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Generation of a chimeric phage by recombination between the CmR-marked Sp15 derivative and Sp5.

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posted on 2009-05-01, 02:44 authored by Md Asadulghani, Yoshitoshi Ogura, Tadasuke Ooka, Takehiko Itoh, Akira Sawaguchi, Atsushi Iguchi, Keisuke Nakayama, Tetsuya Hayashi

(A) PCR scanning analysis of the stx1-flanking region of a chimeric recombinant phage (RP) in a K-12 Sp15Δstx1::CmR transductant. Positions of the PCR primers on the Sp15Δstx1::CmR genome are schematically shown at the top. For comparison, the stx1-flanking region of Sp15Δstx1::CmR in the donor strain O157 Sakai and the corresponding stx2-flanking region of Sp5Δstx2::CmR in its K-12 transductant were analyzed using the same set of primers. In this analysis, a spontaneous Sp5-deletion mutant of the Sp15Δstx1::CmR-containg O157 Sakai was used because Sp15 and Sp5 genomic regions between the P gene and the stx1 (or stx2) gene and between the stx1 (or stx2) gene and the nu1 gene contain highly homologous sequences (see Figure S7 for more details). (B) PCR analysis of the chimeric phage. The stx1-flanking region of the Sp5/Sp15 recombinant phage in K-12 was analyzed using two primer pairs. The positions of the primers on the chimeric phage genome are schematically shown at the top. Primers P_F and T_R are specific to the P and nu1 genes of Sp5, respectively. As a control, an O157 Sakai-derivative containing Sp15Δstx1::CmR was analyzed. PCR products were obtained by the two primer pairs (4.9 kb and 6.4 kb in size, respectively) only from the K-12 derivative carrying the recombinant phage (RP in K-12). (C) PCR analysis of the wrbA locus of a K-12 derivative carrying the chimeric phage (RP in K-12), K-12, and O157 Sakai. Integration of an Sp5-like phage into the wrbA locus (the integration site of Sp5 in O157 Sakai) in the K-12 derivative was confirmed by PCR using two primer pairs. Positions of the primer are schematically shown at the top. Primers LbR and RbF are specific to the left and right ends of the Sp5 genome, respectively.


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