sequence of shiga toxin 2 phage 933w from …jb.asm.org/content/181/6/1767.full.pdfsequence of shiga...

JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Mar. 1999, p. 1767–1778 Vol. 181, No. 6

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Sequence of Shiga Toxin 2 Phage 933W from Escherichia coliO157:H7: Shiga Toxin as a Phage Late-Gene Product†

GUY PLUNKETT III,* DEBRA J. ROSE, TIMOTHY J. DURFEE,‡ AND FREDERICK R. BLATTNER

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, in-cluding pathogenicity and/or virulence determinants. In the enteric pathogen Escherichia coli O157:H7, whichcauses hemorrhagic colitis and hemolytic-uremic syndrome, Shiga toxins 1 and 2 (Stx1 and Stx2) are phageencoded. The sequence and analysis of the Stx2 phage 933W is presented here. We find evidence that the toxingenes are part of a late-phage transcript, suggesting that toxin production may be coupled with, if not depen-dent upon, phage release during lytic growth. Another phage gene, stk, encodes a product resembling eukaryoticserine/threonine protein kinases. Based on its position in the sequence, Stk may be produced by the prophagein the lysogenic state, and, like the YpkA protein of Yersinia species, it may interfere with the signal transduc-tion pathway of the mammalian host. Three novel tRNA genes present in the phage genome may serve toincrease the availability of rare tRNA species associated with efficient expression of pathogenicity determi-nants: 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 withvirulence 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) isa defining characteristic of enterohemorrhagic Escherichia coli(EHEC), along with the capacity to evoke attaching-and-effac-ing intestinal lesions and the presence of a characteristic largeplasmid (50). These strains, particularly E. coli serotype O157:H7, have emerged as an important public health concernworldwide as the causative agents of a severe bloody diarrhealsyndrome, hemorrhagic colitis, and an acute renal disease, he-molytic-uremic syndrome. E. coli O157:H7 is the subject of arecent text (44), as well as a novel (21) and a nonfiction first-hand account of HUS (40). The potent cytotoxins produced bythese bacteria are similar or nearly identical to those producedby Shigella dysenteriae (59, 69). Although the terms “Shiga-liketoxins (SLT)” and “verotoxins (VT)” are still encountered, theterm “Shiga toxin (Stx)” refers to the entire family of relatedtoxins (16, 47); the Stx family contains two subgroups, Stx1 andStx2, which are distinguishable serologically.

In the EHEC strains, as well as many other Stx-producingE. coli (STEC) strains, the toxins are encoded by lysogenic bac-teriophages (70, 71, 90, 94). The EHEC O157:H7 strain EDL933produces both Stx variants Stx1 and Stx2: Stx2 is encoded bythe temperate bacteriophage 933W, while Stx1 is thought to beencoded by a cryptic prophage (70, 71). The isolation of Stx1-encoding phages from this strain has been reported, but thephage called 933J was apparently a contaminant (70); otherisolates, less well characterized, seem to be 933W variants thathave exchanged the Stx1 structural genes for the Stx2 genes,perhaps via a rare recombination event (79).

We have sequenced the entire Stx2 toxin-converting phage933W, and we describe our initial analysis below. This projectis part of an ongoing effort to sequence the entire genome ofthe EHEC O157:H7 strain EDL933; the sequences of the large

virulence plasmid pO157 and the chromosomal pathogenicityisland LEE have also been completed and are described else-where (15, 73).

MATERIALS AND METHODS

Strains and media. EHEC EDL933 was obtained from C. W. Kaspar (FoodMicrobiology & Toxicology, University of Wisconsin—Madison), who obtainedit from the American Type Culture Collection (ATCC 43895). Phage 933W wasroutinely prepared from overnight cultures of EDL933 as spontaneously releasedphage, separated from cells by centrifugation and filtration. The initial phagetiters were ;105 PFU ml21, but the titers fell more than 20-fold after overnightstorage 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 strainsin liquid culture were unsuccessful, but plate lysates were prepared on lawns ofE. coli K-12. Phage were purified by precipitation with polyethylene glycol and/orby equilibrium centrifugation in CsCl density gradients, using standard tech-niques (3). Modified Luria-Bertani agar and broth were supplemented with 10mM CaCl2 (71); phage titers were determined by using E. coli K-12 strain LE392or K802.

Electron microscopy. CsCl-banded phage in 10 mM MgCl2 were adsorbed toPioloform-coated 400-mesh copper grids and negatively stained with 1% (wt/vol)ammonium molybdate. Negatively stained samples were viewed on a PhilipsCM120 STEM instrument at 60 kV.

Preparation and sequencing of nucleic acids. Viral DNA was isolated fromCsCl-banded phage, sheared by nebulization (53), and shotgun cloned into M13Janus (14). The “933 lysate” shotgun was prepared from polyethylene glycol-precipitated cleared culture fluid of an EDL933 overnight growth. The majorityof the sequencing was carried out with Sequenase and 35S label; additional datawas collected with Prism fluorescent dye terminators on ABI373 and ABI377automated fluorescence sequencers. Further sequence data was collected from awhole-genome shotgun of the original-source lysogen EDL933 as part of anongoing effort to sequence-scan the entire genome of this pathogen. Sequencecovering two final ambiguous areas was collected by PCR amplification fromEDL933 genomic DNA. Sequence data was assembled and edited as describedpreviously (22) to yield a circular duplex sequence of 61,663 bp.

Sequence analysis. Open reading frame (ORF) identification, homologysearches, and other analyses were carried out as described for the E. coli K-12genome (11). While a number of database search and sequence alignment toolswere used in the analysis, the percent identity values reported here are from theimplementation of the Clustal method in MegAlign (DNASTAR) (24); unlessexplicitly stated otherwise, sequence comparisons are for amino acid sequences.For some comparisons, E values from BLAST 2.0 (2) are also noted. tRNAswere initially found by visual inspection of the sequence and verified withtRNAscan-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), andthe non-tRNA-containing bacteriophage sequences of lambda (85) and f80 (74).

* 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: [email protected].

† Paper 3519 from the Laboratory of Genetics.‡ Present address: Department of Plant & Microbial Biology, Uni-

versity of California at Berkeley, Berkeley, CA 94720-3102.

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The sequence coverage density in Fig. 3 was calculated and plotted by usingS-PLUS (MathSoft, Inc., Seattle, Wash.).

Nucleotide sequence accession number. The 933W sequence has been depos-ited in GenBank (accession no. AF125520) as a 61,670-bp linear prophagesequence delimited by copies of the 7-bp att core sequence.

RESULTS

Morphology of the 933W virion. We examined the phagesspontaneously released from EDL933, and confirmed the shapeand dimensions reported for 933W and other Stx-convertingphages from serotype O157:H7 or O157:H2 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 havingno tails, very short tails, or short contractile tails, apparentlydepending on how the virions land on the grid. In many of ourimages, the phage particles exhibit clumping by some sort oftail-tail interaction. In such cases, the tails were more readilydiscerned as short contractile tails, about 27 nm long and 13nm wide. There are indications of a baseplate-like structure aswell, but no details could be made out.

Sequence of the 933W genome. The toxin genes of 933W werepreviously sequenced and are the basis of several diagnosticsequence probes (13, 36, 45, 46, 75). A few other sequencesfrom 933W have been previously reported as well (23, 88).

Although we expected a strong overall similarity betweenphage 933W and lambda (70), this was not the case for themajority of the genome. Nonetheless, despite a virion mor-phology quite distinct from both the “classic” lambda phagesand the P22 family, 933W has similarities to lambdoid phagesat both the sequence and gene organization levels. Examina-tion of the sequence reveals a divergent arrangement of ORFsand other features reminiscent of bacteriophage lambda andits relatives. Similarities to several different lambdoid phagescan be noted at both the DNA and protein levels, so that 933W

can be described as a mosaic of different phages. Within thisbackbone of common phage elements, several known or poten-tial pathogenicity determinants are inserted into the so-calleddispensable, nonessential, or accessory regions. A map basedon the sequence of 933W is presented in Fig. 2, and Table 1lists the annotated genes. The various features of the se-quence are described below.

Identification of the prophage attachment site. Like coli-phage lambda, many temperate bacteriophages integrate intotheir host genomes via a site-specific recombination event be-tween short common-core sequences within the phage (attP)and bacterial (attB) attachment sites, generating two compos-ite core sequences (attL and attR) flanking the integrated lin-ear prophage genome. Excision of the prophage involves a sim-ilar site-specific recombination event between attL and attR, togenerate a circular phage genome.

The location of the 933W prophage in EDL933 was deter-mined by examination of the data from a whole-genome shot-gun library of that strain and confirmed by PCR across the attLand attR junctions. The prophage sequence is flanked by twocopies of a 7-bp repeat (GTTTCAA) present only once in E. coliK-12 and only once in the circular phage sequence, which weconclude is the core of the 933W att sites. Integration of 933Wdisrupts the wrbA gene, which encodes the Trp repressor-binding protein, WrbA. During stationary phase, E. coli K-12cells deficient in WrbA are less efficient than wild-type cells intheir ability to repress the trp promoter (99). It was proposedthat the WrbA protein functions as an accessory element inblocking TrpR-specific transcriptional processes that might bephysiologically disadvantageous in the stationary phase of thebacterial life cycle. Of course, it is not known what the physi-ological situation might be in the intestinal tract. In a 933Wlysogen, translation starting from the wild-type wrbA initiatorcodon could yield only a 20-residue peptide, containing thefirst 18 amino acids of WrbA. At the other end of the pro-phage, a phage-encoded start codon overlapping the stop co-don of the integrase (ATGA) might allow the synthesis of a192-amino-acid product retaining most of the WrbA sequence,although the first 13 residues of the wild-type protein would bereplaced by a different 7-amino-acid sequence. This sequencealteration would truncate a conserved domain noted in a“WrbA family” of proteins (domain 4535 of ProDom; 38 res-idues), but the impact, if any, upon function is unknown.

Identification of the virion DNA endpoints. Since the ma-jority of our sequence data for 933W was determined by usinga shotgun library of DNA derived from phage particles, if thepackaged phage DNA had specific endpoints (i.e., like bacte-riophage lambda), we should have generated a unique linearsequence upon its assembly. Moreover, based on our experi-ence with nebulized shotgun libraries derived from other linearDNAs, the actual endpoints of the source DNA would be ex-pected to be proportionally overrepresented, presumably be-cause the end repair of enzymatically generated ends is muchmore efficient than the repair of ends generated by physicalshearing. Instead, assembly of the sequence data yielded a par-tial concatamer with no unique ends or pileups. The identifi-cation of the integrated prophage endpoints indicated that theassembled sequence was a circular permutation of the pro-phage sequence. The 61,670-bp sequence is presented here inthe prophage state, starting and ending with the 7-bp att core.

The lack of definite endpoints in the assembly of virion DNAsequences might indicate the absence of a defined cos-type end(although cohesive ends might have annealed to generate cir-cles or linear concatamers prior to nebulization). The full dataset used to complete the sequence is anything but random,since specific data was collected in a directed manner to deal

FIG. 1. Transmission electron micrograph of bacteriophage 933W. Bar,200 nm.

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VOL. 181, 1999 SEQUENCE OF E. COLI O157:H7 Stx PHAGE 933W 1769


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with gaps and ambiguities. To more specifically address thequestion of the virion DNA endpoints, all of the sequence datacollected from the “933 lysate” shotgun library—which doesrepresent a “random” data set—was aligned with the consen-sus prophage sequence. As shown in Fig. 3, our data supportsa conclusion that the virion endpoints of this phage are notfixed in the genome but instead are distributed over a region ofseveral kilobase pairs. We suggest that this is the result of aheadful packaging of sequences longer than one full genome,as demonstrated for bacteriophages P22, P1, and T1 (8).

Earlier estimates of the 933W genome size, actually esti-mates of the virion DNA length, were longer than the se-quence we have determined (70, 98). These estimates werebased on the lengths of restriction fragments, which might alsoshed light on the unique-versus-headful-packaging question. Inour experience, 933W virion DNA was recalcitrant to restric-tion digestion, but complete digestion with EcoRI or BamHIcould be achieved with prolonged incubation; the positions ofthe EcoRI and BamHI sites in the sequence are shown in Fig.3. The restriction fragments (data not shown) are entirelyconsistent with a circular form of the completed sequence andare indistinguishable from those reported previously. The dis-crepancies in calculated lengths can be accounted for bythe inherent margin of error in determining fragment lengthsbased on electrophoretic mobility; for example, the reportedBamHI fragments of .23, 6.3, 3.05, 0.88, and 0.39 kbp (98) canbe correlated with sequence-derived lengths of 29,558 and21,247, 6,161, 3,031, 890, and 389 and 387 bp. These resultssuggest either a circular virion DNA or a collection of circu-larly permuted linear DNAs where submolar or minority frag-ments from individual molecules (with one end generated bypackaging instead of a restriction cut) do not appear as bands.

Descriptions of selected ORFs. Genes with functional assign-ments are named, usually after the equivalent lambda geneswhen possible. ORFs with no functional assignment or onlytentative assignments based on gene arrangement and locationare designated only by ORF numbers. Genes (ORFs andRNAs) are labelled L0061 to L0142, from left to right as shownin Fig. 2; these labels are from a series of unique identifiers forthe genes of EDL933. References to genes from E. coli K-12include the corresponding identifier labels (b numbers) as-signed to those genes in the complete genome sequence of thatstrain (11).

TABLE 1. Genes (ORFs and tRNAs) of bacteriophage 933W

Label GeneStartposi-tion

Stopposi-tion

Orien-tation

Length ofproduct(aminoacids)

Comments

L0061 int 1363 29 , 444 IntegraseL0062 xis 1691 1392 , 99 Excisionase?L0063 2073 1762 , 103L0064 2498 2133 , 121L0065 3033 2410 , 207L0066 3324 3037 , 95L0067 3544 3326 , 72L0068 3833 3546 , 95L0069 4876 4103 , 257 Ehly homolog (l Ea22)L0070 5474 5193 , 93 l orf-61 homologL0071 5676 5485 , 63 l orf-63 homologL0072 5837 5649 , 62 l orf-60a homologL0073 exo 6508 5831 , 225L0074 bet 7290 6505 , 261L0075 gam 7592 7296 , 98L0076 kil 7937 7668 , 89L0077 cIII 7944 7780 , 54L0078 ssb 8385 8017 , 122L0079 9006 8536 , 156L0080 N 9448 9065 , 127L0081 10101 9937 , 54L0082 stk 11150 10104 , 348L0083 11605 11144 , 153L0084 12014 11673 , 113L0085 cI 12782 12075 , 235L0086 cro 12861 13088 . 75L0087 cII 13227 13523 . 98L0088 O 13556 14494 . 312L0089 P 14491 15192 . 233L0090 ren 15189 15479 . 96L0091 15550 15828 . 92L0092 15997 16356 . 119L0093 16380 16826 . 148 l nin orf-146 homologL0094 16823 17350 . 175 DNA methylase?L0095 17532 18581 . 349L0096 roi 18727 19455 . 242L0097 19455 20060 . 201 l nin orf-204 homologL0098 20057 20251 . 64 l nin orf-68 homologL0099 Q 20205 20678 . 157L0100 ileZ 21120 21195 . Proposed tRNA-IleL0101 argN 21205 21281 . Proposed tRNA-ArgL0102 argO 21295 21371 . Proposed tRNA-ArgL0103 stxA2 21462 22421 . 319 Shiga toxin 2 subunit

A; also sltIIAL0104 stxB2 22433 22702 . 89 Shiga toxin 2 subunit

B; also sltIIBL0105 23189 25126 . 645 E. coli yjhS homologL0106 25307 25753 . 148L0107 S 25830 26045 . 71L0108 R 26050 26583 . 177L0109 ant 26854 27423 . 189L0110 Rz 27577 28041 . 154

Rz1 27797 27982 . 61 Homolog of l ORFoverlapping Rz

L0111 bor 28366 28073 , 97L0112 28775 29581 . 268 Terminase, small

subunit?L0113 29562 31268 . 568 Terminase, large

subunit?L0114 31268 33412 . 714 Portal protein?L0115 33570 34577 . 335L0116 34601 35815 . 404L0117 35871 36260 . 129L0118 36283 36771 . 162L0119 36647 37318 . 223L0120 37318 37968 . 216L0121 37965 39902 . 645 Tail fiberL0122 39784 40173 . 129L0123 40220 40501 . 93L0124 40718 42421 . 567L0125 42418 43686 . 422L0126 43752 43979 . 75

Continued

TABLE 1—Continued

Label GeneStartposi-tion

Stopposi-tion

Orien-tation

Length ofproduct(aminoacids)

Comments

L0127 43985 44602 . 205L0128 lom 44693 45427 . 244L0129 45857 46258 . 133L0130 46352 47008 . 218L0131 47011 47457 . 148L0132 47467 47718 . 83L0133 47729 48994 . 421L0134 49025 57445 . 2,806L0135 57728 57916 . 62L0136 58340 57996 , 114L0137 hokW 58615 58460 , 51 gef-hokC-relF homolog

mokW 58672 58460 , 70 Longer ORF, sameframe as hokW

L0138 sokW 58664 58734 . Antisense RNAL0139 59376 58906 , 156L0140 60200 59301 , 299L0141 60418 60197 , 73L0142 61095 60466 , 209



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In all cases for which data is available, integrative recombi-nation is mediated by a phage-encoded recombinase (inte-grase, or Int protein) which catalyzes the strand cleavage andrejoining, and excision usually requires the cooperative actionof Int and a second phage-encoded excision protein (Xis). Wehave designated L0061, the ORF following attL in the pro-phage, as int; sequence comparisons suggest that it is a memberof the integrase-recombinase family, although very distant fromlambda, P22, and other lambdoid phages. The closest homo-logs are the putative integrases of the E. coli K-12 crypticprophages 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 Intproteins, all of these recombinases can be aligned in theirC-terminal halves to reveal a conserved region implicated asthe active site of this family (1, 4). The proposed 933W Intshows some similarity (10.5 to 15.3% identity; BLAST E val-ues, 2 3 1023 to 3 3 10240) to a great number of other Intsequences, and examination of the alignments reveals that witha single exception, it contains the conserved residues, includingthe tyrosine residue identified as the active amino acid involvedin a transient phosphodiester linkage to the DNA duringstrand cleavage and rejoining. The exception, a tyrosine resi-due instead of a histidine in the highly conserved His-X-X-Argmotif, is also present in the Rac and Qin integrases. Given theobservation that in the P1 Cre integrase this precise substitu-tion reduced the recombination activity about sevenfold in vivo(1), the question arises of how efficient these putative inte-grases are. However, this substitution is also present in theintegrase of the Streptomyces lividans SLP1 element (12), whilethe putative integrase of the Pseudomonas aeruginosa phagefCTX contains an N (asparagine) residue instead of the H(39).

By analogy to lambda and other temperate phages, the933W ORF L0062, immediately upstream of int, may encodethe phage excisionase. A similar sequence is found in the Racprophage (25.3% identical to YdaQ, b1346), which is knownto be excisable, but not in Qin, where an IS2 insertion nearintQ is accompanied or followed by a deletion of flanking se-quences. With a few exceptions, Xis sequences show littlehomology to one another, and even such generalizations as thelambdoid Xis proteins being basic while those from gram-pos-itive bacteria are often acidic (58) are rife with exceptions. Thesuggestion has been made that temperate phage excisionaseshave a helix-turn-helix motif, as scored by the metric of Doddand Egan (26) for recognizing such motifs (83). This metricgenerates SD scores, which are standard deviation units rela-tive to the appropriate mean; scores $ 2.5 SD are indicative ofa helix-turn-helix motif. While most of the lambdoid Xis pro-teins do not score well with the program (25), the best-scoringregions of 933W Xis (score of 0.37 SD at position 33) andYdaQ (score of 20.34 SD at position 19) do align with themotif pointed out by Salmi et al. (83), suggesting that somehelix-turn-helix character may be involved in these proteins aswell. However, in the absence of any experimental data, thepossibility must be noted that excision of 933W (and perhapsRac) does not require an excisionase, as such. Excision of theStaphylococcus aureus phages f13 and f42 requires no phage-encoded product other than the integrase (17), and for coli-phage 186 the transcriptional repressor protein Apl also servesas the phage-encoded excision factor (78).

ORF L0069 is homologous (89.7% identity) to “Ehly2,” theproduct of an ORF associated with an enterohemolysin 2 ac-tivity encoded by phage C3208 in E. coli O26:H11 (6). Both ofthese 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 genesoccur in analogous positions within their respective genomes.In the absence of any demonstrated hemolysin activity by theEhly2 protein itself, this similarity is best viewed as an indica-tion that phage C3208 is also a member of the lambdoid family.If this protein does have a cytotoxic effect, it may contribute tothe virulence of O157:H7.

The 933W sequence spanning ORFs L0073 to L0078 is sim-ilar to the recombination region of lambda (97% identity over3,698 bp), with both the gene order and predicted amino acidsequences of individual genes highly conserved; these ORFsare therefore designated exo (97.3% identity), bet (99.6% iden-tity), 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 ananalog of the lambda regulatory gene N largely on the basis ofits position within the sequence (its product shows 14.0% iden-tity to lambda N; 29.0% identity to P22 gene 24 protein). It wassubsequently found to be very similar (96.9% identity) to the Ngene of H19-B.

The predicted product of ORF L0082 resembles the familyof eukaryotic serine/threonine protein kinases (12.6 to 20.1%identity to more than 100 distinct serine/threonine kinasesfrom a variety of organisms; BLAST E values, 2 3 1024 to 3 310220), and we have designated the gene stk (for “serine/threonine kinase”). The sequence similarities span the con-served regions in the catalytic domain of the eukaryotic proteinkinases, including both the ATP binding and active sites. TheStk sequence is more similar to eukaryotic serine/threonineprotein kinases (e.g., 17.2% identity to STE20 of Saccharomy-ces cerevisiae) than to other prokaryotic protein kinases, in-cluding those of Mycobacterium tuberculosis (10.7% identity),Streptomyces coelicolor (11.3% identity), Myxococcus xanthus(12.6% identity), Bacillus subtilis (12.7% identity), and Yersiniapseudotuberculosis and Y. enterocolitica (11.0% identity).

In bacteriophages lambda and f80, the NinR regions con-tain orf-221, which encodes a phosphoprotein phosphatase re-sembling those of mammalian origin (20). The function of thisphage-encoded activity is unknown, although it presumablyacts to modulate the signal transduction pathways of the E. colihost in a manner similar to the E. coli PrpA and PrpB phos-

FIG. 3. Location of the endpoints of virion DNA. Each of the 222 sequencereads from the 933-lysate shotgun library was aligned with the completed 933Wprophage sequence, and their location is shown above the base pair scale; thevertical offset is only to allow the display of overlapping sequences. Superim-posed over that graph is plotted the relative density of sequence coverage (cal-culated as the midpoint value for each of 200 windows, using a kernel-densitysmoother with a Gaussian kernel). Below the scale are indicated the positions ofEcoRI and BamHI sites in the sequence, as well as the positions of the int(integrase) and L0112 (terminase?) genes.



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phatases, described by Missiakas and Raina (60). While 933Wapparently encodes a protein kinase, the phage does not en-code a homolog of this phosphatase. There is some suggestionthat the Yersinia protein kinase (YpkA) is involved in virulenceby interfering with the signal transduction pathway of themammalian host (38), and bacteriophage 933W may interferewith the host systems in the same manner. The location of stkis 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 ap-pears to be a hybrid of two species of repressors. The amino-terminal 89 amino acids most closely resemble the repressor ofphage HK022 (37.4% similarity), while the rest of the sequenceis almost identical to that of H19-B at both the nucleotide(95.2% identity) and amino acid (96.6% identity) levels. In thewell-characterized lambda repressor, the amino-terminal resi-dues contain the DNA binding helix-turn-helix motif that in-teracts with the operator sequence, while the carboxy-terminaldomain of the protein is involved in dimerization. A similar“hybrid” repressor was recently noted in a comparison of thelysogeny modules from two temperate Streptococcus ther-mophilus bacteriophages (66).

ORF L0086 is the 933W cro homolog and encodes a proteinmost similar to that of HK022 (43.4% identity). This is consis-tent with the cI structure described above, given that both CIand Cro must recognize the same operator sequences. ORFL0087 is the cII homolog, encoding a protein similar to thoseof 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 arenearly identical to those of lambda (94.0% nucleotide se-quence identity), as well as to H19-B (96.7% nucleotidesequence identity). Sequence similarities allow assignmentof L0088 and L0089 as the replication genes O (98.0% identityto lambda; 98.7% identity to H19-B) and P (96.6% identity tolambda; 95.7% identity to H19-B), respectively, and L0090 asren (99.0% identity to lambda; 97.9% identity to H19-B). Thereplication origins of 933W and H19-B have a 39-bp insertrelative to that of lambda, containing two additional iterons.This insert results in an in-frame insertion of 13 amino acids inthe replication protein O of both Stx phages. An in vitro read-through of the UAG termination codon of the O gene has beenfound in bacteriophage lambda (100); the conservation of theO sequences includes this extended carboxy-terminal region.

The 933W sequence to the right of the replication origincontains homologs of several lambda and P22 Nin regionORFs: L0093 (39.0% identity to the product of lambda orf-146; 39.3% identity to P22 NinB), L0097 (91.6% identity to theproduct of lambda orf-204; 88.1% identity to P22 NinG), andL0098 (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 dif-ferent phage genomes, and, except for L0093, there are alsohomologs in H19-B.

ORF L0094 encodes a protein similar to a hypothetical meth-ylase of bacteriophage HP1 (30.1% identity) and the DNA(N6-adenine) methyltransferase of bacteriophage T1 (22.3%identity). Although no functional characterization of this pro-tein is available, such a DNA modification might explain thedifficulties we experienced when attempting to digest 933WDNA with a number of restriction enzymes.

ORF L0099 was identified as the homolog of the late regu-latory gene Q, most closely resembling the functional analog oflambda Q from phage DLP12 (b0551, YbcQ; 77.2% identity);it was subsequently found to be almost identical (96.5% iden-tity) to the Q gene product of H19-B.

The Stx2 subunits are encoded by stx2A and stx2B (aka sltIIAand sltIIB) and were previously characterized (43, 87, 89).

Downstream of the stx genes and seemingly part of the sametranscript, ORF L0105 encodes a protein similar (50.8% iden-tity) to E. coli K-12 YjhS (b4309). Examination of the H19-Bsequence reveals an unannotated 849-bp ORF downstreamfrom the stx1 genes (accession no. AF034975, bases 14651 to15499), whose product is also similar to YjhS (20.5% identity).The function of these genes is unknown, but yjhS is part of afimbrial synthesis and iron transport region which K-12 mayhave 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) andH19-B (91.3% identity). Like a number of other lambdoidholin genes (9), this gene has two Met start codons separatedby one or two codons. However, in 933W neither of the codonsbetween the alternate starts is an arginine or lysine codon, andit is not clear whether a dual-start motif is involved in theregulation of 933W lysis.

L0108 is the R gene (endolysin) analog, also resembling thatof Qin (88.8% identity); although no R gene is annotated in theH19-B sequence, the insertion of 4 bases near the end of thatsequence (C between bases 16927 and 16928; AA between 17254and 17255, and G between 17303 and 17304) would create anR homolog running off the end of the entry, 91.4% identical toL0108.

In other lambdoid phages, S and R are followed by Rz, and933W ORF L0110 is an Rz homolog most like that fromlambda (71.2% identity); a homolog of the overlapping Rz1reading frame (48) is also present. 933W has an additionalORF, L0109, inserted between R and Rz, whose product re-sembles the P22 Ant antirepressor (34.2% identity). The func-tion of this protein in 933W is unknown, although its locationsuggests a possible regulatory role in lysis of the host cell.

The end of the lysis region of 933W is similar to that oflambda (91.0% identity over 940 bp). In addition to Rz (andRz1), this region contains a homolog (L0111; 96.9% identity)of the lambda bor gene. In lambda lysogens, bor expression hasbeen implicated in serum resistance (5), which may confer aselective 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 re-mainder of this genome encode the virion structural and mor-phogenic proteins, as continued analogies would argue, very fewcan be even tentatively identified on the basis of sequence com-parisons. A number of complete or partial phage sequenceshave been determined, and comparisons reveal homologiesbetween different phages for genes encoding enzymes, reg-ulatory proteins, replication proteins, and various “accesso-ry” products. However, the genes encoding the actual struc-tural proteins that comprise the virion seem to be drawn froma much larger pool of potential sequences—as if the possibleways to build morphologically similar phage particles are myr-iad and our sampling has merely skimmed the surface of thegene pool.

Based on the 933W virion DNA endpoint analysis, onemight expect the region starting with ORF L0112 to containthe genes involved in DNA packaging. Although the analogousgenes 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 ter-minase small subunit (bacteriophage P1 PacA, BLAST E value0.78); L0113, the terminase large subunit (bacteriophage T4gp17; BLAST E value 0.005); and L0114, the prohead portalprotein (bacteriophage P22 gp1; BLAST E value 0.002). The



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sequence matches have very poor scores, and these assign-ments must be thought of as provisional.

One of the few structural-gene candidates that can be iden-tified is L0121, a putative tail fiber gene; its product shows17.0% identity to the Stf tail fiber of lambda and containsmotifs described in various phage tail fiber proteins (84). ThisORF is also one of several phage genes encoding structuralcomponents of bacteriophage virions in which the characteris-tic collagen-like repeats (Gly-X-Y)n have been noted (91). Thepredicted 933W tail fiber protein displays extensive homo-logies to collagen (e.g., 26.8% identity to human alpha type Icollagen), with stretches of 40 and 38 repeats of the collagenmotif. The repeats have the bias toward proline at the secondand third positions of the motif that has long been known tooccur in vertebrate collagens. Furthermore, the collagen se-quences in 933W tail fibers may well have the triple-helixstructure found in animal collagen: all well-characterizedphage tail fiber proteins are trimeric (18), including the phagel fibers that have sequence similarity to the 933W protein. Wenote that the position of L0121 in the genome of 933W is notanalogous to that of the lambda stf gene, but, given the differ-ence in tail morphology between these phages, analysis byanalogy is probably at its weakest for tail structural and mor-phogenic genes.

In addition to the bor homolog in the lysis region, two other933W ORFs may be involved in virulence. L0128 encodes ahomolog (28.6% identity) of the lambda Lom protein (77, 82),which encodes a member of a family of outer membrane pro-teins associated with virulence in two species. Expressed inlysogens, these proteins confer the ability to survive in macro-phages.

L0137 encodes a member of the hok-gef-relF family of killerproteins from “toxin-antitoxin” systems (90.4% identity toRelF; 78.4% identity to Gef). In these systems, the best char-acterized of which are involved in plasmid maintenance, anunstable antisense RNA prevents expression of the lethal pro-tein by binding to the more stable mRNA (33, 76). Examina-tion of the sequence surrounding L0137 reveals that all of thesequence elements and potential secondary-structure features

described for these systems (30, 35) are conserved in 933W. Byanalogy to the Hok system, L0137 is designated hokW (for“host killing, 933W”) and the antisense RNA is designatedsokW (for “suppression of killing, 933W”). If these genes areexpressed in the lysogenic state, loss of the prophage would beselected against in a manner similar to the selection againstloss of plasmids. Interestingly, in the O157:H7 strain EDL933,at least four such systems are present: in addition to phage933W, the large virulence plasmid pO157 carries a Hok systemhomolog (15), and data from the genomic sequence indicatesthat Gef and RelF homologs are also present (10). The targetspecificity of these systems resides in the interactions betweenthe antisense RNA and the mRNA, and for the plasmid andphage sequences these seem to be different. Whether the chro-mosomal loci interact with either of those systems to reinforcethe maintenance of the various pathogenicity determinants isunknown.

tRNAs. In the region between the late regulatory gene Q andthe Shiga toxin genes, a tRNAIle-like sequence was previouslyfound (88). Our reexamination of the sequence reveals genesfor two additional tRNA sequences, and the proposed clover-leaf secondary structures of all three tRNAs are shown in Fig.4. Most invariant or semi-invariant eubacterial tRNA residuesare present in these sequences, and they may encode func-tional tRNAs although this has not been demonstrated.

tRNA1 (designated ileZ) has the anticodon CAU. The se-quence of the tRNA closely resembles that of the tRNAIle

species encoded by the E. coli K-12 genes ileY (92.1% identi-cal) and ileX (89.5% identical), and none of the differencesaffect residues known to be involved in the function or identityof the tRNAs. The identification of these as Ile tRNAs rests onthe experimental characterization of the chromosomal ileXlocus. If the wobble position C34 remained unmodified, theCAU anticodon would correspond to the Met codon AUG.However, in the tRNAIle encoded by bacteriophage T4 andE. coli ileX (as well as a number of other organisms), the C34is modified to lysidine (2-lysylcytidine, k2C). This modificationbestows AUA (Ile) decoding capacity and is required for rec-ognition by the isoleucyl-tRNA synthetase (54, 62, 63). The

FIG. 4. Bacteriophage 933W apparently encodes three tRNAs. Each of the tRNA-like sequences is shown folded into the cloverleaf tRNA secondary structure.



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other known determinants for E. coli tRNAIle are present inileZ: the anticodon loop bases A37 and A38, the discriminatorbase A73, and the C4 z G69, U12 z A23, and C29 z G41 basepairs (67, 72).

tRNA2 has the anticodon UCG, which is not found in anyknown E. coli tRNAs, and in fact the sequence is not similar toany other specific tRNA species. On the basis of the anticodon,this sequence is designated argN. Uridine at the first position ofthe anticodon is modified in all cases so far sequenced at theRNA level in E. coli (97, 101); therefore, modification of U34may restrict recognition by this species to a subset of the CGNfamily of Arg codons. In E. coli K-12, the CGU, CGC, andCGA codons are read by the ICG anticodon while the CGGcodon is read by CCG. This sequence does contain the A20and C35 determinants for E. coli tRNAArg, but the residue atposition 73 is U instead of A or G (57, 72). In addition, thealmost invariant base pair R15 z Y48 is replaced by T15 z G48,which might prevent formation of the “Levitt pair” involved intRNA tertiary structure (51)—although the E. coli tRNACys

has G15 z G48 (55), which is an identity element for this tRNA(56).

tRNA3 (argO) has the anticodon UCU; with an unmodifiedU as the first nucleotide of the anticodon, this could corre-spond to the codons AGA (Arg), AGG (Arg), AGU (Ser), andpossibly AGC (Ser). As with tRNA2, this sequence does notclosely resemble any tRNA sequences in the sequence data-bases. However, the tRNAArg species encoded by bacterio-phage T4, E. coli argU (dnaY), and Salmonella argU (fimU) allhave UCU anticodons, and these species favor the argininecodon AGA due to modification of U34 to 5-methoxycarbon-ylmethyluridine (mcm5U) (93). The argO sequence containsthe A20 and C35 determinants for E. coli tRNAArg; A73 isreplaced 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. colichromosome, since a number of other bacteriophages andpathogenicity-associated islands are inserted at or near tRNAgenes (19, 37). This is not the case for 933W, although thesimilar E. coli K-12 chromosomal ileY locus is the insertiontarget of coliphage 186 (78). However, the proximity of thetRNA genes to the stx genes might reflect the outcome ofanother recombination event during which these sequenceswere initially acquired by the phage. If this is the case, are thetRNAs functional or are they just along for the ride? Exami-nation of codon usage (Table 2) suggests that the phage-en-coded 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 thetRNAs, regardless of their origin. There are no double “killerarginines” (102, 103) in the 933W genome, and so it seemsunlikely that the phage makes any explicit regulatory useof differential tRNA availability. However, the alteration oftRNA base modifications has been reported to affect virulencefactor expression in Shigella flexneri (27, 28) and Agrobacteriumtumefaciens (34), and this may actually be the result of analteration in the efficient translation of key proteins.

Promoters, operators, and terminators. A number of othernoncoding sequence elements were examined in the 933Wsequence, especially where homologs in better-characterizedphages allowed “analysis by analogy.” These features are anno-tated 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 immediatelydownstream (to the left) of L0081, in a short sequence of dyadsymmetry conserved between 933W and H19-B; no assignmentcould be made for OL or pL. Since the H19-B and 933W Ngenes are almost identical and nut sites are N specific, the nutsites should be within the sequences conserved between thetwo phages; a nutL candidate was identified, although it is notclear which of two “boxB”-like sequences would be involved.

The cI-cro intergenic region should include OR, pRM, andpR. Given the similarity of the amino-terminal domains of the933W and HK022 repressors, sequence comparisons with theequivalent region from HK022 (68) allowed us to tentativelyidentify all three features in 933W. The ready identification ofOR makes the failure to detect OL all the more puzzling, andit may be that regulation of transcription of the left and rightarms 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 and933W. pRE, by analogy to other lambdoid phages, would beactivated by 933W (and H19-B) CII; however, the recognitionsite for this protein is not known, and no candidates are pro-posed. A nutR candidate was found, and, as was the case fornutL, there are two candidate “boxB” sequences; perhaps in933W and H19-B the N recognition determinants involve anextended sequence relative to those in other lambdoid systems(31, 32, 86).

The near identity between the 933W and H19-B Q genesextends 293 bp beyond the coding sequences (i.e., ending up-stream of the tRNA genes), defining the region where the pR9promoter and qut site should be located. The A(N)3T(S)2–3motif in the nontranscribed DNA strand, noted by Ring and

TABLE 2. Isoleucine and arginine codon usagea

Gene

Codon usageb

Ile(AUU)

Ile(AUC)

Ile(AUA)

Arg(CGC)

Arg(CGU)

Arg(CGG)

Arg(CGA)

Arg(AGA)

Arg(AGG)

E. coli K-12 genome 30.3 25.1 4.4 22.0 20.9 5.4 3.6 2.1 1.2E. coli K-12 all ORFs 30.8 24.5 5.2 21.4 20.3 5.7 4.0 2.7 1.7

933W genome 22.9 19.3 10.1 15.2 19.2 8.9 6.5 9.9 6.0933W all ORFs 24.2 23.2 12.5 14.5 14.0 9.0 7.9 11.6 6.6

933W stxA 9.4 6.3 46.9 6.3 28.1 9.4 6.3 18.8 6.3933W stxB 11.1 11.1 0.0 11.1 0.0 0.0 0.0 0.0 0.0933W stk 22.9 2.9 48.7 0.0 8.6 5.7 0.0 25.8 11.5

a For E. coli K-12 (4,290 ORFs, 1,363,501 codons) and bacteriophage 933W (78 ORFs, 18,246 codons), codon usage was calculated for each genome (i.e., all codons)and for each ORF individually.

b Values are expressed as codon frequency per 1,000 codons; for “all ORFs,” the mean of the individual ORF values is shown.



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Roberts (80), can be found in several locations downstream ofthe presumptive pR9 promoter, but only one of these occurs inclose proximity to the promoter and before the first potentialterminator. We propose that this is part of the qut site for both933W and H19-B. Once Q protein modifies the RNA polymer-ase complex at qut, sequence divergence should not alter itsantitermination activity for the late transcripts; therefore, thelate regions of both phages are likely to be under Q regulation.

In E. coli, tRNA genes are often found in clusters withtypical prokaryotic 235 and 210 promoter elements as well asa GC-rich discriminator domain common to all E. coli genessubject to stringent control, and downstream of almost alltRNA genes is found a rho-independent terminator-like struc-ture (42). Both of these features are found flanking the 933WilvZ-argN-argO cluster, and the predicted transcript would be atrimeric precursor RNA resembling those readily processed bythe E. coli RNA processing machinery.

DISCUSSION

The Q gene product of lambdoid phages functions as atranscription antiterminator that regulates the expression oflate phage genes by modifying the transcription complex initi-ated at the late promoter pR9. The protein acts at the qut siteoverlapping the promoter, and the Q analogs of differentphages are specific for their own qut sites (81). Based on thearrangement of the 933W genes, the stx2 genes are part of anapparent Q-dependent late transcript, as diagrammed in Fig. 5.If this is the case, the toxin would be expressed only (or at leastmaximally) during lytic growth of the phage.

Muhldorfer et al. (61) examined the regulation of the stx2operon in experiments involving a low-copy-number plasmidcarrying a translational fusion of stx2A to a phoA reportergene. They concluded that a phage factor played a positiveregulatory role in the expression of stx2 and that this factorcould be provided in trans by either 933W or H19-B but notlambda. The increased expression was mitomycin C and recA1

dependent, as expected for a mechanism requiring prophageinduction. These results are entirely consistent with the phagefactor being the Q gene: when provided in trans by the phageafter induction and transition to lytic growth, Q could act toantiterminate pR9 transcripts on the reporter plasmid (the con-

structs included the entire Q-stx2A intergenic region). Thesimilarity of the 933W and H19-B genes is such that we wouldexpect the H19-B Q gene to function as well as that from 933Win this system.

The results of our analyses seem to be at odds with the re-port by Sung et al. (95) that a promoter for stx2 was located only118 bp upstream of the stx2A coding sequence, which wouldput the promoter (pSlt-II or pStx2) within argO. It may be thatsome constitutive level of Stx2 expression is provided by thatpromoter but that Stx2 production is significantly increasedafter phage induction as a more efficient promoter becomesavailable.

A number of pathogenicity factors, including several toxins,are encoded by lysogenic phages (7, 19). A linking of toxinproduction to prophage induction in such cases might openanother means of increasing the toxin yield. Infections are un-likely to occur in monoculture, and while other bacteria al-ready carrying the prophage (uninduced) would be immune tosuperinfection, nonlysogens in the vicinity could be infectedand produce additional phage (and toxin) in what can be en-visioned as an amplification by recruitment. It has been shownthat the cholera toxin-encoding phage CTXf infects Vibrio cho-lerae more efficiently within the gastrointestinal tracts of micethan under laboratory conditions (96). A coupling of toxin re-lease 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 bemore likely to be subsequently released into the environment.

Bacteriophage H-19B, isolated from E. coli O26:H11 strainH19 (90), is morphologically quite distinct from 933W andmore closely resembles lambda (98). This phage also displaysgreater sequence homologies to lambda (as detected by hy-bridization), and the virion DNA has cohesive termini (41).Nonetheless, both 933W and H-19B have stx genes in analo-gous positions and have similar regulatory elements. The cryp-tic Stx1 phage in EDL933 is still essentially uncharacterized,and there remains a possibility that it resembles H-19B. If theStx prophages in the EDL933 genome have overlapping regu-latory specificities, the coexistence of both elements couldpresent an interesting additional layer of complexity.

FIG. 5. The stx genes of O157:H7 are integrated into the late operon of a lambdoid bacteriophage. The late operons of lambda and 933W are diagrammed forcomparison, indicating ORFs, tRNAs, and real or predicted promoters (p) and terminators (t). Transcripts (known for lambda, proposed for 933W) in the presenceor absence of the cognate gpQ antitermination activity are indicated by arrows below the gene maps. The proposed tRNA promoter (pT) is not associated with a qutsite, and transcripts presumably terminate at tT. The reported Stx promoter (pS) also lacks a qut site, and the length of that transcript is unknown.



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ADDENDUM

Sequence data from the Stx1-encoding lambdoid bacterio-phage H19-B (64) was compared to our sequence in the courseof our analyses, and similarities are noted above. While thispaper was being revised, Neely and Friedman (65) publishedtheir analysis of this H-19B sequence. They reached conclu-sions similar to our own regarding the regulation of Stx pro-duction and phage release and showed that the H-19B Q geneproduct can activate the expression of 933W stx2 genes as wellas its own stx1 genes.

ACKNOWLEDGMENTS

This work was supported by NIH grant AI41329-01 and by a re-search grant from the Ronald McDonald House Charities.

We thank the technical staff of the University of Wisconsin Ge-nomes Project for help with sequencing, Randall Massey and GraysonScott of the University of Wisconsin Medical School Electron Micro-scope Facility for electron microscopy, and Bill McClain for usefuldiscussions about the tRNAs. For bearing with his return to the benchafter too many years at the computer, one of us (G.P.) also thanksHeather Kirkpatrick of this laboratory.

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