Role of staphylococcal phage and SaPI integrase in intra- and interspecies SaPI transfer

Abstract

SaPIbov2 is a member of the SaPI family of staphylococcal pathogenicity islands and is very closely related to SaPIbov1. Typically, certain temperate phages can induce excision and replication of one or more of these islands and can package them into special small phage-like particles commensurate with their genome sizes (referred to as the excision-replication-packaging [ERP] cycle). We have studied the phage-SaPI interaction in some depth using SaPIbov2, with special reference to the role of its integrase. We demonstrate here that SaPIbov2 can be induced to replicate by different staphylococcal phages. After replication, SaPIbov2 is efficiently encapsidated and transferred to recipient organisms, including different non-Staphylococcus aureus staphylococci, where it integrates at a SaPI-specific attachment site, att(C), by means of a self-coded integrase (Int). Phages that cannot induce the SaPIbov2 ERP cycle can transfer the island by recA-dependent classical generalized transduction and can also transfer it by a novel mechanism that requires the expression of SaPIbov2 int in the recipient but not in the donor. It is suggested that this mechanism involves the encapsidation of standard transducing fragments containing the intact island followed by int-mediated excision, circularization, and integration in the recipient.

FIG. 1.

Induction of SaPIbov2 replication by MC exposure. Bacterial cultures were exposed to MC and then incubated in broth at 32°C. Samples were removed at the indicated time points and used to prepare minilysates. Lysates were separated by agarose gel electrophoresis and transferred. The Southern blot hybridization pattern of the samples analyzed, obtained using a SaPIbov2-specific probe, is shown.

FIG. 2.

Precise excision and circularization of SaPIbov2 mediated by the Int protein. (A) Detection of Int mediated SaPIbov2 excision and formation of att_B. DNA from MC-induced strains were extracted (at 0 and 90 min) and PCR amplified using specific primers Ipl-1m and Ipr-2c recognizing the flanking sequence of the SaPIbov2 island. (B) Detection of int-mediated SaPIbov2 circularization. Samples obtained as previously described were PCR amplified with a pair of primers used divergently at both termini of the SaPIbov2 island (primers Ipl-16cB and Ipr-28m). JP2130 is a wild-type strain, and JP2487 is an int mutant strain.

FIG. 3.

Int-mediated mechanism involved in SaPIbov2 transfer.

FIG. 4.

Integration-specific PCR: PCR analysis using primers Ipr-13mP and Ipr-2c to test the site-specific integration of the SaPIbov2 island in the coagulase-negative staphylococci mediated by Int activity. JP1522 is an S. xylosus SaPIbov2-positive strain, JP2632, JP2633, JP2557, and JP2558 are S. epidermidis SaPIbov2-positive strains, and JP2130 is an S. aureus positive control.