Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations - PubMed (original) (raw)

Specificity of staphylococcal phage and SaPI DNA packaging as revealed by integrase and terminase mutations

Carles Ubeda et al. Mol Microbiol. 2009 Apr.

Abstract

SaPI1 and SaPIbov1 are chromosomal pathogenicity islands in Staphylococcus aureus that carry tst and other superantigen genes. They are induced to excise and replicate by certain phages, are efficiently encapsidated in SaPI-specific small particles composed of phage virion proteins and are transferred at very high frequencies. In this study, we have analysed three SaPI genes that are important for the phage-SaPI interaction, int (integrase) terS (phage terminase small subunit homologue) and pif (phage interference function). SaPI1 int is required for SaPI excision, replication and packaging in a donor strain, and is required for integration in a recipient. A SaPI1 int mutant, following phage induction, produces small SaPI-specific capsids which are filled with partial phage genomes. SaPIbov1 DNA is efficiently packaged into full-sized phage heads as well as into SaPI-specific small ones, whereas SaPI1 DNA is found almost exclusively in the small capsids. TerS, however, determines DNA packaging specificity but not the choice of large versus small capsids. This choice is influenced by SaPIbov1 gene 12, which prevents phage DNA packaging into small capsids, and which is also primarily responsible for interference by SaPIbov1 with phage reproduction.

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Figures

Fig. 1

Fig. 1. Effect of int mutation on the SaPI1 ERP cycle

80α lysogens containing either SaPI1 int::tetM (at left) or SaPI1 WT (at right) were mitomycin induced and 5 ml samples removed for DNA analysis at 0, 45, and 90 min. These were used to prepare screening lysates that were analyzed by agarose gel electrophoresis and stained with ethidium bromide or Southern blotted with a SaPI1 probe. B – bulk DNA; S – SaPI1 monomer-sized DNA.

Fig. 2

Fig. 2. Electron micrographs of negatively stained phage and SaPI particles

φ80α lysogens were MC induced, then incubated in CY broth until lysis. Lysates were centrifuged to remove debris, then filtered and the filtrates pelleted by centrifugation, allowed to resuspend in phage buffer and analyzed by electron microscopy. A – 80α alone; B – 80α-SaPI1 WT; C – 80α-SaPI1-int::tetM; D – 80α-SaPIbov1 DterSSaPIbov1. L – mature (large) phage particle; S – Mature SaPI (small) particle; LP – phage pro-capsid; SP – SaPI pro-capsid.

Fig. 3

Fig. 3. Packaging specificity

φ80α lysogens were induced, then incubated in CY broth until lysis. DNA extracted from phage particles purified by centrifugation, was analyzed by gel electrophoresis and gel was blotted with a SaPI-specific probe or phage-specific probes downstream or upstream of the 80α pac site. Composition of the strains used for the preparation of lysates is indicated above.

Fig. 4

Fig. 4. Effects of SaPIbov1 mutations on SaPIbov1 and phage DNA packaging

Samples were prepared and analyzed as in Fig. 3. 1- φ80α; 2- φ80α SaPIbov1 wt; 3- φ80α SaPIbov1 Δ gene 12; 4- φ80α SaPIbov1 Δ gene 12 + pCN51:gene12; 5- φ80α SaPIbov1 Δ ter; 6- φ80α SaPIbov1 Δ gene 6; 7- φ80α SaPIbov1 Δ gene 10; 8- φ80α SaPIbov1 Δ gene 11;

Fig. 5

Fig. 5. Effect of SaPI1 on 80α replication

Cultures of RN10616 (φ80α, SaPI1-) or RN10628 (φ80α, SaPI1+) were grown to a Klett=30, UV-induced, and aliquots were removed at the indicated times and used to prepare minilysates. Equivalent amounts of total DNA were loaded in each well of an agarose gel and separated by electrophoresis. Gels were stained and photographed, and then transferred and probed with an 80α probe from the replication region (generated with primers SMT98 and SMT99). The upper panel shows the stained gel; the lower panel is an autoradiogram of the Southern blot. The first lane contains purified 80α genomic DNA. Similar results were obtained following 80α infection of a SaPI1-negative and SaPI1-positive strain (data not shown). P- phage monomer-sized band; S - SaPI1 monomer-sized band.

Fig. 6

Fig. 6. Effect of cloned SaPIbov1 GP12 on 80α DNA replication and packaging

Cultures of RN4220 derivatives were infected with 80α at a multiplicity of 3 and incubated in CY broth for 1 h, at which time, mini-lysates were prepared and separated on agarose. The gels were stained and photographed (left panel), then Southern blotted (right panel) with an 80α c1 gene probe. Bulk – phage and chromosomal DNAs; SaPI – SaPI-sized monomers released from intracellular phage capsids; Lane 1, pCN51 (vector); 2, pCN51::gene12; 3, SaPIbov1Δ12 + pCN51; 4, SaPIbov1Δ12 + pCN51::gene12; 5, SaPIbov1; 6, SaPI1 + pCN51; 7, SaPI1 + pCN51::gene12. Cultures used for lower gel were grown with 0.1 mM CdCl2.

Fig. 7

Fig. 7. Homologs of SaPIbov1 GP12 (phage interference function)

Residues defining the SaPI1 group of Pifs are mostly in red. In blue are residues in SaPI5 and SaPImw2 that are specifically different from the other SaPIs. Residues shown in green differ from consensus sequences in undefined ways.

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