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And Inter-Generic Transfer of Pathogenicity Island-Encoded Virulence Genes by Cos Phages The ISME Journal (2015) 9, 1260–1263 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej SHORT COMMUNICATION Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages John Chen1, Nuria Carpena2,3, Nuria Quiles-Puchalt3,4, Geeta Ram1, Richard P Novick1 and Jose´ R Penade´s3 1Skirball Institute Program in Molecular Pathogenesis and Departments of Microbiology and Medicine, New York University Medical Center, New York, NY, USA; 2Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain; 3Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK and 4Universidad Cardenal Herrera CEU, Moncada, Valencia, Spain Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution. The ISME Journal (2015) 9, 1260–1263; doi:10.1038/ismej.2014.187; published online 14 October 2014 Classically, transducing phages use the pac site- phages, of which lambda is the prototype, do not headful system for DNA packaging. Packaging is engage in generalized transduction. For this reason, initiated on concatemeric post-replicative DNA by cos-site phages have been preferred for possible terminase cleavage at the sequence-specific pac site, phage therapy, since they would not introduce a genome slightly longer than unit length is pack- adventitious host DNA into target organisms. aged, and packaging is completed by non-sequence- The Staphylococcus aureus pathogenicity islands specific cleavage (reviewed in Rao and Feiss, 2008). (SaPIs) are the best-characterized members of the Generalized transduction results from the initiation phage-inducible chromosomal island family of of packaging at pac site homologs in host chromo- mobile genetic elements (MGEs; Novick et al., somal or plasmid DNA, and typically represents 2010). SaPIs are B15 kb mobile elements that B1% of the total number of phage particles. In the encode virulence factors and are parasitic on alternative cos site mechanism packaging is also specific temperate (helper) phages. Helper phage initiated on concatemeric post-replicative DNA by proteins are required to lift their repression (Tormo- terminase cleavage at a sequence-specific (cos) site. Ma´s et al., 2010, 2013), thereby initiating their Here, however, packaging is completed by terminase excision, circularization and replication. Phage- cleavage at the next cos site, generating a precise induced lysis releases vast numbers of infectious monomer with the cohesive termini used for sub- SaPI particles, resulting in high frequencies of sequent circularization (Rao and Feiss, 2008). transfer. Most SaPI helper phages identified to date Although cos site homologs may exist in host are pac phages, and many well-studied SaPIs are DNA, it is exceedingly rare that two such sites packaged by the headful mechanism (Ruzin et al., would be appropriately spaced. Consequently, cos 2001; Ubeda et al., 2007). Recently, we have reported that some SaPIs, of which the prototype is SaPIbov5 (Viana et al., 2010), carry phage cos Correspondence: J Chen or RP Novick, Skirball Institute Program sequences in their genomes, and can be efficiently in Molecular Pathogenesis and Departments of Microbiology and Medicine, New York University Medical Center, New York, NY packaged and transferred by cos phages to S. aureus 10016, USA or JR Penade´s, Institute of Infection, Immunity and strains at high frequencies (Quiles-Puchalt et al., Inflammation, University of Glasgow, 120 University Place, Sir 2014). Here we show that this transfer extends to Graeme Davies Building, Glasgow G12 8TA, UK. non-aureus staphylococci and to Listeria E-mail: [email protected] or [email protected] or [email protected] monocytogenes. Received 15 May 2014; revised 22 July 2014; accepted 4 August Since the pac phages transfer SaPIs to non-aureus 2014; published online 14 October 2014 staphylococci and to the Gram-positive pathogen cos-mediated gene transfer J Chen et al 1261 Listeria monocytogenes (Maiques et al., 2007; Table 1 Intra- and intergeneric SaPIbov5 transfera Chen and Novick, 2009), we reasoned that cos phages might also be capable of intra- and inter- Donor strain generic transfer. We tested this with SaPIbov5, into b which we had previously inserted a tetracycline Phage SaPI Recipient strain SaPI titre resistance (tetM) marker to enable selection, and 4 f f12 SaPIbov5 S. aureus JP4226 8.3 Â 10 with lysogens of two helper cos phages, 12 and S. epidermidis JP829 2.4 Â 104 fSLT, carrying SaPIbov5 (strains JP11010 and S. epidermidis JP830 4.7 Â 104 JP11194, respectively; Supplementary Table 1). L. monocytogenes SK1351 6.6 Â 103 The prophages in these strains were induced with L. monocytogenes EGDe 2.1 Â 104 4 mitomycin C, and the resulting lysates were S. xylosus C2a 7.1 Â 10 À 1 adjusted to 1 mgml DNase I and RNase A, filter f12 SaPIbov5 Dcos S. aureus JP4226 o10 sterilized (0.2 mm pore), and tested for SaPI transfer S. epidermidis JP829 o10 with tetracycline selection, as previously described S. epidermidis JP830 o10 (Ubeda et al., 2008). To test for trans-specific or L. monocytogenes SK1351 o10 trans-generic transduction, coagulase-negative sta- L. monocytogenes EGDe o10 S. xylosus C2a o10 phylococci species and L. monocytogenes strains were used as recipients for SaPIbov5 transfer, f12 DterS SaPIbov5 S. aureus JP4226 o10 respectively, as previously described (Maiques S. epidermidis JP829 o10 et al., 2007; Chen and Novick, 2009). As shown in S. epidermidis JP830 o10 L. monocytogenes SK1351 o10 Table 1, SaPIbov5 was transferred to S. xylosus, L. monocytogenes EGDe o10 S. epidermidis and L. monocytogenes strains at S. xylosus C2a o10 frequencies only slightly lower than to S. aureus. PCR analysis demonstrated that the complete island fSLT SaPIbov5 S. aureus JP4226 4.1 Â 103 S. epidermidis JP829 1.1 Â 103 was transferred to the recipient strains and integrated S. epidermidis JP830 2.1 Â 103 at the cognate attB site in the host chromosome L. monocytogenes SK1351 3.6 Â 102 (Figure 1 and Supplementary Table 2). In contrast, L. monocytogenes EGDe 3.1 Â 103 deletion of the SaPIbov5 cos site (strains JP11229 S. xylosus C2a 4.0 Â 103 and JP11230) did not affect SaPI replication fSLT SaPIbov5 Dcos S. aureus JP4226 o10 (Supplementary Figure 1), but completely eliminated S. epidermidis JP829 o10 SaPIbov5 transfer (Table 1). To rule out the possibility S. epidermidis JP830 o10 that other mechanisms of gene transfer were involved L. monocytogenes SK1351 o10 in this process, we generated a f12 phage mutant in L. monocytogenes EGDe o10 the small terminase (terS) gene (strain JP11012), S. xylosus C2a o10 using plasmid pJP1511 (Supplementary Table 2). Abbreviation: SAPI, Staphylococcus aureus pathogenicity island. The TerS protein is essential for f12 and SaPIbov5 aThe means of results from three independent experiments are shown. DNA packaging, but not for phage-mediated lysis Variation was within ±5% in all cases. (Quiles-Puchalt et al., 2014). As expected, this bNo. of transductants per ml induced culture. mutation abolished SaPIbov5 transfer (Table 1). Taken together, these results show that intra- and no previous studies have described the natural intra- or intergeneric transfer of the island was cos phage intergeneric transfer of pathogenicity islands by cos mediated. Furthermore, SaPI proteins, such as phages. As bacterial pathogens become increasingly integrase (Int) and potentially toxins, can be antibiotic resistant, lytic and poorly transducing expressed and functional in non-aureus strains. phages, such as cos phages, have been proposed for Because plaque formation is commonly used to phage therapy, on the grounds that they would not determine phage host range, we next determined the introduce adventitious host DNA into target organisms ability of phages f12 and fSLT to parasitize and and that the phages are so restricted in host range that form plaques on S. xylosus, S. epidermidis and the resulting progeny are harmless and will not L. monocytogenes strains. As shown in Supplementary result in dysbiosis of human bacterial flora. Because Figure 2, phages f12 and fSLT can parasitize and plaque formation was once thought to determine the form plaques on their normal S. aureus hosts, but host range of a phage, the evolutionary impact of are completely unable to lyse the non-aureus phages on bacterial strains they can transduce, but strains. Therefore, as previously observed with pac are unable to parasitize, has remained an unrecog- phages (Chen and Novick, 2009), these results nized aspect of phage biology and pathogen evolu- indicate that the overall host range of a cos phage tion. Our results add to the recently recognized may also be much wider if it includes infection concept of ‘silent transfer’ of pathogenicity factors without plaque formation. carried
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