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24B in the Escherichia coli genome, description of a novel integrase and evidence for a functional anti-repressorMicrobiology Research Group, Division of Integrative Biology, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK
Correspondence
Heather E. Allison
hallison{at}liv.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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phage. However, contrary to the superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage 
24B. Here, the 24B integrase gene is identified, and the preferred site of integration defined. Although an E. coli int gene was identified close to the 24B integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the E. coli genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to E. coli and are therefore unlikely to support phage integration. A 24B gene, possessing similarity to the well-characterized P22 ant gene, was identified. RT-PCR was used to demonstrate that ant is transcribed in a 24B E. coli lysogen, and expression of an anti-repressor is the likely explanation for the absence of immunity to superinfection. Demonstration of the ability of 24B to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the stx gene may lead to an increase in toxin production and consequently increased virulence.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Some integrases are involved in the integration and excision of phage genomes, whilst others are essential for the maintenance of plasmid copy number or elimination of chromosomal dimers (Chen et al., 2000; Sherratt et al., 2004; Van Duyne, 2001). The first tyrosine recombinase extensively studied in vitro was that of bacteriophage (Craig, 2001; Ptashne, 2004). The minimal substrates for integration are a 21 bp segment of host DNA (attB) that surrounds the point of crossover (OB) and 
240 bp of specific bacteriophage sequence (attP) that flanks the bacteriophage DNA crossover point (OP). A combination of phage- and bacterial-encoded proteins regulates complete integration of the bacteriophage genome into the host chromosome (Biswas et al., 2005).
Stx-phages are lambdoid phages that confer the ability to produce Shiga toxin (Stx) on their bacterial host. A partially successful attempt was made elsewhere to characterize the integrases and integration sites of seven Stx-phage isolates via a DNA-amplification strategy that employed oligonucleotide primer sets specific for the integrase genes from bacteriophages and 933W (Johansen et al., 2001). Bacteriophage 933W was one of the earliest characterized Stx-phages for which there is a complete sequence (O'Brien et al., 1984). Of the seven isolates induced and challenged with the primers, none was positive for -like integrase and only one was positive for 933W-like integrase, highlighting the need for a more universal integrase detection technique. The 933W-like integrases are the most common type associated with the Stx-phages sequenced to date (Johansen et al., 2001). The integrase from the short-tailed Stx-phage 24B, which has a relatively broad host range (James et al., 2001) and unusually is capable of multiple lysogeny (Allison et al., 2003), could not be identified using this published strategy, even though it shares a considerable amount of genomic DNA sequence with Stx-phage 933W (data not shown). In view of the sequence diversity found amongst integrases and the mosaicism inherent in bacteriophage populations (Hatfull et al., 2006; Johansen et al., 2001), there is considerable scope for Stx-phage integration directed by as yet uncharacterized factors.
An initial comparison of 28 integrase protein sequences revealed the presence of four conserved amino acid residues (Arg, His-X-X-Arg and Tyr), known as the RHRY motif, in the more highly conserved C-terminal domain. There is good evidence that the RHRY motif forms the active site of the enzyme. Subsequently, a comparison of 105 recombinases identified two boxes of amino acid similarity that were present in all known integrases: box I (A202–G227 in ), encompassing the His and both Arg residues; and box II (T306–D344 in ), flanking the Tyr residue (Esposito & Scocca, 1997; Nunes-Duby et al., 1998). These conserved regions were used to align the amino acid sequence of 32 inducible enteric phage integrases, resulting ultimately in their classification into eight distinct groups, for which a bank of 11 degenerate primer sets was designed to amplify all known integrases (Balding et al., 2005).
Bacteriophages frequently integrate within, or in the vicinity of, tRNA genes; however, in the limited number of Stx-phages described thus far, tRNA insertion sites are rarely preferred (Campbell, 2003; Herold et al., 2004). The model Stx-phage 933W and the non-inducible (remnant) phage VT2-Sakai, both encoding Stx2, are integrated into their respective hosts within the wrbA gene, which encodes a tryptophan repressor-binding protein (Plunkett et al., 1999), whilst remnant Stx1-phages CP-933V and VT1-Sakai are integrated into yehV (a putative transcriptional regulator) (Makino et al., 1999). The two Stx2-phages have completely identical integrases, as do the two Stx1-phages, with 62.6 % amino acid identity shared between the two groups. Other known Stx-phage integration sites include sbcB (Stx2-phage; Makino et al., 1999; Ohnishi & Hayashi, 2002), ssrA (cryptic CP-1639; Creuzburg et al., 2005), yecE (P27; Recktenwald & Schmidt, 2002), varphi297 (De Greve et al., 2002), various phages from an O157 : NM strain as well as various O26 strains (Bielaszewska et al., 2006, 2007, respectively), and Z2755 (phage-6220; Koch et al., 2003). The first two encode exonuclease I and 10Sa tmRNA, respectively, whilst the others encode hypothetical proteins of unknown function (Herold et al., 2004). The temperate bacteriophages characterized to date have a distinct preference for a single specific integration site within the host chromosome, although it cannot be ruled out that secondary sites may be occupied under some circumstances. Indeed, the temperate bacteriophages and P2 utilize various insertion sites when the primary site is unavailable (Barreiro & Haggard-Ljungquist, 1992; Rutkai et al., 2003).
It has been demonstrated unequivocally, through the use of differentially labelled, isogenic recombinant phages (24B : : Cat and 24B : : Kan), that double lysogens of a lambdoid Stx-phage are possible (Allison et al., 2003). Previously, this phenomenon had only been observed in via immunity mutant experiments or homologous recombination events (Calef, 1967; Freifelder & Kirschner, 1971). Clearly, the occurrence of multiple lysogens in a single host is likely to enhance the evolution and dissemination of bacteriophage-encoded genes throughout bacterial populations, with particular applied relevance for Stx-phages responsible for increasing the pathogenic potential of Escherichia coli hosts. The underlying mechanisms responsible for multiple lysogeny in E. coli infected with Stx-phage 24B are unknown, and it was the objective of this study to specifically identify the 24B integrase and the locations of the attB and the attP sites that are required for site-specific recombination. In addition, the factors that overcome the immunity system to permit multiple infections of isogenic temperate phages are investigated.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. E. coli K-12 strain MC1061-Rif was the host of choice for bacteriophage infection and lysogen production. JM109- pir was the propagation strain for suicide plasmids, while Invitrogen One Shot TOP10 cells were used for all other recombinant work. Bacteria were routinely cultured in phage buffer [2.5 %, w/v, Luria–Bertani (LB) broth (Merck) with 0.01M CaCl2] with or without 1.5 % (w/v) agar (Merck). Bacteriophage suspensions were routinely stored in the phage buffer at 4 °C. Where appropriate, the following antibiotics were added: rifampicin (3500 µg ml–1), ampicillin (100 µg ml–1), kanamycin (50 µg ml–1), chloramphenicol (50 µg ml–1), spectinomycin (100 µg ml–1), tetracycline (10 µg ml–1).
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). Double and multiple lysogens were created as described previously (Allison et al., 2003); briefly, a colony of a confirmed lysogen (obtained after either a primary or a secondary infection) was cultured to OD600 0.5 in phage buffer at 37 °C with shaking, and then infected with a differentially labelled phage to produce a double lysogen, resistant to two different antibiotics and capable of producing both phages. Multiple lysogens were produced as above, except that a double lysogen was infected with a differentially labelled phage.
PCR amplification parameters.
DNA was amplified using 1.25 U Expand High Fidelity DNA polymerase (Roche Diagnostics) in a reaction mix containing 1 mM dNTP mix, 1.5 mM MgCl2, proprietary buffer, 200 nM oligonucleotide primers (Table 2) and 100 ng template DNA. Cycling conditions consisted of an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing for 1 min at various temperatures (Table 2) and extension at 68 °C for 1 min kb–1, followed by a final extension at 68 °C for 7 min.
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24B-infected MC1061-Rif was digested with EcoRI, BamHI or HincII (New England Biolabs) restriction endonucleases according to the manufacturer's recommendations. Each digest (1 µg) along with 5 µl DIG-labelled DNA marker IV (Roche Diagnostics) was separated on a 0.75 % TAE agarose gel (40 mM Tris base, 20 mM glacial acetic acid, 1 mM EDTA, 0.75 %, w/v, agarose) for 4 h at 4 V cm–1. The DNA was transferred to a nylon membrane according to manufacturer's protocols (Roche Diagnostics). The DNA was UV cross-linked to the membrane using a UV Stratalinker 2400 (Stratagene) at 120 mJ. The membranes were placed in proprietary pre-hybridization solution for 1 h at 42 °C. The proprietary hybridization solution was then spiked with DIG-labelled probes (20 ng ml–1), which were allowed to hybridize overnight at 42 °C. Washing and blocking of the membrane was carried out to the manufacturer's guidelines. Bands were resolved by chemiluminescent detection using CDP-Star reagent (Roche Diagnostics).
Colony blots.
Agar plates supporting the overnight growth of putative transformants were incubated at 4 °C for 1 h and the colonies were then transferred to nylon membrane disks (Hybond-N, Amersham Pharmacia Biotech). The membranes were placed sequentially on Whatman 3MM paper soaked in denaturing solution (0.5 M NaOH, 1.5 M NaCl) for 15 min, neutralization solution (0.5 M Tris/HCl, pH 7.5, 3 M NaCl) for 15 min, and finally 2x SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.0) for 10 min. The DNA was UV cross-linked to the nylon membrane. Cell debris was removed mechanically between two sheets of Whatman 3MM paper soaked in distilled H2O. Hybridization and chemiluminescent detection were carried out as described above for Southern blotting.
Creation of an isogenic MC1061 intS mutant.
The CPS-53 (KpLE1) putative prophage integrase intS was amplified using intS Fwd AgeI and intS Rev SalI primers (Table 2
) using Expand High Fidelity polymerase. The DNA was purified using the Eppendorf Perfectprep gel extraction kit (Eppendorf) and cloned into Zero Blunt cloning vector (Invitrogen) according to the manufacturers' protocols. Plasmid from the resulting transformants was purified using the Qiagen Miniprep kit and subjected to PCR using intS Inverse Fwd AccI and intS Inverse Rev AccI oligonucleotides (Table 2). The resulting amplification product was purified and 1 µg digested with the restriction endonuclease AccI (New England Biolabs) for 1 h at 37 °C. The antibiotic-resistance cassette aadA, encoding resistance to spectinomycin, was amplified with aadA Fwd AccI and aadA Rev AccI oligonucleotides using Expand High Fidelity polymerase (Table 2). After subsequent purification, 1 µg amplification product was digested with the restriction enzyme AccI for 1 h at 37 °C. The spectinomycin-resistance cassette possessing AccI 3' overhangs was then cloned into the Zero Blunt backbone containing the intS flanking regions (268 and 224 bp) and compatible AccI cohesive ends. The resulting plasmid was purified using the Qiagen Miniprep kit, and the mutated intS was excised from the plasmid backbone by sequential digestion using the restriction endonucleases AgeI and SalI. Following heat-inactivation of the restriction endonucleases, the construct was ligated into AgeI/SalI-digested pJP560 suicide vector3. This plasmid is dependent upon pir for replication and was routinely propagated in the pir+ E. coli strain JM109. The intS-containing pJP5603 construct pPCMF
intS was introduced into MC1061 via electroporation (2.5 V, 25 µF, 200 
) by means of the Bio-Rad Gene Pulser (Bio-Rad Laboratories). Selection for kanamycin and spectinomycin resistance permitted the identification of clones produced by a single recombination event, which were confirmed by PCR and Southern hybridization. Passage of a single recombinant followed by screening for the loss of kanamycin resistance facilitated the identification of an intS knockout in MC1061intS, and ablation of the gene was confirmed by PCR.
Complementation of intS.
The putative prophage integrase intS was amplified with intS Fwd AgeI and intS Rev SalI primers and Expand High Fidelity DNA polymerase (Table 2) and cloned into the low-copy-number vector pKT230 (Table 1) to produce pPCMFintScomp according to standard cloning protocols (Sambrook et al., 1989). MC1061intS was used as the E. coli K-12 host.
Recombinant mutant phage construction.
The plasmid NTP707 was digested with the restriction endonuclease PstI, whilst the spectinomycin antibiotic-resistance cassette aadA was amplified with aadA Fwd PstI and aadA Rev PstI (Table 2) and digested with PstI. The two fragments were ligated together and the resultant plasmid, NTP707-aadA, was transformed into E. coli strain MC1061, which was subsequently infected with 24B : : Kan. Bacteriophages resulting from this infection were recovered by membrane filtration (0.2 µm pore-size, Millipore), and used to infect naïve MC1061. Lysogens produced by the infection were plated onto LB agar containing spectinomycin and replica-plated onto both LB agar plus kanamycin and LB agar plus tetracycline. Colonies displaying spectinomycin resistance but kanamycin and tetracycline sensitivity were deemed to be putative 24B : : Spec recombinant phage lysogens, and were further examined for the ability to produce plaques, as described previously (Allison et al., 2003; James et al., 2001).
RT-PCR.
Total RNA was isolated using the Qiagen RNeasy RNA isolation kit according to the manufacturer's guidelines. RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies), and the quality was assessed on a Syngene GeneGenius imaging system (Syngene Europe). Bacterial genomic DNA carryover was removed by incubation with RQ1 RNase-free DNase according to the manufacturer's guidelines (Promega).
Total RNA (5 µg) was combined with specific oligonucleotide primers (200 nM; Table 2), 1 mM dNTP mix, proprietary reaction buffer and 50 U BioScript reverse transcriptase (Bioline) in a total volume of 30 µl. The primers and RNA were heated to 70 °C for 5 min and cooled on ice prior to addition of the remaining reaction constituents. The mixture was incubated at 42 °C for 30 min, followed by a further 30 min at 50 °C to increase cDNA yields (according to the manufacturer's recommendations). The reaction was terminated by incubation at 70 °C for 10 min. The resultant cDNA was used directly as a template for subsequent PCR amplifications.
Sequence analysis and alignments.
GenBank database searches were carried out using various BLAST programs on the NCBI website (http://www.ncbi.nlm.nih.gov). In addition, the Colibri web server was used to locate specific short sequence patterns in the E. coli K-12 genome (http://genolist.pasteur.fr/Colibri). Sequence alignments were carried out with GeneDoc Multiple Sequence Alignment Editor and Shading Utility (http://www.psc.edu/biomed/genedoc; Nicholas et al., 1997), BioEdit Sequence Alignment Editor (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html; Hall, 1999) and CLUSTALW Multiple Sequence Alignment Program (http://www.ebi.ac.uk/clustalw/; Thompson et al., 1994).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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24B in the E. coli genome, the fact that the phage integrase would be located near to the site of integration in the bacterial lysogen was exploited (Campbell, 1994). To specifically identify the integrase of Stx-phage 24B, we used a bank of 11 degenerate PCR primers that had been designed to amplify all known integrase genes (Balding et al., 2005). When applied to a suspension of 24B phage particles, a DNA fragment of the expected size was generated with only one of the primer pairs, and this product was purified and sequenced (data not shown). BLASTN analysis of this sequence revealed 100 % identity to the intS integrase gene of the remnant prophage element CPS-53 (KpLE1), located within the genome of E. coli K-12 strain MG1655 (Blattner et al., 1997). A specific, DIG-labelled intS probe was amplified using the int Probe Fwd/Rev primers (Table 2) and subsequently used to probe restriction endonuclease-digested (EcoRI, BamHI or HincII) chromosomal DNA from naïve MC1061 or MC1061 24B lysogen cells by Southern hybridization. This enabled detection of a single DNA fragment in both the naïve MC1061 and the 24B lysogen chromosomes; however, the latter was 1 kb larger in size in both the EcoRI and BamHI digests (Fig. 1a). This size discrepancy suggested that the integration of bacteriophage 24B had occurred in the vicinity of the host intS gene (Fig. 1b). Thus, one or more of the multiple integration events previously reported for 24B in E. coli (Allison et al., 2003) might in fact have been directed by the host-cell intS gene. If the creation of double lysogens by this phage was in fact directed, at least in part, by IntS, then double lysogens of 24B are not a consequence of the phage, but are due to host functions, and the phage model of immunity to superinfection is not confounded. In order to experimentally address the role that IntS plays in the production of double 24B lysogens, an isogenic MC1061 mutant was created in which the endogenous intS gene was ablated. This mutant was constructed by replacing intS with a spectinomycin-resistance cassette, aadA. Although the yield of lysogens from infection of MC1061intS with 24B was significantly lower than that obtained when wild-type MC1061 was the host (P<0.05 in a parallel infection assay; data not shown), both single and double lysogens could nevertheless be produced in MC1061intS. Furthermore, lysogen yields could not be returned to the original values through complementation of the intS deletion by reintroduction of intS on the low-copy-number plasmid pKT230 (Bagdasarian et al., 1981). Lysogens are routinely detected in wild-type MC1061 from a single infection event of a naïve population at a rate of 1 in every 6724 cells. Double lysogens are routinely detected from a single infection of a single lysogen at a rate of 1 in every 4931 cells, and triple lysogens are routinely detected from a single infection of a double lysogen at a rate of 1 in every 339 cells (Table 3). Consequently, it can be concluded that the presence of an additional copy of intS on the E. coli genome does not explain the phenomenon of multiple lysogeny with 24B.
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24B integrase gene sequence, and to define the exact site of phage insertion, EcoRI-digested E. coli MC1061intS 24B lysogen genomic DNA fragments of 4.7 kb, corresponding to the increased band size (Fig. 1a), were excised and cloned into EcoRI pre-cut pGEM cloning vector. The resulting transformants were screened for ampicillin and spectinomycin resistance, and the presence of aadA in resistant colonies was confirmed by PCR amplification with aadA Fwd/Rev AccI (Table 2). In this way, 4.7 kb of DNA sequence containing the entire 24B int gene, the attB site and most of the intS gene interrupted by the spectinomycin cassette was obtained (Fig. 1b). Alignment of the DNA sequences from the genome sequence of E. coli K-12 strain MG1655 (Blattner et al., 1997) and the doubly resistant transformant enabled the identification of the integration point. As expected from the original Southern blot analysis, the insertion occurs almost 250 bp 5' of intS and within 75 bp of the site preferred by the bacteriophages Sf6 and HK620, both of which possess an intS-like integrase (Casjens et al., 2004; Clark et al., 2001). The extent of complementary sequence overlap between the 24B attP and the MC1061 attB sites was determined to be 24 bp (Fig. 2a). The 24B integrase identified here (accession no. EF397940) has only been found once before, on Stx-phage-86 (96 % identity; accession no. AB255463), which has yet to be described in the scientific literature. The amounts of overall amino acid sequence identity and similarity shared between the 24B int gene and intS were only 14 and 24 %, respectively. BLASTX analysis revealed a match to the conserved domain database for bacteriophage P4 integrase, whilst the closest matches in GenBank, other than phage-86, were to prophages of Photobacterium profundum and Shewanella frigidimarina (46 and 38 % identity, respectively; Fig. 3).
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24B DNA integrase sequence enabled the production of a DIG-labelled 24B-int-specific probe, which was allowed to hybridize to the DNA from two single lysogens (MC1061 carrying 24B : : Cat or 24B : : Kan) and from two double lysogens (MC1061 infected first with 24B : : Cat then 24B : : Kan and vice versa) digested with either EcoRI or AvaI. In total, from the four different DNA preparations, the int probe bound to DNA fragments that indicated the presence of four distinct integration sites (Fig. 4). The DNA fragments corresponding to the three novel insertion sites were excised and cloned to produce three subgenomic libraries. The presence of the int-containing sequence in each of these libraries was confirmed by PCR using 24B int Fwd/Rev primers (Table 2). An aliquot from each plasmid library was then used as template for a subsequent PCR amplification using one primer specific for phage sequence, Integ Fwd, and one specific to pGEM sequence, pGEM Fwd/Rev (Table 2). A 350 bp product was amplified from the library prepared from AvaI fragments of approximately 3 kb (Fig. 4, fragment II*), and the amplified product was sequenced. Approximately 150 bp were found to correspond to a second distinctive location in the host genome identified by a 100 % BLASTN match to an E. coli K-12 MG1655 hypothetical protein, yfbO (Blattner et al., 1997). The degree of homology between the core attP sequence and the second insertion site was less than the 24 bp overlap identified at the first site, but there was a consensus sequence motif (TATCYUTTTAWMTAA). The sequence TATC--TTTA--TAA, based upon this consensus sequence, was used to search the Colibri E. coli database for potential additional sites in the published E. coli K-12 genome sequence. This analysis yielded six possible sites, identified as putative integration sites A–F (PIA–F, lettered in order of their appearance in the genome), including the two already identified above. The first integration site, near the MC1061 intS gene, was identified in silico as site PID, and will be referred to as site I. The second site, identified by PCR amplification of the library possessing the 3 kb AvaI fragment, was identified as PIC in silico, and will now be referred to as site II. To investigate if any of the other sites identified in silico were occupied, PCR primers were designed to enable amplification across each of the putative integration sites: PIA, PIB, PIE and PIF (Table 2). Of these, only PCR amplification of DNA prepared from a double lysogen (MC1061 infected first with 24B : : Cat then 24B : : Kan) using the PIB-specific primer pair produced no DNA product, suggesting that this site was occupied. PCR amplification of the same DNA sample with PIB Fwd and Integ Fwd produced a 400 bp band. Sequence analysis of this band revealed 100 % identity (BLASTN) to a region of the E. coli K-12 MG1655 genome that lies 377 bp upstream of yfaL, which encodes an adhesion protein, and 265 bp downstream of nrdA, a gene that encodes a reductase 
-subunit (Blattner et al., 1997). This site will now be referred to as site III (Fig. 4).
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, transformants produced from the library created with 10 kb EcoRI fragments from a double lysogen (MC1061 infected first with 24B : : Kan then 24B : : Cat; Fig. 4, fragment IV*) were subjected to colony blot hybridization with a 24B int-specific DIG-labelled probe. The labelled int probe bound to several colonies, which were picked and confirmed to possess the target sequence by PCR using 24B int-specific primers (Table 2). The plasmids from three transformant clones were purified and sequenced. BLASTN analysis revealed that the final integration site indicated in Fig. 4 fell within the sgcA gene, which encodes a putative transport protein encoded on the KpLE2 phage-like element in E. coli K-12 MG1655 (Blattner et al., 1997). Alignment of the sequence to the other three insertion sites reduced the consensus from a motif containing 11 conserved residues to one containing only seven; however, the percentage similarity of site IV to site I (the original 24 bp overlap) was 54 %, while site III and site II were 50 and 62.5 % similar, respectively, to site I (Fig. 3b). Site IV was not identified in silico. The sequences of all insertion sites in the naïve E. coli MC1061 genome were compared to those found in the sequenced E. coli strain MG1655 (Blattner et al., 1997) using primer pairs Naïve 1–4 Fwd/Rev (Table 2) and found to possess 100 % identity to those sequences in strain MG1655. The putative integration sites PIA and PIF are located within regions thought to encode essential functions for E. coli (Gerdes et al., 2003), and are therefore unlikely to be utilized for phage integration to produce lysogens.
A third recombinant phage, 24B : : Spec, was constructed and used to infect an existing double lysogen culture. Putative triple lysogens were confirmed by screening for resistance to kanamycin, chloramphenicol and spectinomycin, as well as the ability to release all three marked phages upon induction. The 24B triple lysogen was subjected to further phage infection using all three of the labelled recombinant phages, in an attempt to identify an occupied PIE site, the only in silico-identified putative integration site in a non-essential gene for which actual 24B integration has not been demonstrated. This site does not share any known association with genes predicted to provide an essential function. However, this site was never found to be occupied.
Lysogen production by 24B has therefore been demonstrably extended to the ability to form triple lysogens, and four different sites of bacteriophage integration, which are scattered across the chromosome, have been identified within the E. coli host genome (Fig. 2b). However, the question of how the superinfection immunity model (Ptashne, 2004; Allison et al., 2003) is overcome remained.
Identification of an expressed anti-repressor gene
The entire sequence of the 24B immunity region, from OL through to cro (accession no. EF517298), was determined and found to be identical to the immunity region of the fully sequenced Stx-phage 933W. There are a few notable differences between the 24B immunity region and that of the bacteriophage immunity region, which have been described elsewhere (Fattah et al., 2000; Koudelka et al., 2004; Tyler et al., 2004). Briefly, OL possesses only two operator binding regions, the spacing of the promoters pR and pRM between the three binding sites within OR is different, and the third operator binding region lies within the coding region of the cI gene. There are two alternatives to explain the formation of double lysogens. Either the immunity region is sufficiently different from such that it has reduced function or there is an anti-repressor. Earlier work (Fattah et al., 2000; Koudelka et al., 2004; Tyler et al., 2004) suggests that the first option is unlikely. By examining sequences of other anti-repressor proteins (encoded by ant genes), a gene possessing 37 % homology to the P22 anti-repressor gene ant (accession no. NP_059643), was identified in unannotated large fragments of the 24B genome sequence. The Ant protein of P22 has the ability to inactivate the P22 repressor protein, which is analogous to the C1 repressor protein, essential for maintenance of lysogeny and homoimmunity to superinfection by this bacteriophage (Botstein et al., 1975; Susskind & Botstein, 1975). The P22 anti-repressor is also known to be capable of neutralizing the repressor (Schaefer & McClure, 1997). Alignment of the 24B anti-repressor protein with its P22 counterpart revealed significant sequence similarity between the C-terminal domains (82 % identity for the final 104 C-terminal residues), while the sequences of the N termini were distinct (Fig. 5). In contrast, alignment of 24B Ant to the 933W anti-repressor, which appears to be truncated and incomplete, produced an apparent low level of shared amino acid sequence (12 % sequence identity). In order to establish whether the ant gene is transcribed in 24B lysogens, total RNA from mid-exponential naïve and lysogen cultures was harvested. In addition, total RNA of mid-exponential E. coli MC1061 cultures undergoing 24B infection at two m.o.i. values (0.1 and 1.0) was also harvested. The RNA samples were subjected to RT-PCR with internal ant-specific primers (Table 2), expected to produce a 456 bp fragment. The use of 24B int Fwd/Rev primers, which should produce a fragment 414 bp in length, served as an internal positive control for the RT-PCR. In this way, the presence of ant mRNA transcripts was identified in the lysogen/infection samples, and these were absent in the control RNA harvested from the naïve E. coli cultures (Fig. 6). Thus, this 24B gene is actively expressed during bacteriophage infection and, as demonstrated for phage P22 (Susskind & Botstein, 1978), could explain the avoidance of homoimmunity leading to the creation of multiple lysogens by the lambdoid Stx-phage described here.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) via the horizontal transfer of virulence determinants (Blaisdell et al., 1996). Within bacteriophage populations, the modular theory of evolution describes the exchange of regions of the genome, usually of comparable or similar function, between integrated phages, whether they are inducible or remnant (Botstein, 1980). This process of modular substitution provides phages with substantial genomic diversity, and in turn can lead to the emergence of novel pathogens (Saunders et al., 2001; Sherratt et al., 2004). These recombination events between phages will occur while they are harboured within a bacterial host cell or when bacteriophages within a bacterial host are exposed to new phages during a fresh infection event (Brussow et al., 2004). The presence of multiple bacteriophage genomes within a host is limited by two major mechanisms: prophage loss or decay, and immunity to superinfection by a homoimmune phage (Brussow et al., 2004), the latter of which is the focus of this study. The ability of a temperate Stx-encoding bacteriophage (24B) to form double lysogens has been described previously (Allison et al., 2003), and this provides an intracellular environment for recombination to further enhance the continued expansion of diversity amongst Stx-phages (Allison, 2007). Here, we begin to characterize this phenomenon by describing four distinct integration sites utilized by 24B in an E. coli K-12 genome, and extend our observation of double lysogeny to achieve the production of a triple lysogen. Multiple insertion sites for bacteriophages in bacterial genomes have been described previously only when the primary site is either absent or occupied, i.e. they are not utilized concurrently (Barreiro & Haggard-Ljungquist, 1992; Rutkai et al., 2003).
Although 24B does possess an integrase gene with an active site similar to that of the intS gene of E. coli strain MG1655, and 24B does integrate near the integration site utilized by intS, the integration site for 24B is distinct from the integration site recognized by the E. coli intS gene product. Creation of an intS knockout in the genome of E. coli strain MC1016 did reduce the efficiency of lysogen formation, but double lysogens could still be formed. Furthermore, complementation of the intS gene did not restore the original phenotype, so it must be concluded that additional polar effects of the intS knockout are responsible for the measured drop in lysogen formation efficiency. The genes located immediately downstream of intS are yfdG, yfdH and yfdI, and they are predicted to be membrane proteins that play a role in outer-membrane biogenesis (Riley et al., 2006). The expression of these genes may have been affected in the intS knockout mutant, with concomitant effects on cell fitness and susceptibility to Stx-phage infection. Phage 24B does appear to prefer a single site (I) for integration, and this is near the E. coli chromosomal copy of intS. Site I possesses 24 bp of 100 % sequence identity to the attP site of 24B, and without fail it was occupied in all of the lysogen DNA preparations examined by Southern hybridization. The supplementary sites, which are scattered across the E. coli genome, do share a degree of conserved consensus sequence within the 24 bp overlap region of the primary site, and in all cases this consensus sequence has an overall identity in excess of 50 %. Although a further three potential integration sites were identified in the Colibri database (PIA, -E and -F), PIA and PIF interrupt the expression of two genes, insE5 and yfiF, respectively, predicted to encode essential functions for E. coli (Gerdes et al., 2003). A defined, minimal consensus sequence cannot yet be reported, as the production of at least five knockout mutants (I, II, III, IV and PIE) would be required before site-directed mutation experiments could be attempted in order to determine the minimal sequences necessary for integration. It is also clear from our data that the integrated 24B prophages in double and multiple lysogens are not tandemly linked to one another but are scattered across the genome, each having integrated into the chromosomal molecule independently. It was also found that a single infection event can result in a double integration event, as shown in Fig. 4, lanes 1 and 2. This chloramphenicol-resistant lysogen has, at some point, undergone two separate integration events with the same phage. The DNA sequence of 24B int identified here shares 100 % identity within the conserved boxes (Fig. 3) associated with catalytic activity at the protein level, and 96 % nucleotide identity overall with the int gene of the recently submitted Stx2-phage-86 (accession no. NC 008464) isolated from an enterohaemorrhagic E. coli serotype O86 : H– strain, which also possesses a 24 bp attP overlap region identical to the one carried by 24B. It is also interesting to note that the only area that these two phages appear to share is the integrase gene with its cognate att site. The sequence differences between the two integrase genes are external to the conserved box regions vital for catalytic activity. The location of phage-86 integration within the O86 : H– strain genome has not been described; however, given the integrase and attP overlap similarities with 24B, it can be suggested that both phages utilize the same site.
The precise mechanism underlying the non-functional 24B superinfection immunity system is as yet unclear. Alignment of the immunity regions of 24B and the model Stx-phage 933W (Plunkett et al., 1999) revealed 100 % identity in sequence conservation between the two phages (accession nos EF517298 and AF125520, respectively). Nevertheless, in contrast to bacteriophage , for both sequences the OR3 DNA-binding site is located within the cI ORF, possibly influencing the availability of the binding site to the repressor or Cro proteins in situ, and the OL3 DNA-binding site is completely absent (Koudelka et al., 2004; Tyler et al., 2004). Consequently, the combination of these factors may result in reduced efficiency of repression; however, the production of double 933W lysogens has not been reported. Alternatively, sequence data from 24B reveals the presence of a gene homologous to the ant gene of bacteriophage Lahn1 (Allison, 2007). Another homologue of this gene forms part of the immI anti-repressor operon in P22 and, when expressed, is responsible for the deactivation of the repressor protein along with the ensuing derepression of phage gene transcription (Botstein et al., 1975; Susskind & Botstein, 1975, 1978). The P22 ant gene product has also been shown to be effective against repressor cI (Susskind & Botstein, 1975). The presence of the ant gene product could go some way to explaining the absence of immunity or complete immunity to a superinfecting homoimmune phage. The data presented here do not provide definitive evidence that ant suppresses immunity to superinfection, but do provide supporting evidence that the 24B ant could be involved. In P22, ant expression is tightly regulated by two different proteins (Mnt and Arc) as well as a complementary transcript, sar (Susskind & Botstein, 1975). The sequences surrounding the 24B ant gene do not encode comparable control factors. In fact, the 24B ant gene is located 6.5 kb downstream of the cro gene and 1.2 kb upstream of the Q gene (data not shown). Our demonstration that the 24B anti-repressor gene is transcribed in a lysogenised host supports the hypothesis that the cI repressor protein is inactivated to an extent whereby an additional infection event is permitted, even if the infecting phage possesses an identical immunity region to an existing prophage. Although there is a 24B ant homologue in the 933W genome, it does not appear to be intact, as very little sequence identity is shared with 24B ant, and in 933W this homologue is located just downstream of the lysis gene. Further work will be required to confirm that the Ant protein interacts with the 24B repressor and to understand how and when ant is expressed.
In addition to insertion site identification, Southern hybridization analyses revealed the presence of a band that could only have been produced by AvaI restriction endonuclease digestion if the phage genome was circularized (Fig. 4), with the recognized AvaI sites lying at each distal end of the integrated prophage sequence, as extrapolated from 24B genome restriction analysis. The method of lysogen genomic DNA extraction excluded the isolation of extracellular free phage. Therefore, the extra-chromosomal phage DNA in a lysogen must either result from a surprising number of spontaneously induced, unreleased viral particles or, alternatively, from circularized plasmid-like pseudolysogens. Both circumstances are events that might have a deleterious outcome during a Shiga-toxin-producing E. coli (STEC) infection. In the first instance, there would be a very high level of spontaneous induction (which has been reported for Stx-phages 933W and H-19B; Livny & Friedman, 2004), and since stx gene expression is linked to the expression of the late genes, this might result in higher than expected levels of toxin. A pseudolysogenic state will amplify the number of stx genes within a bacterial cell that could be used as a template for Stx expression, a situation that might also result in higher than expected levels of Stx production. Increased Stx production might be expected to result in more severe STEC-mediated disease and higher rates of complication with sequelae such as haemolytic uraemic syndrome (HUS) (Siegler et al., 2001).
The ability of a single bacterium to harbour multiple phages, whether closely related or not, could have a profound impact on pathogen evolution, providing an increased gene pool for genetic exchange in situ (Allison, 2007) and possibly even supporting enhanced spontaneous mutation rates of duplicated/redundant phage genes that may ultimately result in the formation of new phage components that alter host range, etc. Furthermore, the presence of several stx gene copies within a host might in turn lead to amplification of toxin load and, upon lysis, manifestation of more severe disease pathology. The mechanisms that underpin the immunity patterns observed need to be further investigated, but evidence for an actively transcribed anti-repressor is an important first step. The potential of the anti-repressor to influence immunity and the effect of divergence of the immunity region structure from the established model are key factors that impact upon the evolution of Stx-phages and their role as drivers of the emergence of new Stx-producing bacterial pathogens.
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ACKNOWLEDGEMENTS |
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24B. Edited by: D. L. Gally
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Allison, H. E., Sergeant, M. J., James, C. E., Saunders, J. R., Smith, D. L., Sharp, R. J., Marks, T. S. & McCarthy, A. J. (2003). Immunity profiles of wild-type and recombinant Shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens. Infect Immun 71, 3409–3418.
Bagdasarian, M., Lurz, R., Ruckert, B., Franklin, F. C., Bagdasarian, M. M., Frey, J. & Timmis, K. N. (1981). Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16, 237–247.[CrossRef][Medline]
Balding, C., Bromley, S. A., Pickup, R. W. & Saunders, J. R. (2005). Diversity of phage integrases in Enterobacteriaceae: development of markers for environmental analysis of temperate phages. Environ Microbiol 7, 1558–1567.[CrossRef][Medline]
Barreiro, V. & Haggard-Ljungquist, E. (1992). Attachment sites for bacteriophage P2 on the Escherichia coli chromosome: DNA sequences, localization on the physical map, and detection of a P2-like remnant in E. coli K-12 derivatives. J Bacteriol 174, 4086–4093.
Bielaszewska, M., Prager, R., Zhang, W., Friedrich, A. W., Mellmann, A., Tschape, H. & Karch, H. (2006). Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic Escherichia coli O157 : NM. Appl Environ Microbiol 72, 1900–1909.
Bielaszewska, M., Prager, R., Kock, R., Mellmann, A., Zhang, W., Tschape, H., Tarr, P. I. & Karch, H. (2007). Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26 infection in humans. Appl Environ Microbiol 73, 3144–3150.
Biswas, T., Aihara, H., Radman-Livaja, M., Filman, D., Landy, A. & Ellenberger, T. (2005). A structural basis for allosteric control of DNA recombination by integrase. Nature 435, 1059[CrossRef][Medline]
Blaisdell, B. E., Campbell, A. M. & Karlin, S. (1996). Similarities and dissimilarities of phage genomes. Proc Natl Acad Sci U S A 93, 5854–5859.
Blattner, F. R., Plunkett, G., III, Bloch, C. A., Perna, N. T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K. & other authors (1997). The complete genome sequence of Escherichia coli K-12. Science 277, 1453–1462.
Botstein, D. (1980). A theory of modular evolution for bacteriophages. Ann N Y Acad Sci 354, 484–490.[Medline]
Botstein, K., Lew, K. K., Jarvik, V. & Swanson, C. A. (1975). Role of antirepressor in the bipartite control of repression and immunity by bacteriophage P22. J Mol Biol 91, 439–462.[CrossRef][Medline]
Boyd, E. F. & Brussow, H. (2002). Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends Microbiol 10, 521[CrossRef][Medline]
Brussow, H., Canchaya, C. & Hardt, W.-D. (2004). Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev 68, 560–602.
Calef, E. (1967). Mapping of integration and excision crossovers in superinfection double lysogens for phage lambda in Escherichia coli. Genetics 55, 547–556.
Campbell, A. (1994). Comparative molecular biology of lambdoid phages. Annu Rev Microbiol 48, 193–222.[CrossRef][Medline]
Campbell, A. (2003). Prophage insertion sites. Res Microbiol 154, 277–282.[Medline]
Casjens, S., Winn-Stapley, D. A., Gilcrease, E. B., Morona, R., Kuhlewein, C., Chua, J. E. H., Manning, P. A., Inwood, W. & Clark, A. J. (2004). The chromosome of Shigella flexneri bacteriophage Sf6: complete nucleotide sequence, genetic mosaicism, and DNA packaging. J Mol Biol 339, 379–394.[CrossRef][Medline]
Chen, Y., Narendra, U., Iype, L. E., Cox, M. M. & Rice, P. A. (2000). Crystal structure of a Flp recombinase–Holliday junction complex: assembly of an active oligomer by helix swapping. Mol Cell 6, 885–897.[Medline]
Clark, A. J., Inwood, W., Cloutier, T. & Dhillon, T. S. (2001). Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages. J Mol Biol 311, 657–679.[CrossRef][Medline]
Craig, N. L. (2001). Mobile DNA II. Washington, DC: American Society for Microbiology.
Creuzburg, K., Kohler, B., Hempel, H., Schreier, P., Jacobs, E. & Schmidt, H. (2005). Genetic structure and chromosomal integration site of the cryptic prophage CP-1639 encoding Shiga toxin 1. Microbiology 151, 941–950.
De Greve, H., Qizhi, C., Deboeck, F. & Hernalsteens, J. P. (2002). The Shiga-toxin VT2-encoding bacteriophage varphi297 integrates at a distinct position in the Escherichia coli genome. Biochim Biophys Acta 1579, 196–202.[Medline]
Esposito, D. & Scocca, J. J. (1997). The integrase family of tyrosine recombinases: evolution of a conserved active site domain. Nucleic Acids Res 25, 3605–3614.
Fattah, K. R., Mizutani, S., Fattah, F. J., Matsushiro, A. & Sugino, Y. (2000). A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity. Genes Genet Syst 75, 223–232.[CrossRef][Medline]
Freifelder, D. & Kirschner, I. (1971). The formation of homoimmune double lysogens of phage lambda and the segregation of single lysogens from them. Virology 44, 633–637.[CrossRef][Medline]
Gerdes, S. Y., Scholle, M. D., Campbell, J. W., Balázsi, G., Ravasz, E., Daugherty, M. D., Somera, A. L., Kyrpides, N. C., Anderson, I. & other authors (2003). Experimental determination and system level analysis of essential genes in Escherichia coli MG1655. J Bacteriol 185, 5673–5684.
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
Hatfull, G. F., Pedulla, M. L., Jacobs-Sera, D., Balázsi, G., Ravasz, E., Daugherty, M. D., Somera, A. L., Kyrpides, N. C., Anderson, I. & other authors (2006). Exploring the mycobacteriophage metaproteome: phage genomics as an educational platform. PLoS Genet 2, e92[CrossRef][Medline]
Herold, S., Karch, H. & Schmidt, H. (2004). Shiga toxin-encoding bacteriophages – genomes in motion. Int J Med Microbiol 294, 115–121.[CrossRef][Medline]
James, C. E., Stanley, K. N., Allison, H. E., Flint, H. J., Stewart, C. S., Sharp, R. J., Saunders, J. R. & McCarthy, A. J. (2001). Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage. Appl Environ Microbiol 67, 4335–4337.
Johansen, B. K., Wasteson, Y., Granum, P. E. & Brynestad, S. (2001). Mosaic structure of Shiga-toxin-2-encoding phages isolated from Escherichia coli O157 : H7 indicates frequent gene exchange between lambdoid phage genomes. Microbiology 147, 1929–1936.
Kaniga, K., Delor, I. & Cornelis, G. R. (1991). A wide-host-range suicide vector for improving reverse genetics in Gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109, 137–141.[CrossRef][Medline]
Koch, C., Hertwig, S. & Appel, B. (2003). Nucleotide sequence of the integration site of the temperate bacteriophage 6220, which carries the Shiga toxin gene stx(1ox3). J Bacteriol 185, 6463–6466.
Koudelka, A. P., Hufnagel, L. A. & Koudelka, G. B. (2004). Purification and characterization of the repressor of the Shiga toxin-encoding bacteriophage 933W: DNA binding, gene regulation, and autocleavage. J Bacteriol 186, 7659–7669.
Livny, J. & Friedman, D. I. (2004). Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system. Mol Microbiol 51, 1691–1704.[CrossRef][Medline]
Makino, K., Yokoyama, K., Kubota, Y., Yutsudo, C. H., Kimura, S., Kurokawa, K., Ishii, K., Hattori, M., Tatsuno, I. & other authors (1999). Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157 : H7 derived from the Sakai outbreak. Genes Genet Syst 74, 227–239.[CrossRef][Medline]
Nicholas, K. B., Nicholas, H. B., Jr & Deerfield, D. W., II (1997). GeneDoc: analysis and visualization of genetic variation. EMBnet News 4 (2). http://www.embnet.org/files/shared/EMBnetNews/embnet_news_4_2.pdf.
Nunes-Duby, S. E., Kwon, H. J., Tirumalai, R. S., Ellenberger, T. & Landy, A. (1998). Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res 26, 391–406.
O'Brien, A. D., Newland, J. W., Miller, S. F., Holmes, R. K., Smith, H. W. & Formal, S. B. (1984). Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea. Science 226, 694–696.
Ohnishi, M. & Hayashi, T. (2002). Genetic diversity of enterohemorrhagic Escherichia coli. Nippon Rinsho 60, 1077–1082 (in Japanese).[Medline]
Penfold, R. J. & Pemberton, J. M. (1992). An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene 118, 145–146.[CrossRef][Medline]
Plunkett, G., III, Rose, D. J., Durfee, T. J. & Blattner, F. R. (1999). Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157 : H7: Shiga toxin as a phage late-gene product. J Bacteriol 181, 1767–1778.
Ptashne, M. (2004). A Genetic Switch: Phage Lambda Revisited, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Recktenwald, J. & Schmidt, H. (2002). The nucleotide sequence of Shiga toxin (Stx) 2e-encoding phage 
P27 is not related to other Stx phage genomes, but the modular genetic structure is conserved. Infect Immun 70, 1896–1908.
Rice, P. A. (2005). Resolving integral questions in site-specific recombination. Nat Struct Mol Biol 12, 641[CrossRef][Medline]
Riley, M., Abe, T., Arnaud, M. B., Berlyn, M. K., Blattner, F. R., Chaudhuri, R. R., Glasner, J. D., Horiuchi, T., Keseler, I. M. & other authors (2006). Escherichia coli K-12: a cooperatively developed annotation snapshot – 2005. Nucleic Acids Res 34, 1–9.
Rutkai, E., Dorgai, L., Sirot, R., Yagil, E. & Weisberg, R. A. (2003). Analysis of insertion into secondary attachment sites by phage and by int mutants with altered recombination specificity. J Mol Biol 329, 983–996.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Saunders, J. R., Allison, H. E., James, C. E., McCarthy, A. J. & Sharp, R. (2001). Phage-mediated transfer of virulence genes. J Chem Tech Biotech 76, 662–666.[CrossRef]
Schaefer, K. L. & McClure, W. R. (1997). Antisense RNA control of gene expression in bacteriophage P22. I. Structures of sar RNA and its target, ant mRNA. RNA 3, 141–156.[Abstract]
Sherratt, D. J., Soballe, B., Barre, F. X., Filipe, S., Lau, I., Massey, T. & Yates, J. (2004). Recombination and chromosome segregation. Philos Trans R Soc Lond B Biol Sci 359, 61–69.
Siegler, R. L., Pysher, T. J., Tesh, V. L. & Taylor, F. B., Jr (2001). Response to single and divided doses of Shiga toxin-1 in a primate model of hemolytic uremic syndrome. J Am Soc Nephrol 12, 1458–1467.
Susskind, M. M. & Botstein, D. (1975). Mechanism of action of Salmonella phage P22 antirepressor. J Mol Biol 98, 413–424.[CrossRef][Medline]
Susskind, M. M. & Botstein, D. (1978). Molecular genetics of bacteriophage P22. Microbiol Rev 42, 385–413.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Tyler, J. S., Mills, M. J. & Friedman, D. I. (2004). The operator and early promoter region of the Shiga toxin type 2-encoding bacteriophage 933W and control of toxin expression. J Bacteriol 186, 7670–7679.
Van Duyne, G. D. (2001). A structural view of Cre-loxP site-specific recombination. Annu Rev Biophys Biomol Struct 30, 87–104.[CrossRef][Medline]
Willshaw, G. A., Smith, H. R., Scotland, S. M., Field, A. M. & Rowe, B. (1987). Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes. J Gen Microbiol 133, 1309–1317.
Received 18 July 2007;
revised 13 September 2007;
accepted 24 September 2007.
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