Due to the aberrant GC content of 89K and its absence in the reference P1/7 strain and a number of international virulent strains, it appears that 05ZYH33 acquired the 89K PAI horizontally. 89K then integrated into the chromosome at the attB site in the rplL gene, generating two direct repeats (attL and attR) at both ends of the island. It is well documented that integrative and conjugative elements (ICEs) are able to excise from the chromosomes to form circular intermediates prior to conjugative transfer into recipient cells (Burrus et al., 2002) . We confirmed that 89K did excise from the 05ZYH33 chromosome to produce a circular form and the host genome was repaired upon excision because PCR products were obtained for both primer pairs P2/P3 and P1/P4 (Fig. 2B) . Subsequent sequence analysis confirmed the expected structures of attP and attB generated upon site-specific excision ( Fig. 2C and D) . Noticeably, the amplicons obtained with primer pairs P2/P3 and P1/P4 showed a substantially weaker signal compared to that with P1/P2 and P3/P4, indicating that 89K excision is a rare event. To measure the rate of 89K excision, we developed a real-time quantitative PCR (qPCR) assay and determined that the frequency of 89K excision from an 05ZYH33 overnight culture was 0.032%, a rate similar to that observed for SLG excision from the Streptomyces lividans 66 chromosome (He et al., 2007) . These data demonstrated that 89K can spontaneously excise from the chromosome and form a circular A. Aberrant average G+C content of 89K, which is much less than that of the chromosome. B. Gene organization of 89K. The terminal direct repeats attL and attR are indicated by red triangles. Genes predicted to likely be involved in the mobilization of 89K are shown in yellow. For more gene details, see Chen et al. (2007) . A. Schematic representation of the site-specific integration and excision of 89K. The left and right junctions (attL and attR), which are shown as red rectangles, are formed by recombination between the chromosomal attB site and the 89K attP site. The flanking genes are shown by solid arrows indicating the direction of transcription. The location and orientation of primers used for detection of integrated and excised 89K are indicated by thin arrows. B. Detection of a circular extrachromosomal form of 89K and the empty chromosomal attB site following precise excision, by PCR analysis using primer pairs which are presented upon the lanes, with 05ZYH33 genomic DNA as the template. C. Printout of the sequencing chromatogram of PCR products amplified with primer pair P2/P3, showing the attP site (boxed) formed by the joining of the two ends of 89K. D. Printout of the sequencing chromatogram of PCR products amplified with primer pair P1/P4, showing the empty chromosomal attB site (boxed) upon 89K excision.