Our first attempts at using the protocol described by Datsenko and Wanner 20 to construct recombinant prophages were unsuccessful. After several trials, we decided to modify the protocol. The modifications allowed the use of this methodology with the special characteristics of our strains, which carry inducible Shiga toxin-converting prophages.

The most obvious drawback was the spontaneous activation of the phage's lytic cycle during the process. This affirmation is supported by the isolation of phage DNA from the supernatants of the bacterial cultures without previous induction (data not shown). This increased prophage excision and/or phage release, which significantly reduced the efficacy of recombinant clone formation. Two possible causes of this failure were considered. Possibly, excision of the prophage DNA from bacterial DNA occurred without phage particle formation. In this case, the target gene (stx) where the amplimer must recombine would be lost, thus hindering correct recombination. Alternatively, after excision, phage particles were formed and released from the cells by lysis. In this case, a significant proportion of bacterial cells would be lysed, reducing the number of cells susceptible to transformation. Both of these scenarios would lead to a reduction in the efficacy of obtaining recombinant clones. The first cause could not be checked, but the second one was experimentally confirmed by the moderate reduction of the OD₆₀₀ observed in the cultures of the lysogens during the process, compared with the OD₆₀₀ of E. coli C600 or DH5α cultures (non-lysogens) used as a control.

The spontaneous induction of the lytic cycle could be due to several causes that would activate the SOS response. For example, the double application of the protocol to prepare electrocompetent cells (firstly to transform plasmid pKD46 and secondly to transform the PCR amplimer) which may activate the stress response in the bacterial cell. In any case, we assayed several modifications of the original protocol to optimize our application. These modifications of each step and the results obtained are described in this section.

The tet and cat genes were introduced in the stx₂ gene of prophages: ØA9, ØA312, ØA534, ØA549, ØA557, ØVTB55 and 933W. The genes were placed at position 251 bp of the stx₂-A subunit and 264 bp upstream of the end of the stx₂-B subunit. The identification and characterisation of the respective gene in each recombinant prophage was achieved by PCR and sequencing.

The induction of stx₂-phages from the six recombinant lysogens had slightly different kinetics than the original lysogens. Nevertheless, all recombinant lysogens conserved their lytic capacity after incorporating the antibiotic resistance gene. As previously described 3334, plaques produced by stx-phage are usually poorly visible or turbid on the top agar layer. Therefore, confirmation of the presence of infective recombinant phages was achieved by plaque blot hybridization with specific cat or tet probes (data not shown).

The antibiotic resistance cassette appears to remain stable within the phage genome, as observed after four steps of subculture without antibiotic selection. However the long-term stability of the marker gene in the phage genome without antibiotic selection remains to be elucidated.

To evaluate the capacity of the recombinant phages to infect and convert new E. coli strains, suspensions of the recombinant phages were prepared. Transduction of E. coli DH5α and E. coli C600 was performed. All phages produced new transductants, which conferred resistance to the appropriate antibiotic on DH5α or C600. Presence of the recombinant prophages in the host strain was confirmed by PCR and plaque hybridization analysis (Figure 2).

Some other authors have produced recombinant stx-phages 91516 for different purposes. Schmidt et al. 9 used phage φ3538 to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. Allison et al., 15 used recombinant phages to show the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. Acheson et al., 16 generated a recombinant Shiga toxin 1-converting phage H-19B to facilitate the study of intestinal transmission of stx₁-phages.

In the present work we have generated different recombinant phages with two antibiotic resistance genes to use them for different purposes. The use of these phages will allow analyse transduction in different matrices, as food or water samples. It would also be interesting to evaluate the phage induction and the transduction after different processes applied for food or water treatments, such as high temperature or high hydrostatic pressure (HHP), which has been reported as a method that can generate an increase in the induction of the lytic cycle of certain stx-phages 35.

The recombinant phages would be also useful tools to evaluate the ability of stx-phages to generate double lysogens and to evaluate whether the double lysogeny is really favoured in STEC, as some observations done in water environments or in strains isolated from humans and animals would suggest 15333637.

The bacteriophages ØA9, ØA312, ØA534, ØA549, ØA557, ØVTB55 used in the experiments were induced from selected STEC strains described elsewhere 36 and consisting of serotypes O157:H7 and O2:H27.

E. coli laboratory strains DH5α and C600 were used as host strains in some of the experiments described below. The aforementioned bacteriophages were introduced in these receptor strains as mentioned elsewhere 36 to generate the lysogens: C600(A9), DH5α(534), DH5α(557), DH5α(VTB55), DH5α(312) and DH5α(549).

Bacteriophage 933W 26 was induced from E. coli C600(933W), as described below, and used as a reference stx-bacteriophage.

Bacterial strains were grown in Luria-Bertani (LB) broth and on LB agar. The LB medium was supplemented with: ampicillin (Ap) (100 μg/ml); chloramphenicol (Cm) (5 μg/ml); and tetracycline (Tc)(5 μg/ml), when needed. SOB and SOC media 45 were used to prepare electrocompetent cells and for recovery after transformation. TSB medium supplemented with 5 mM CaCl₂ was used for the preparation of phage lysates

Plasmid pKD46 (Genbank AY048746) was used for the expression of Red recombinase 20. Plasmid pACYC184 (Genbank X06403) 46 was used to obtain the tetracycline resistance gene (tet). Plasmid pKD3 (Genbank AY048742) 20 was used to obtain the chloramphenicol acetyl transferase gene (cat), which confers resistance to chloramphenicol. Plasmid pBC-SK+ (Stratagene Inc. Amsterdam, the Netherlands) was used as a control for the transformation. All vectors were purified using the Qiagen Plasmid Midi purification kit (Qiagen Inc., Valencia, USA).

PCR reactions were performed using a GeneAmp PCR system 2400 (Perkin-Elmer, PE Applied Biosystems, Barcelona, Spain.). One μl of cellular suspension obtained from three-four colonies was used for the PCR amplification. The oligonucleotides used in this study are described in Table 3.

Construction of the amplimers containing tet and cat genes, inserted in the stx₂ gene, was performed as follows. The primer pairs Tc5'-Tc3'and Cm5-Cm3 (Table 3) were used for the amplification of the tet and cat genes respectively, with an annealing temperature of 54°C and elongation time of 45 s. Primer pairs amplified from the stxA₂ subunit initial codon to the 5' region of each resistance cassette (5' fragment) and from the 3' region of each resistance cassette to the final codon of the stxB₂ subunit (3' fragment) (Fig 1). The conditions used for all primer combinations were an annealing temperature of 41°C and elongation time of 45 s. The amplimers obtained were S2Aup/Tc5-stx (290 bp), S2Aup/Cm5-stx (291 bp) and GK4/Tc3-stx (281 bp) and GK4/Cm3-stx (281 bp).

Amplimers of the 5' fragment, the respective resistance gene and the 3' fragment for each resistance gene were annealed at their overlapping region (underlined letters in Table 3). They were then amplified by PCR as a single fragment with the external primers S2Aup and GK4 with an annealing temperature of 41°C and an elongation time of 2 min. The fusion product was amplified again using primer pair S2Aup/GK4. It was then excised from the gel and purified using the Qiaquick Gel Extraction Kit (Qiagen Inc., Valencia, USA). The final product was used for the transformation in the lysogens.

A DNA fragment of the stx₂A gene obtained with primers UP378/LP378, the tet gene and the cat gene, resulting from amplification with the respective primers (Table 3), were labeled with digoxigenin and used as probes. The probes were labeled by incorporating digoxigenin-11-deoxy-uridine-triphosphate (Roche Diagnostics, Barcelona, Spain) during PCR, as described in Muniesa et al., 36.

Plaque blot was performed using Nylon-N+ membranes (Hybond N+, Amersham Pharmacia Biotech, Spain) 47.

Colony hybridization was performed as previously described 37. The membranes were washed in a solution consisting of 1% SDS, 2× SSC for five min at 50°C. They were then washed in 0.1% SDS, 2× SSC for 5 min at room temperature and finally in 2× SSC for 5 min at room temperature.

Membranes were pre-hybridized with standard pre-hybridization solution at 68°C for 2 hours. Stringent hybridization was achieved with the DIG DNA Labelling and Detection Kit (Roche Diagnostics, Barcelona, Spain), according to the manufacturer's instructions. The DIG-labeled probes were prepared as described above.

Electroporation-competent cells were prepared from 10–50 ml of cultures in SOB medium and concentrated by centrifugation at 3000 × g for 5 min. They were then washed in 2 ml of ice-cold double distilled water. After four washing steps, the cells were suspended in 15–100 μl of ice-cold double-distilled water. The cells were mixed with the corresponding amount of DNA (plasmid or PCR-amplified, see results) in an ice-cold microcentrifuge tube and transferred to a 0.2 cm electroporation cuvette (Bio-Rad, Inc.). The cells were electroporated at 2.5 kV with 25 F and 200 ohm resistance. Immediately after electroporation, 1 ml of SOC medium 47 was added to the cuvette. The cells were transferred to a 17 by 100 mm polypropylene tube and recovered in SOC medium for 1–4 hours at either 30°C (for temperature-sensitive plasmids) or 37°C, without shaking. Cells were concentrated ten-fold from a 1 ml culture before plating on selective media.

A protocol modified from the one-step inactivation method using the Red recombinase system, as proposed by Datsenko and Wanner 20, was performed to obtain recombinant phages. Antibiotic resistance cassettes were inserted inside the truncated stx₂ gene of each phage to obtain recombinant phages carrying the resistance markers. For this purpose, plasmid pKD46 encoding the Red recombinase was transformed by electroporation, as described above. This was performed in electrocompetent cells prepared from 5 ml of the culture (approximately 5 × 10⁹ CFU/ml) of lysogens: C600(A9), DH5α(534), DH5α(557), DH5α(VTB55), DH5α(312), DH5α(549) and C600(933W). Transformation of the vector was confirmed by PCR, using the primer pair RR46up/lp. In a second step, the transformation of 30 μl of each PCR amplimer (corresponding to 0.1–0.5 μg of amplified DNA) containing the stx₂ gene, truncated by insertion of the respective resistance gene and prepared as described above (see PCR techniques), was performed in electrocompetent cells. These cells were prepared from 50 ml cultures (approximately 5 × 10¹⁰ CFU/ml) of each lysogen containing the pKD46 plasmid, grown at 30°C in SOB medium with ampicillin and 0.1 M of L-arabinose to an OD₆₀₀ of 0.6. After recovery in SOC medium and incubation for 4 hours, recombinant clones were selected on medium containing the appropriate antibiotic. Presumptive colonies were confirmed by PCR, using the rho primer and the respective primer for each antibiotic cassette. Cycling times and temperatures were according to the properties of the primer pairs. Positive clones were also further confirmed by sequencing.

PCR amplimers of the stx gene containing each antibiotic resistance gene and PCR amplimers obtained to characterise recombinant phages were sequenced. The oligonucleotides used for sequencing are described in Table 3.

Sequencing was performed with the ABI PRISM Big Dye III v.1 Terminator cycle Sequencing Ready reaction Kit (Perkin Elmer, Applied Biosystems, Spain) in an ABI PRISM 3700 DNA Analyzer (Perkin Elmer, Applied Biosystems, Spain), according to the manufacturer's instructions. All sequencing was performed in duplicate.

Nucleotide sequence analysis and searches for homologous DNA sequences in the EMBL and Genbank databases were performed with the GCG Wisconsin Package Version 10.2, Genetics Computer Group, Madison, Wisc. BLAST analyses were carried out with the tools available on the web 48.

Lysogens were grown from single colonies in TSB medium supplemented with 5 mM CaCl₂ at 37°C to the exponential growth phase. Growth was measured with a spectrophotometer (Spectronic 501, Milton Roy. Belgium). At OD₆₀₀ = 0.5, Mitomycin C was added to the cultures to a final concentration of 0.5 μg/ml. Cultures were then further incubated overnight. The induced cultures were centrifuged at 10,000 × g for 10 min and the supernatants were filtered through low protein-binding 0.22-μm-pore-size membrane filters (Millex-GP, Millipore, Bedford, MA).

To evaluate the transduction capacity of the recombinant phages, they were used to convert E. coli DH5α and/or C600 as previously described 9. Antibiotic resistant colonies were tested by colony blot and confirmed by PCR amplification of the truncated stx₂ gene containing the antibiotic marker.