3.2.1. Culture of the Host Strain and Phage Infection
1. Typical host strains recommended are E. coli DH5a, E. coli C600, or E. coli O157:H7, ATCC 43888 (Stx2~). However, other host strains could be used for a given experiment.
2. The host strain is inoculated in LB until an optical density of 0.1 is attained at 600 nm (containing approx 2 x 108 cells/mL).
In order to determine the presence in sewage of bacteriophages carrying the Stx2 gene, which is detectable by DNA amplification and able to infect the host strain, bacteriophage enrichment cultures are performed as follows.
1. Sewage samples are centrifuged at 12,000g for 30 min to remove particulate material, filtered through a 0.22-^m low-protein-binding membrane (Millex-GV, Millipore) to remove bacteria, and treated with DNase and RNase for 30 min at 37°C to eliminate free DNA and RNA.
2. Then volumes of 100, 10, 1, 0.1, 0.01, and 0.001 mL of the sample are added to 100 mL of liquid cultures in logarithmic growth phase of the host strain, which does not possess the gene for the Stx2 (see Notes 1 and 2).
3. LB culture medium is then added to a final volume of 250 mL.
4. After overnight incubation, aliquots of the enrichment cultures are centrifuged at 12,000g for 30 min.
5. The bacteriophages present in the supernatants are filtered through a 0.22-^m low-protein-binding membrane and then treated once more with 10 U of Dnase/mL for 30 min. All these steps are focused on eliminating bacteria, cell debris, and free DNA present in the supernatant of the enrichment cultures, which could interfere with the following steps. In this step, 10 ml of the supernatant may be stored at -20°C for posterior analysis of the proteins present in the supernatant (see Note 3).
DNA is extracted from bacteriophages according to Sambrook et al. (12). The same protocol is used for phages directly isolated from sewage or for phages obtained from the supernatant of an enrichment culture.
1. Bacteriophages present in a 1-mL sample are digested by addition of 20 mMEDTA, 0.5% SDS, and 50 ^g/mL of proteinase K and incubated at 45°C for 1 h.
2. Extraction of DNA is performed with phenol/chloroform/isoamyl alcohol (25:24:1) and centrifugation at 16,000g for 5 min (see Note 4).
3. After centrifugation, the upper part is recovered and the DNA present is precipitated with 10% 3 M sodium acetate and 2 vol of frozen absolute ethanol. Precipitation is allowed for at least 2 h at -70°C (see Note 5).
4. After precipitation, a pellet containing phage DNA is recovered by centrifugation at 16000g for 30 min.
5. DNA is washed with 200 ^L of 70% ethanol and centrifuged at 16,000g for 15 min.
6. After centrifugation, the supernatant is discarded. The washing step is repeated twice (see Note 6).
7. The washed DNA is dried (air-dried or dried with a speed vacuum if available) and dissolved in 20 ^L of double-distilled water. One microliter of the DNA solution is used for the PCR amplification.
1. First-round PCR DNA amplification for specific detection of the Stx2 gene is performed with the two primers described above at a concentration of 30 mM each, at an annealing temperature of 55°C for 1 min for 30 cycles. Primers were selected from the DNA sequence of the Stx2 gene, aligned with previously published sequences, and evaluated against the sequences of the EMBL data bank by the FastA program of the Genetics Computer Group package. Other primers can be used for different purposes.
2. One microliter of the first-round PCR is used for the nested PCR at the same conditions as the first-round PCR (see Note 7).
3. Five microliters of the amplified DNA mixture is analyzed for amplification products by electrophoresis on a 2% agarose gel stained with ethidium bromide.
4. To estimate the number of phages carrying the Stx gene, the MPN method (13) could be used. For each given volume, all positive results of nested PCR are considered in relation to the total number of experiments assayed for the same volume. For example, from three different samples from site A, detection was made of three positives of 10 mL, two positives of 1 mL, one of 0.1 mL, and no positives for 0.01 and 0.001. The combination to be applied to the MPN is 3:2:1:0:0. These values will give an estimation of 147 phages containing Shiga toxin 2 in a 100-mL sample. Performance of more replicas of the same volume will give a more accurate final estimation.
Several methods could be used to label a probe. Short oligonucleotides could be purchased directly labeled. Another useful method is labeling with DIG-11-dUTP. For this purpose the amplimer of a PCR (or nested PCR) reaction is directly labeled with DIG.
1. From a positive control DNA, a standard PCR reaction is performed but adding 1 ^L of the nucleotide mix for labeling, which has a lower concentration of dTTP (10 mM dATP, 10 mM dGTP, 10 mM dCTP, 7 mM dTTP).
2. To substitute for this lack of dTTP, 3 ^L of DIG-11-dUTP (1 mM) are added.
3. The other PCR reagents are used in the standard concentrations, and the final volume is adjusted with double-distilled water.
4. The PCR reaction is carried on by using the corresponding PCR program according to the primers.
5. The DIG-labeled PCR product will be a bit longer than the nonlabeled product, thus confirming efficiency of labeling. The probe should be purified before use with a suitable PCR purification kit.
Hybridization to confirm PCR and/or nested PCR results could be performed by dot blot or Southern blot (14) of the amplification mixture. If a custom-made oligonucleotide is used, it should be selected from the DNA sequence of the Stx2 gene and placed inside the sequence of the obtained amplimer.
1. For dot blot, 5 ^L of each PCR product is blotted onto a nylon membrane and fixed by exposure of the membrane to UV light for 3 min at each side.
2. For Southern blot, DNA is transferred to the nylon membrane from the agarose gel by capillary blotting (12). Once transferred, DNA is fixed to the membrane by exposure of the membrane to UV light as described in step 1 just above.
1. Introduce the membrane in a plastic bag or hybridization tube with forceps, and add 1 vol of hybridization buffer (7 mL buffer/10 cm2 of the nylon membrane).
2. Equilibrate the membrane with hybridization buffer at the hybridization temperature for 1 h. Close the bag with a thermal sealer.
3. Add 3.5 ^L of DIG-labeled probe per 7 mL of hybridization buffer. Incubate at the hybridization temperature overnight.
4. Place the membrane in a Petri dish and wash the membrane twice by gently shaking in 2X SSC + 0.01% SDS for 5 min at room temperature.
5. Place the membrane into the hybridization tube or bag again. Wash the membrane twice in 0.04X SSC + 0.01% SDS for 15 min at the hybridization temperature.
6. Equilibrate the membrane for 5 min at RT in washing buffer by gently shaking.
7. Equilibrate the membrane for 30 min at RT with buffer 2.
8. Add 10 ^L of the antibody anti-DIG 10 mL of buffer 2. Incubate the membrane for 30 min at RT with gentle shaking.
9. Wash the membrane twice by shaking with washing buffer for 15 min at RT.
10. Equilibrate the membrane for 5 min in buffer 3 (see Note 8).
11. Place the membrane in a plastic bag and add 10 mL of buffer 3 and 40 ^L of NBT/BCIP. Seal the bag with a thermal sealer. Store the membrane in the dark at RT until the signal is visible. Time is dependent on the intensity of the reaction. The reaction is stopped by washing the membrane with distilled water.
3.7. Detection of the Protein Encoded by the Gene Carried by Bacteriophages in the Supernatant of the Enrichment Cultures
After infection of the host strain, the phages may have transferred the gene coding for the toxin to the chromosome of the bacterial cell. Thus, the bacteria were able to produce the protein after the infection. This phenomenon is known as lysogenic con version. The protein produced can be detected in the supernatant of the enrichment culture by using the specific antibody against the toxin.
The presence of the toxin in the supernatants of bacteriophage enrichment cultures could be analyzed by Immunoblot or Western blot (15). Immunoblot is easy and faster and is recommended for positive/negative results. If the objective is to identify the protein, then a Western blot is recommended. In both cases, protein should be recovered from the supernatant of the enrichment cultures.
3.7.1. Recovery of the Protein Fom the Supernatant of the Enrichment Cultures
1. Ten milliliters of each supernatant are concentrated 10-fold by passage through 10-K cutoff filtration membrane microconcentrators (Microsep, Millipore) by centrifugation at 16,000g at 4°C.
2. Then 15 ^L of each concentrated supernatant could either be directly blotted onto the nitrocellulose membrane for immunoblot or subjected to SDS-PAGE in slab gels for Western blot.
3.7.2. Protein Electrophoresis
1. A 12% polyacrylamide slab gel is prepared and samples are mixed 1:1 with SDS loading buffer.
2. The samples should be boiled for 5-7 min before loading in the gel. Keep one of the lanes for a prestained marker.
3. Meanwhile, prepare the vertical electrophoresis and fill it with running buffer. Run the electrophoresis at 100 V for around 1.5 h (see Note 9).
3.7.3. Protein Electrotransfer to the Membrane (Western Blot)
Transference is achieved by using a semidry transfer to the nitrocellulose membrane (0.45 ^m) following the manufacturer's instructions.
1. Briefly, cut the nitrocellulose membrane and two extra pieces of thick blotting paper the same size as the gel.
2. Equilibrate the gel, the paper, and the membrane by soaking them for 15 min in the transfer buffer.
3. Place one piece of paper on the surface of the cell, then the membrane, then the gel on top of the transfer membrane, and finally another sheet of the presoaked filter paper.
4. Add 10 mL of transfer buffer between each element and close the cell.
5. Run the transference for 1 h at 100 V.
1. After transference, the membrane is blocked at 4°C overnight with blocking reagent.
2. Next, the nitrocellulose sheet is incubated by swirling in an orbital incubator for 1.5 h at RT with undiluted hybridoma culture supernatant (11E10, ATCC CRL 1907, which produces an IgG [Mab 11E10] toward the A subunit of Stx2 [primary antibody]).
3. This should be followed by washing three times with washing buffer, rocking for 10 min at RT.
4. Bound MAb is detected after incubation for 1.5 h with a 1:1000 dilution of an anti-mouse alkaline phosphatase-conjugated antibody.
5. After three washings, secondary antibody is detected by adding NBT/BCIP at 40 ^L for each 2 mL of the immunodetection buffer.
6. Store the membrane in the dark at RT until the signal is visible on the membrane. Time is dependent on the intensity of the reaction.
7. The reaction is stopped by washing the membrane with distilled water (see Notes 10 and 11).
1. Enrichment cultures performed with high volumes of sample may sometimes give negative results although smaller volumes give positive results. This is because other lytic bacteriophages present in the sample could cause total lysis of the host cell, interfering with the multiplication of the Stx2 phages.
2. The density of the host strain is important. An overgrowth of the host strain will make multiplication of the phages difficult.
3. In case the proteins produced after phage conversion need to be analyzed, it is important to keep aliquots of the supernatant of the enrichment culture at -20°C until detection of the protein; otherwise proteases could produce degradation of the protein.
4. For DNA extraction, the extraction steps with phenol/chloroform/isoamyl alcohol should be repeated if a thick white interphase caused by protein debris is observed.
5. For DNA extraction: If recovery of DNA is not high enough, it could be increased by addition of 6 ^L glycogen for each mL of sample together with sodium acetate and absolute ethanol.
6. For DNA extraction: Washing steps with 70% ethanol should be repeated if a large white precipitate is observed (caused by salt precipitation, which could interfere with the PCR).
7. Nested PCR should be handled with care because of contamination problems. False-positive results may be avoided by using several negative controls, such as negative controls from the DNA extraction process, from the first step of PCR, and from the nested PCR itself as well as from the enrichment cultures of the host strain without phage infection. Material used for nested PCR should be sterilized before use and the reaction mixture performed in a sterile bench.
8. For hybridization with DIG-labeled probes: If spotty background is observed, do not include MgCl2 in buffer 3. Change the plates at each washing step.
9. Protein detection: Caution: Acrylamide used for the polyacrylamide gels is neurotoxic. Handle with care.
10. Immunodetection: To develop the signal with NBT/BCIP, a long incubation may be used if necessary. However, longer incubation could also increase the background, disturbing signal detection.
11. Immunodetection: signal obtained with alkaline phosphatase may disappear from the membrane after some weeks. Thus, the results should be photographed as soon as they are obtained.
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