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Journal of Bacteriology, June 2004, p. 3472-3479, Vol. 186, No. 11
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.11.3472-3479.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of a Eukaryotic-Like Tyrosine Protein Kinase Expressed by the Shiga Toxin-Encoding Bacteriophage 933W

Jessica S. Tyler and David I. Friedman*

Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109-0620

Received 15 December 2003/ Accepted 11 February 2004


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ABSTRACT
 
The Shiga toxin (Stx)-encoding bacteriophage 933W contains an open reading frame, stk, with amino acid sequence similarity to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs). Eukaryotic PKs are related by a common catalytic domain, consisting of invariant and nearly invariant residues necessary for ATP binding and phosphotransfer. We demonstrate that rather than a Ser/Thr kinase, stk encodes a eukaryotic-like tyrosine (Tyr) kinase. An affinity-purified recombinant Stk (rStk) autophosphorylates and catalyzes the phosphorylation of an artificial substrate on Tyr residues and not on Ser or Thr residues. A change of an invariant lysine within the putative catalytic domain abolishes this kinase activity, indicating that Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs. We provide evidence suggesting that stk is cotranscribed with cI from the phage promoter responsible for maintaining CI expression during lysogeny. The stk gene was identified in prophages obtained from independently isolated Stx-producing Escherichia coli clinical isolates, suggesting that selective pressure has maintained the stk gene in these pathogenic bacteria.


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INTRODUCTION
 
The lambdoid phage 933W contains an open reading frame (ORF), stk, with significant homology at the amino acid level to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs) (48). On this basis, it was considered a eukaryotic-like kinase (48). This Shiga toxin (Stx) type 2-encoding bacteriophage was isolated from the Stx-producing Escherichia coli (STEC) strain EDL933 (43). Infection with STEC can produce a range of clinical manifestations, including watery diarrhea and hemorrhagic colitis, and the infection can progress to life-threatening sequelae (29, 53). Production and release of Stx, the major virulence factor of STEC (39), are enhanced by induction of phage lytic growth (40, 64, 65). It is of interest that a phage contributing to the virulence of a pathogenic bacterium contains a putative eukaryotic-like Ser/Thr kinase. Bacterial effectors that phosphorylate and dephosphorylate eukaryotic proteins, when transferred to the eukaryotic host cell, have been shown to interfere with host cellular activities and are essential to the virulence of some bacterial pathogens (6, 13, 17).

Eukaryotic PKs target Ser/Thr or tyrosine (Tyr) residues, whereas most prokaryotic PKs target histidine (His) residues. Originally thought to be exclusively in eukaryotic cells, Ser/Thr and Tyr PKs have been identified in prokaryotic cells and have been shown to participate in a variety of cellular processes, including development, signal transduction, and virulence (reviewed in references 4, 5, and 71). Although functionally similar, previously identified bacterial Tyr PKs use different phosphotransfer mechanisms from the mechanism used by their eukaryotic functional homologs (14, 42, 68, 69). Aside from Stk, the T7 Ser/Thr PK required for phage growth under suboptimal conditions is the only identified phage-encoded eukaryotic-like kinase (23).

Bacteriophage 933W belongs to the lambdoid family of phages, as do all known Stx-encoding phages (12, 27, 48, 61). These phages share similarities in genetic organization, regulatory mechanisms, and a common pool of genes (9, 21). Although genes at most positions may vary in sequence between the various lambdoid phages, similarity in function is maintained. At some positions in their genomes, different lambdoid phages have genes that encode proteins of unrelated function (9, 21). Such genes are often considered accessory genes because they are not required for lytic growth or maintenance of lysogeny, but in some cases, they confer an advantage to the bacterial host (10). One genome region that varies between lambdoid phages is located downstream of and adjacent to cI, the gene encoding the repressor protein (Fig. 1A) (10). This region in 933W contains the stk gene (48), and in {lambda} it contains the rexAB genes. In {lambda}, RexAB proteins are coordinately expressed with the CI repressor from a transcript initiating at the phage pRM promoter (52, 57). In addition, rexB can be expressed independently of rexA by transcription initiating at a rexB-associated promoter (33). The Rex functions are thought to benefit the lysogen in several ways: e.g., excluding infection by other phages and preventing programmed cell death (16, 46). The similar genome locations and orientations of rexAB and stk led to the prediction that Stk is expressed by the 933W prophage (48).



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  		FIG. 1. (A) Map of the regulatory region of the bacteriophage 933W genome (not drawn to scale) based on sequence analysis (48). The ORFs are drawn as arrows pointing in the direction of transcription inferred from studies on transcription of analogous genes of {lambda}. Putative phage promoter pRM was previously assigned by sequence analysis (48). Phage promoters pR and pL were identified by primer extension, and the right and left repressor operator sites, OR and OL, respectively, were identified by mutational and biochemical studies in this laboratory (Tyler et al., unpublished). For comparison, a map of the regulatory region of bacteriophage {lambda} is included below the phage 933W map. (B) Alignment of the amino acid sequence of the deduced catalytic domain of Stk with the amino acid sequence of the catalytic domain of bovine cyclic AMP-dependent PK (cAPK-{alpha}), a Ser/Thr PK, and the human Src PK, a Tyr PK. This comparison is based on amino acid sequence alignment of Stk with the Ser/Thr PK catalytic domain consensus sequence conducted by BLAST (1). The 12 subdomains typical of eukaryotic PKs (19) are designated by Roman numerals. Conserved residues important for catalysis are indicated: identical residues are boxed in black, and similar residues are boxed in gray (adapted from reference 20). Dashes were inserted in gaps of sequence homology to optimize the alignment. Forward slashes mark the deduced boundaries of each subdomain. The arrow indicates Lys42, which was changed to alanine in the mutant protein, rStk-K42A, by altering the coding sequence.

We show that the 933W prophage expresses a functional eukaryotic-like PK that is encoded by the stk gene. Although Stk contains motifs that are characteristic of eukaryotic Ser/Thr PKs and was annotated as such when the 933W genome was sequenced (48), we find that Stk is a Tyr-specific PK. We also demonstrate that stk is conserved in genomes of lambdoid-like prophages of independently isolated STEC clinical isolates.


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MATERIALS AND METHODS
 
Media. E. coli strains were cultured in Luria broth or M9 minimal medium supplemented with 0.1% Casamino Acids at 37°C (unless otherwise stated) (34). Ampicillin, chloramphenicol, spectinomycin, and mitomycin C were used at final concentrations of 100, 25, 80, and 2 µg/ml, respectively (unless otherwise noted).

Strain construction. Table 1 lists bacterial strains, phages, and plasmids. Table 2 lists sequences of the primers indicated below.


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  		TABLE 1. Strain and phage list


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  		TABLE 2. List of primers used in this study

(i) Lysogens. 933W lysogens were constructed essentially as described previously (18). Strains K37 and DY330 were lysogenized with phage 933W, yielding strains K9675 and K9857. The phage isolated from strain E32511, designated {phi}E32511, was obtained by mitomycin C induction.

The {lambda} Red recombination system was used to construct strains with gene replacements as previously described (15, 70).

stk::c-myc fusion genes. The cat-sacB cassette (15) was amplified by PCR from strain DY406. The c-myc tag was amplified by PCR from plasmid pA3 M (2). The C-terminal c-Myc fusion to Stk was inserted into the 933W prophage in strain K9857 in a two-step process (70). The stk stop codon was replaced with the cat-sacB cassette and subsequently replaced with a c-myc amplicon, generating the stk::c-myc translation fusion. Following construction of the strain with the insertion of c-myc in the 933W prophage, a phage 933Wstk::c-myc lysate was obtained by mitomycin C induction. The resulting 933Wstk::c-myc lysate was used to lysogenize K37, yielding strain K10567.

Plasmid construction. The integrity of the inserts in the constructed plasmids was verified by DNA sequencing (University of Michigan Sequencing Core). The phage 933W stk ORF was PCR amplified from strain K9675, using primers 5'-stk-NcoI and 3'-stk-XhoI. The gene encoding the recombinant Stk (rStk) mutant kinase rStk-K42A was constructed by PCR splicing by overlap extension (24), using DNA from strain K9675 as a template. The wild-type stk and mutant stk-K42A PCR products were digested with NcoI and XhoI (New England Biolabs) and then inserted into pTYB4 (New England Biolabs). The constructed plasmids, pTYB4-stk and pTYB4-stk-K42A, were transformed into BL21-CodonPlus(DE3)-RIL (Stratagene), yielding strains K10383 and K10397, respectively.

The wild-type stk amplicon was also inserted into pET-33b(+) (Novagen), using the NcoI and XhoI sites, generating plasmid pJES110, which was transformed into BL21 (DE3), yielding strain K10208. This strain was then transformed with pJES113, a pGB2-plac vector (40) in which a cassette of the putative tRNA genes, ileZ, argN, and argO, from phage 933W was inserted by using the ClaI and KpnI sites, generating strain K10217.

The phage 933W cI repressor gene was PCR amplified from strain K9675, using primers ClaI-cI and KpnI-cI. The resulting product was inserted into pGB2-plac (40) using the ClaI and KpnI sites. The constructed plasmid was transformed into strain K10567, generating strain K10661.

Isolation of Stk proteins. rStk proteins, rStk and rStk-K42A, fused to an intein tag at their carboxy terminus, were isolated from cultures of K10383 and K10397 by using the New England Biolabs IMPACT-CN system protocol. Purified protein was concentrated in Centricon YM-30 concentrators (Amicon).

Recombinant protein rStk-His, fused to a His6 tag at its carboxy terminus, was purified with Ni-nitrilotriacetic acid agarose (Qiagen, Valencia, Calif.) under denaturing conditions following the manufacturer's instructions.

Phosphatase treatment of rStk. rStk was treated with a modified form of the Yersinia enterocolitica protein tyrosine phosphatase (PTP) (72) (New England Biolabs) following the manufacturer's instructions.

Antibody generation. Antibodies to rStk-His were generated in rabbits by Cocalico Biologicals, Inc.

Kinase assay. In vitro kinase assays were performed as described previously (28), using approximately 0.5 to 1 µg of rStk or rStk-K42A. Artificial substrate proteins, myelin basic protein (MBP) and histone II protein (HII), were supplied by J. Dixon.

Phosphoamino acid analysis. rStk and HII protein were labeled in an in vitro kinase assay with [{gamma}-32P]ATP as described above and subsequently used for phosphoamino acid analysis as described previously (8). Internal standards for phosphoserine, phosphothreonine, and phosphotyrosine (Sigma) were visualized by staining with 0.2% ninhydrin in acetone.

Western blot analyses. Western blot analyses were conducted essentially as described previously (3). Stk-c-Myc protein was detected by Western blot analyses with mouse anti-c-Myc clone 9E10 monoclonal antibody (Santa Cruz Biotechnology, Inc.) and goat anti-mouse immunoglobulin G1 (IgG1) horseradish peroxidase (HRP)-conjugated clone sc-2060 antibody (Santa Cruz Biotechnology, Inc.). Tyr-phosphorylated protein was detected with mouse antiphosphotyrosine clone 4G10 monoclonal antibody (Upstate Biologicals, Inc.) and goat anti-mouse IgG1 HRP-conjugated clone sc-2060 monoclonal antibody (Santa Cruz Biotechnology, Inc.) essentially as described previously (44). Stk protein was detected with the rabbit anti-Stk-His polyclonal antibody at a dilution of 1:10,000 and donkey anti-rabbit Ig HRP-linked monoclonal antibody (Amersham Biosciences). NusA protein was detected by using rabbit anti-NusA polyclonal antibody (11) and donkey anti-rabbit Ig HRP-linked monoclonal antibody (Amersham Biosciences). The ECL enhanced chemiluminescence Western blotting system (Roche) was used to detect secondary antibodies.

Isolation of genomic DNA. Genomic DNA from STEC strains was isolated by the modified cetyltrimethylammonium bromide method (3).

Southern blot analysis. Genomic DNA digested with HindIII and EcoRI (New England Biolabs) was separated by gel electrophoresis and then transferred to a nylon membrane. A radiolabeled DNA probe of approximately 550 bp of stk was generated by PCR amplification with the Southern blot primers listed in Table 2 using [{alpha}-32P]dCTP (Amersham Biosciences) (55). Low-stringency Southern blot analyses were conducted essentially as described previously (55).

Sequencing of stk genes and flanking regions. PCR products amplified from genomic DNA were used as templates for sequencing (University of Michigan Sequencing Core), unless otherwise stated. stk genes were PCR amplified with primers 5'-stk-NcoI and 3'-stk-XhoI, and the isolated products were sequenced with primers 5'-stk seq and 3'-stk seq. The cI genes and the intergenic regions between stk and cI were PCR amplified with primers 5'-933W-cI and stk upstream seq, and the isolated product was sequenced with primers 5'-933W-cI and cI seq. Genomic DNA was used as the template for sequencing the region following the 3' end of stk, using the primer stk-N seq.

Isolation and characterization of phage DNA. Phage lysates were made from cultures of strains K9675 and K10664 by mitomycin C induction. DNA obtained using the Qiagen (Valencia, Calif.) Lambda Midi kit was digested with EcoRI and HindIII (New England Biolabs), separated on a 0.8% agarose gel, and visualized by staining with ethidium bromide.


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RESULTS AND DISCUSSION
 
Analysis of stk amino acid sequence. Based on alignments with the catalytic domains of known PKs, the Stk ORF, predicted to be a Ser/Thr PK (48), can be divided into the 12 subdomains typical of eukaryotic PKs as defined by Hanks (Fig. 1B) (19, 20). Stk contains all invariant residues and sequence motifs important in catalysis, with exception of the GxGxxGxV motif in subdomain I (19). This motif is required to anchor ATP so that the {gamma}-phosphate is correctly oriented for phosphotransfer (31). However, subdomain I of Stk has a glycine residue, Gly22, followed by valine residue Val27 five residues to the carboxy side. Alignment of the Stk sequence with the catalytic domain of eukaryotic PKs indicates that Stk has a variant of the consensus motif in which Gly22 corresponds to the second glycine in the glycine-rich segment (Fig. 1B). This arrangement is not unique, because variations of the glycine-rich motif have been observed in other PKs (45).

Although the catalytic domains of all eukaryotic PKs share conserved sequences, two regions contain signature motifs that have been used to distinguish Ser/Thr PKs from Tyr PKs (20). One is the catalytic loop, located in subdomain VIB (19), which is predicted to direct the phosphotransfer event using similar mechanisms in both classes of PKs (30). However, the catalytic loops of the two classes are not functionally interchangeable (30, 35). Subdomain VI of Stk contains the sequence DIKPNN, which is more similar to the Ser/Thr PK catalytic loop consensus sequence, DxKPxN, rather than the consensus sequence of receptor Tyr PKs, DxAARN, or that of cytoplasmic Tyr PKs, DxRAAN (20, 59). The other highly conserved motif is found in subdomain VIII (19). The residues in this region have been implicated in peptide substrate recognition because they are thought to position the appropriate hydroxyamino acid for phosphotransfer through interactions with the catalytic loop and the substrate (30, 59). Tyr PKs possess a highly conserved proline residue in this motif, whereas Ser/Thr PKs contain a Ser or Thr residue at an equivalent position (20). Stk subdomain VIII, which contains a Tyr at an equivalent position, does not seem similar to either motif. At this time, it is unclear how Stk accommodates peptide substrates for phosphotransfer to Tyr residues. Although consensus sequences distinguishing Ser/Thr PKs from Tyr PKs can usually be used as indicators of specificity, there are exceptions in which the residues within these two defined regions do not fit the appropriate consensus sequences (58).

PK activity of Stk. Affinity-purified rStk was used for in vitro kinase assays to determine if stk encodes a protein kinase. In vitro, rStk catalyzed the phosphorylation of artificial substrates MBP and HII (Fig. 2B, lanes 2 and 3). In the absence of rStk, MBP and HII were not phosphorylated (data not shown). In vitro rStk autophosphorylation was weak, both when incubated alone and in the presence of MBP (Fig. 2B, lanes 1 and 2). However, rStk autophosphorylation was significantly enhanced when incubated with HII (Fig. 2B, lane 3). We speculate that HII interaction with rStk induces conformational changes in the kinase that open additional autophosphorylation sites. As is the case with eukaryotic PKs, addition of EDTA to chelate the Mg2+ cofactor present in the buffer abrogated rStk autophosphorylation (Fig. 2B, lane 4).



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  		FIG. 2. Purification and kinase activity of rStk and the rStk-K42A mutant. (A) The rStk and mutant rStk-K42A proteins were affinity purified and visualized by SDS-PAGE (Coomassie stained). (B) Both proteins were incubated with [{gamma}-32P]ATP in an in vitro kinase assay, and the products of the reactions were resolved by SDS-PAGE and examined by autoradiography. Transphosphorylation activity of the two rStk proteins was assayed with the artificial substrates MBP and HII. In lane 4, EDTA was added prior to initiation of the assay to chelate the Mg2+ cofactor.

To determine if Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs, we constructed a mutant stk gene that encodes a variant Stk protein with a single amino acid substitution of alanine for Lys42 in subdomain II. Lys42 corresponds to an invariant lysine (Fig. 1B) that has been implicated in anchoring and orienting ATP (20, 31). Replacement of this conserved lysine with any other amino acid abolishes kinase activity in nearly all eukaryotic PKs (20). In vitro, rStk-K42A did not autophosphorylate nor did it catalyze the phosphorylation of MBP (Fig. 2B, lanes 5 and 6) or HII (data not shown). Hence, as predicted by alignment of the Stk sequence with the catalytic domain sequence of eukaryotic PKs, Stk Lys42 is required for the enzymatic activity of rStk. These results provide compelling evidence that Stk uses a phosphotransfer mechanism similar to that used by eukaryotic PKs.

Phosphoamino acid analysis of rStk phosphorylation. To determine the specificity of Stk phosphorylation, rStk and HII phosphoradiolabeled in vitro by rStk were subject to phosphoamino acid analysis. Although Stk was predicted to be a Ser/Thr PK (48), both rStk and HII were phosphorylated on Tyr residues and not on Ser or Thr residues (Fig. 3A).



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  		FIG. 3. Amino acid specificity of rStk autophosphorylation and transphosphorylation. (A) Phosphoamino acid analysis of in vitro phosphoradiolabeled rStk and histone II. The migration distances of internal standards for phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y), are marked with circles. (B) Western blot analyses of purified rStk, rStk-K42A, and Y. enterocolitica PTP-treated rStk (rStk + PTP), probed with the monoclonal phosphotyrosine antibody 4G10 ({alpha}-PY) and a polyclonal Stk antiserum ({alpha}-Stk).

Tyr-specific rStk autophosphorylation in vivo was confirmed by Western blot analysis. The antiphosphotyrosine antibody strongly interacted with purified rStk but failed to interact with purified rStk-K42A, the kinase with the mutant catalytic domain (Fig. 3B). Treatment of rStk with a modified form of the Y. enterocolitica protein tyrosine phosphatase (PTP) (72) yielded a form of rStk that migrated to a position in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel similar to that of the mutant protein rStk-K42A (Fig. 3B). Therefore, the differences in migration of rStk and rStk-K42A can be attributed to the state of tyrosine phosphorylation of the protein (Fig. 2A and 3B).

Most of the known prokaryotic Tyr PKs are members of a family of membrane-bound autokinases that share a distinct mechanism of autophosphorylation (14, 26, 38, 42, 62). Many of these PKs are required for the synthesis of capsular polysaccharides and exopolysaccharides in a variety of gram-negative and gram-positive bacteria (26, 36, 38, 62). They resemble eukaryotic PKs because they have conserved residues that are important for catalysis in eukaryotic PKs (14). However, these PKs use Walker A and B motifs (66) at the carboxy terminus of the protein to facilitate ATP binding, instead of a conserved lysine in the subdomain II found in eukaryotic PKs (14, 36, 42, 69). This difference in ATP binding distinguishes this family of prokaryotic Tyr PKs from eukaryotic Tyr PKs. Stk is the only identified prokaryotic Tyr PK, of which we are aware, that uses a phosphotransfer mechanism similar to that used by eukaryotic PKs.

As discussed, two motifs within the PK catalytic domain differ between the Ser/Thr and Tyr PK families (20). Crystallographic studies of the catalytic domains of eukaryotic PKs indicate that these signature motifs are important in positioning the hydroxyl group of Ser/Thr or Tyr residues of the substrate for phosphotransfer (25, 31, 59). The fact that Stk functions as a Tyr PK but has an active site that resembles a Ser/Thr PK raises the question of how Stk correctly positions the substrate amino acid for phosphotransfer. In this regard, we note that two Tyr PKs identified in the unicellular organism Dictyostelium discodium do not possess both of the signature motifs that are highly conserved in Tyr kinases (58). Our observation that Stk, which resembles eukaryotic PKs, is also missing motifs characteristic of eukaryotic Tyr PKs provides additional evidence that these motifs are not necessarily required for tyrosine-specific phosphotransfer.

Analysis of stk expression. Western blot analyses were performed to determine if stk is expressed by the 933W prophage. That the prophage expresses Stk was predicted both from the location and orientation of the stk gene in the 933W genome (48) as well as by analogy with the regulation of expression of the rex genes of {lambda}. Proteins from K10567, a lysogen with a 933W prophage carrying an in-frame fusion of stk with a c-myc tag, were probed for Stk-c-Myc expression with a high-affinity anti-c-Myc monoclonal antibody. The Western blot of protein isolated from K10567 shows that Stk-c-Myc is expressed by the 933W prophage (Fig. 4, lane 2).



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  		FIG. 4. Expression of the fusion protein Stk-c-Myc from the 933W prophage containing a translation fusion of stk and c-myc. Western blot analyses, using an anti-c-Myc monoclonal antibody (9E10) as the probe, of equal amounts of soluble protein isolated from 933W lysogens. The following strains were examined: K9675 (lane 1), which carries the stk gene; K10567 (lane 2), which carries the stk::c-myc fusion; and derivatives of K10567 containing pGB2-plac, K10660 (lane 3), or pGB2-plac-cI, K10661 (lane 4). Strain K10687 (lane 5) is a derivative of K10661 cured of pGB2-plac-cI. NusA protein was used as an internal control.

Studies with {lambda} demonstrate that during lysogeny, transcription of cI initiates at the pRM promoter, which is responsible for maintenance of CI expression from the repressed prophage (Fig. 1A). CI binding at the right {lambda} operator (OR) can both stimulate and repress transcription from pRM; thus, repressor expression is controlled autogenously during lysogeny (41, 50, 60). Like {lambda}, the 933W OR has three binding sites (48; J. S. Tyler, M. J. Mills, and D. I. Friedman, unpublished data). Under presumed physiological conditions, CI binding to two of the {lambda} OR binding sites activates transcription from pRM (49). However, at very high concentrations of repressor, CI binding to all three {lambda} OR sites represses transcription from pRM (50). The rexAB genes of {lambda}, like the stk gene of 933W, are located just downstream of the cI gene. Both rex genes are expressed by the repressed prophage by transcription initiating at pRM (52, 57), while rexB is also expressed from a promoter in rexA (Fig. 1A) (32, 33). Because the stk and rexAB genes are similarly located and oriented in their respective phage genomes, it was predicted that stk is coordinately expressed with cI by transcription initiating at pRM (48). To assess if stk is transcribed from the 933W pRM promoter and presumably coordinately expressed with cI, we analyzed Stk synthesis by a 933W lysogen under conditions in which pRM would be repressed but lysogeny would be maintained. Strain K10567, the 933W lysogen containing a 933W prophage with the stk::c-myc gene replacing stk, was transformed with a vector containing the 933W cI gene cloned downstream of the plac promoter, generating strain K10661. As a control, K10567 was also transformed with vector alone, generating strain K10660. Western blot analysis of protein obtained from K10660 and K10661 grown under inducing conditions for the plac promoter, with an anti-c-Myc antibody, were used to assess CI control of stk expression. These studies demonstrated that overexpression of the 933W CI protein inhibits synthesis of Stk-c-Myc by the prophage (Fig. 4, lane 4). Curing strain K10661 of the plasmid containing the cI gene resulted in restored expression of Stk-c-Myc from the prophage (Fig. 4, lane 5). We assume, as with {lambda}, high levels of repressor result in binding to all three OR operator sites, blocking transcription from pRM. Hence, these data suggest that Stk-c-Myc fails to be synthesized by strain K10661 because expression of Stk by the 933W prophage requires transcription initiating at the phage pRM promoter.

Conservation of stk. We next addressed the question of how widely stk is distributed by using Southern blot analyses of genomic DNA from STEC clinical isolates that were collected from a number of different outbreaks. DNA obtained from these STEC isolates was examined by low-stringency Southern blot analyses with a DNA probe targeted to the coding sequence of the Stk catalytic domain. In addition to the reference isolate EDL933, stk-like sequences were detected in four other independently obtained STEC clinical isolates (Fig. 5A and Table 1). stk-like genes were also identified in two uncharacterized clinical isolates (data not shown). In addition, we have identified the stk gene in the genome of clinical isolate 905 (data not shown) (54). Sato et al. (56) report the presence of the stk gene in the genome of the lambdoid phage Stx2{phi}-I, which was isolated from an O157:H7 clinical isolate associated with an outbreak of hemorrhagic colitis in Japan in 1996. In separate experiments, we failed to identify stk in six other clinical isolates (data not shown). We note that isolates containing an stk-like gene are not all of the same serotype and were collected over a course of approximately 15 years. This suggests that stk gives a selective advantage to the pathogenic bacteria, perhaps by contributing to their virulence. We note that not all disease-causing STEC isolates share the same collection of virulence genes (47). Therefore, the finding that some STEC isolates do not contain stk does not eliminate the possibility that Stk could contribute to virulence in those bacteria that contain the gene.



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  		FIG. 5. Identification of the stk gene in genomes of independently obtained STEC isolates. (A) Low-stringency Southern blot analysis of restriction enzyme-digested genomic preparations of STEC clinical isolates, with a probe targeted to the sequence encoding the catalytic domain of Stk. The bacterial isolate sources of the genomic DNA, with the number of the lane in which they are run, are as follows: EDL933 (lane 1), 93-111 (lane 2), OK-1 (lane 3), 86-24 (lane 4), 493-89 (lane 5), E32511 (lane 6), G5101 (lane 7), 3256-97 (lane 8), TB226A (lane 9), 928/91 (lane 10), 3007-85 (lane 11), B2F1 (lane 12), 3215-99 (lane 13), and DA-5 (lane 14). Descriptions of the clinical isolates are listed in Table 1. Included as a negative control, DNA isolated from strain K37, a nonlysogenic laboratory strain, did not show any evidence of hybridization with the probe in separate experiments (data not shown). (B) Genomic DNA from phages {phi}933W and {phi}E32511 digested with EcoRI and HindIII.

DNA sequencing showed that the stk-like genes found in genomes of the other STEC isolates all have 100% identity to stk of phage 933W (data not shown). To determine if the stk genes in these isolates reside in a lambdoid phage genome, the DNA flanking the stk gene in each isolate was sequenced. Analysis of the sequences downstream of stk revealed that in all of the stk+ isolates, this region is identical to the intergenic region between the stk and N genes of phage 933W (data not shown), a region that includes the phage pL promoter and the associated OL operator (Tyler et al., unpublished). Furthermore, sequence analysis of the region flanking the 5' end of stk revealed that all of these stk+ isolates contain the cI gene of 933W (data not shown). Therefore, in all of these isolates, stk is carried in a 933W prophage or a phage with the immunity region of 933W. We attempted to isolate the phages from the clinical isolates to determine how related they are to the original 933W phage. Only the stk-containing phage from isolate E32511, designated {phi}E32511, could be isolated. The products of restriction enzyme digestions were used to compare the genomes of {phi}E32511 and 933W. The EcoRI and HindIII restriction patterns were significantly different (Fig. 5B), indicating that although these phages share the same immunity region, including stk, they are likely different phages.

Analysis of the genetic structure of identified Stx-encoding phages indicated that although all known phages are members of the lambdoid family (27, 61), with shared genetic organization and regulatory mechanisms, there is still a great degree of diversity among these phages, with respect to morphology, genome sequence, host range, and accessory functions (37, 48, 51, 63, 67). Therefore, conservation of stk in the genomes of STEC-isolated phages or phage-like elements suggests that there has been selective pressure to maintain the stk gene, pressure that occurs presumably on the prophage in the growing lysogen. It is likely that factors encoded by a prophage that provide a selective advantage to the bacterial host enhance the propagation of the resident phage (reviewed in reference 7). Several bacterial pathogens express phage-encoded factors that contribute to their virulence and presumably facilitate propagation of the host bacterium. Similar to Stk, many of these factors are produced by the repressed prophage (reviewed in reference 22). Studies with derivatives of 933W with stk deleted failed to show any obvious difference in lytic growth or induction from the prophage state (data not shown). Therefore, it appears that Stk does not contribute to the propagation of 933W per se. Although the results of the latter studies are not definitive, taken together with the finding that Stk is expressed by the prophage, they bolster the argument that the stk gene has been conserved because it enhances growth of the lysogen, perhaps by contributing to the virulence of STEC.


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ACKNOWLEDGMENTS
 
We thank Victor DiRita, Larry Argetsinger, Karen O'Brien, Steve Juris, and Jack Dixon for help and advice. Victor DiRita is also thanked for comments on the manuscript.

This work was supported by Public Health grant AI11459-10. J.S.T. was supported in part by NIH training grant T32-GM08353.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Michigan, 5641 Medical Science Building II, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0620. Phone: (734) 763-3142. Fax: (734) 764-3562. E-mail: davidfri{at}umich.edu. Back


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Journal of Bacteriology, June 2004, p. 3472-3479, Vol. 186, No. 11
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.11.3472-3479.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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