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Microbiology vol. 153, part 10, pp. 3235-3245
Media and growth conditions. GL agar and CYGP broth were described previously (Novick, 1991). Preparation of phage lysates and procedures for phage titration and transduction were also as described previously (Novick, 1991). Bacterial inocula were prepared by overnight growth at 37 °C on GL agar with antibiotics as required for plasmid maintenance. Cultures for phage studies and for DNA isolation were prepared by growth in CYGP broth (Novick, 1991) at 37 °C with shaking (240 r.p.m.). Overnight cultures in CYGP were used for certain experiments.
Isolation of SaPI2 DNA. Strain RN10619 was grown in CYGP to an OD540=0.4 and induced by adding mitomycin C (1 μg ml-1). Cultures were grown at 32 °C with slow shaking (80 r.p.m.). Lysis usually occurred within 3 h. Lysates were sterilized with 0.22 μm filters, then centrifuged at 28,000 r.p.m. for 3 h (Spinco SW28 rotor), and the pellet was resuspended in phage buffer overnight. Phage DNA was extracted with phenol/chloroform and purified bby using a PCR purification kit (Qiagen). DNA content and purity were estimated by separation on 0.5 % agarose, staining with ethidium bromide and scanning with an Alpha-Innotech imager.
PCR analysis. All primers were obtained commercially (Integrated DNA Technologies). PCR reactions were performed with a Perkin-Elmer 9600 thermocycler in a volume of 50 μl using the reaction buffer supplied by the manufacturer. Deoxynucleotide triphosphates were used at 50 μM. Cycling times were according to the properties of the primer pairs. Ordinarily, reactions were carried out for 35 cycles with an annealing temperature of 55 °C. For junctional PCR, genomic DNA was used as template. Primers used for PCRs are listed in Table S3. RN4282 DNA was used as template for tst and SaPI1-specific PCRs, RN3984 for SaPI2, RN8482 for SEB, MSSA476 for SaPI4, Mu50 for SaPIm4 and RF122 for SaPIbov1.
Sequencing and sequence analysis. Sequencing was performed by the Skirball Microchemistry Facility, using the Sanger method with an ABI model 3730XL automatic sequencer. The purified SaPI2 DNA extracted from phage was used directly to sequence SaPI2. Sequencing was started by primer walking from tst in both directions until the 21 nt attachment sites in both directions were reached. All sequences were reconfirmed in both directions using small PCR products amplified directly from RN3984 genomic DNA. DNA sequences were analysed using DNASTAR. The DNA sequence database was searched using standard BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).
Southern blot hybridization. EcoRV digests of chromosomal DNA were separated on a 0.8 % agarose gel, transferred to Hybond-N+ nylon membrane (Amersham) with a vacuum blotter (USB), UV cross-linked and hybridized overnight with /α-32P]dATP-labelled probes. Probes were PCR products using chromosomal DNA of SaPI-containing strains (Table S1) as templates, and primers as listed in Table S3. Washed blots were exposed to Phosphorimager screens, which were read by a Molecular Dynamics Phosphorimager.
Western blot analysis. Standard CYGP medium (Novick, 1991) without glucose was used for exoprotein preparation. Culture samples (1-10 ml) were centrifuged in an Eppendorf centrifuge. The supernatant was recentrifuged to remove any residual organisms, then precipitated with a 10 % volume of 50 % trichloracetic acid, and the proteins were separated by SDS-PAGE according to the method of Laemmli (1970). All samples were equalized to the density of the culture. Western blotting was carried out by a standard protocol using rabbit anti TSST-1 (Toxin Technology) as primary antibody, and anti-rabbit Ig with horseradish peroxidase (Amersham) as secondary antibody. Signals were detected by ECL (Amersham).
Figs. S1-S7. Southern blot analyses of restriction digests of chromosomal DNA with strain numbers corresponding to those in Tables S2 and S5.
Fig. S1. EcoRV digest, tst probe.
Fig. S2. Strains and digests as indicated, tst probe.
Fig. S3. EcoRV digest, int-I probe.
Fig. S4. EcoRV digest, int-II probe.
Fig. S5. EcoRV digest, int-III probe.
Fig. S6. EcoRV digest, int-IV probe.
Fig. S7. EcoRV digest, int-V probe.
Fig. S8. Alignment of the right ends of SaPI2 and SaPI4. Homologous regions are shown (shaded dark grey) where recombinational exchange could insert tst into SaPI4, which lacks identifiable virulence genes.
Fig. S9. attS core sequences at the SaPI-chromosomal junctions at position 44' (see Fig. 4 in the paper). Single nucleotide substitutions occur at positions 1 and 16 and there are single nucleotide mismatches at positions 1 and 13 in the SaRIfusB junctions, and at position 16 in the SaPI6Δ junctions. These nucleotides are in italic type.
Table S1. Strains used in this study.
Table S2. List of clinical TSS strains.
Table S3. Oligonucleotide primers.
Table S4. SaPI2 features and PGs.
Table S5. Features of clinical TSS strains.
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