host tRNA pool, allowing the rare
codons to be more efficiently decoded (88). This may
provide sufficient selective advantage to retain the tRNAs, regardless
of their origin. There are no double "killer arginines" (102,
103) in the 933W genome, and so it seems unlikely that the
phage makes any explicit regulatory use of differential tRNA
availability. However, the alteration of tRNA base modifications has been reported to affect virulence factor expression in
Shigella flexneri (27, 28) and
Agrobacterium tumefaciens (34), and this may
actually be the result of an alteration in the efficient translation of
key proteins.
|
Promoters, operators, and terminators. A number of other noncoding sequence elements were examined in the 933W sequence, especially where homologs in better-characterized phages allowed "analysis by analogy." These features are annotated in the GenBank entry, and a few are briefly noted here.
The 933W N-L0081 intergenic region should contain tM, nutL, OL, and pL. A candidate for tM is present immediately downstream (to the left) of L0081, in a short sequence of dyad symmetry conserved between 933W and H19-B; no assignment could be made for OL or pL. Since the H19-B and 933W N genes are almost identical and nut sites are N specific, the nut sites should be within the sequences conserved between the two phages; a nutL candidate was identified, although it is not clear which of two "boxB"-like sequences would be involved. The cI-cro intergenic region should include OR, pRM, and pR. Given the similarity of the amino-terminal domains of the 933W and HK022 repressors, sequence comparisons with the equivalent region from HK022 (68) allowed us to tentatively identify all three features in 933W. The ready identification of OR makes the failure to detect OL all the more puzzling, and it may be that regulation of transcription of the left and right arms of 933W are achieved by entirely unrelated means. The cro-cII intergenic region should include nutR, pRE (pE), and tR1. This region is 100% conserved between H19-B and 933W. pRE, by analogy to other lambdoid phages, would be activated by 933W (and H19-B) CII; however, the recognition site for this protein is not known, and no candidates are proposed. A nutR candidate was found, and, as was the case for nutL, there are two candidate "boxB" sequences; perhaps in 933W and H19-B the N recognition determinants involve an extended sequence relative to those in other lambdoid systems (31, 32, 86). The near identity between the 933W and H19-B Q genes extends 293 bp beyond the coding sequences (i.e., ending upstream of the tRNA genes), defining the region where the pR' promoter and qut site should be located. The A(N)3T(S)2-3 motif in the nontranscribed DNA strand, noted by Ring and Roberts (80), can be found in several locations downstream of the presumptive pR' promoter, but only one of these occurs in close proximity to the promoter and before the first potential terminator. We propose that this is part of the qut site for both 933W and H19-B. Once Q protein modifies the RNA polymerase complex at qut, sequence divergence should not alter its antitermination activity for the late transcripts; therefore, the late regions of both phages are likely to be under Q regulation. In E. coli, tRNA genes are often found in clusters with typical prokaryotic35 and 10 promoter elements as well as a
GC-rich discriminator domain common to all E. coli
genes subject to stringent control, and downstream of almost all tRNA
genes is found a rho-independent terminator-like structure
(42). Both of these features are found flanking the 933W
ilvZ-argN-argO cluster, and the predicted transcript would
be a trimeric precursor RNA resembling those readily processed by the
E. coli RNA processing machinery.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The Q gene product of lambdoid phages functions as a transcription antiterminator that regulates the expression of late phage genes by modifying the transcription complex initiated at the late promoter pR'. The protein acts at the qut site overlapping the promoter, and the Q analogs of different phages are specific for their own qut sites (81). Based on the arrangement of the 933W genes, the stx2 genes are part of an apparent Q-dependent late transcript, as diagrammed in Fig. 5. If this is the case, the toxin would be expressed only (or at least maximally) during lytic growth of the phage.
|
Mühldorfer et al. (61) examined the regulation of the stx2 operon in experiments involving a low-copy-number plasmid carrying a translational fusion of stx2A to a phoA reporter gene. They concluded that a phage factor played a positive regulatory role in the expression of stx2 and that this factor could be provided in trans by either 933W or H19-B but not lambda. The increased expression was mitomycin C and recA+ dependent, as expected for a mechanism requiring prophage induction. These results are entirely consistent with the phage factor being the Q gene: when provided in trans by the phage after induction and transition to lytic growth, Q could act to antiterminate pR' transcripts on the reporter plasmid (the constructs included the entire Q-stx2A intergenic region). The similarity of the 933W and H19-B genes is such that we would expect the H19-B Q gene to function as well as that from 933W in this system.
The results of our analyses seem to be at odds with the report by Sung et al. (95) that a promoter for stx2 was located only 118 bp upstream of the stx2A coding sequence, which would put the promoter (pSlt-II or pStx2) within argO. It may be that some constitutive level of Stx2 expression is provided by that promoter but that Stx2 production is significantly increased after phage induction as a more efficient promoter becomes available.
A number of pathogenicity factors, including several toxins, are
encoded by lysogenic phages (7, 19). A linking of toxin production to prophage induction in such cases might open another means
of increasing the toxin yield. Infections are unlikely to occur in
monoculture, and while other bacteria already carrying the prophage
(uninduced) would be immune to superinfection, nonlysogens in the
vicinity could be infected and produce additional phage (and toxin) in
what can be envisioned as an amplification by recruitment. It has been
shown that the cholera toxin-encoding phage CTX infects Vibrio cholerae more efficiently within the
gastrointestinal tracts of mice than under laboratory conditions
(96). A coupling of toxin release with phage release
might also favor DNA transfer events, including the acquisition of new
pathogenicity determinants, by other bacteria under conditions where
the bacteria would be more likely to be subsequently released into the environment.
Bacteriophage H-19B, isolated from E. coli O26:H11 strain H19 (90), is morphologically quite distinct from 933W and more closely resembles lambda (98). This phage also displays greater sequence homologies to lambda (as detected by hybridization), and the virion DNA has cohesive termini (41). Nonetheless, both 933W and H-19B have stx genes in analogous positions and have similar regulatory elements. The cryptic Stx1 phage in EDL933 is still essentially uncharacterized, and there remains a possibility that it resembles H-19B. If the Stx prophages in the EDL933 genome have overlapping regulatory specificities, the coexistence of both elements could present an interesting additional layer of complexity.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by NIH grant AI41329-01 and by a research grant from the Ronald McDonald House Charities.
We thank the technical staff of the University of Wisconsin Genomes Project for help with sequencing, Randall Massey and Grayson Scott of the University of Wisconsin Medical School Electron Microscope Facility for electron microscopy, and Bill McClain for useful discussions about the tRNAs. For bearing with his return to the bench after too many years at the computer, one of us (G.P.) also thanks Heather Kirkpatrick of this laboratory.
| |
ADDENDUM |
|---|
Sequence data from the Stx1-encoding lambdoid bacteriophage H19-B (64) was compared to our sequence in the course of our analyses, and similarities are noted above. While this paper was being revised, Neely and Friedman (65) published their analysis of this H-19B sequence. They reached conclusions similar to our own regarding the regulation of Stx production and phage release and showed that the H-19B Q gene product can activate the expression of 933W stx2 genes as well as its own stx1 genes.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Laboratory of Genetics, University of Wisconsin, 445 Henry Mall, Madison, WI 53706. Phone: (608) 262-2534. Fax: (608) 263-7459. E-mail: ecoli{at}genetics.wisc.edu.
Paper 3519 from the Laboratory of Genetics.
Present address: Department of Plant & Microbial Biology,
University of California at Berkeley, Berkeley, CA 94720-3102.
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