Transducing particles of Staphylococcus aureus pathogenicity island SaPI1 are comprised of helper phage-encoded proteins

Abstract

The relationship between the composition of SaPI1 transducing particles and those of helper phage 80alpha was investigated by direct comparison of virion proteins. Twelve virion proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry; all were present in both 80alpha and SaPI1 virions, and all were encoded by 80alpha. No SaPI1-encoded proteins were detected. This confirms the prediction that SaPI1 is encapsidated in a virion assembled from helper phage-encoded proteins.

FIG. 1.

Electron micrographs of 80α and SaPI1 preparations analyzed in this study. CsCl was removed by dialysis in 10 mM Tris-HCl, pH 7.8, 20 mM NaCl, and 1 mM MgCl_2. Samples were applied to a 200 mesh, formvar-coated copper grid, stained with 1% phosphotungstic acid, and magnified 10,000× using a JEOL-JEM-1230 transmission electron microscope equipped with an UltraScan 4000SP charge-coupled-device camera. (A) Phage 80α particles. (B) SaPI1-enriched virions. (C) 80α fraction containing apparent procapsids.

FIG. 2.

Identification of proteins in 80α and SaPI1-enriched virion preparations. (A) Negative image of a Sypro-Ruby-stained 10% Criterion XT SDS-PAGE bis-Tris polyacrylamide gel (Bio-Rad, Hercules, CA). Phage samples were denatured for 15 min at 95°C in the loading buffer provided by the manufacturer and vortexed for 20 min prior to gel loading. Lanes a and e, Precision Plus Protein standard (10 kDa to 250 kDa); lane b, 80α; lane c, SaPI1; lane d, 80α procapsid fraction. Numbers indicate those bands excised (1 to 8 from both 80α and SaPI1 samples) and analyzed as described in the text. (B) Identification of virion proteins from the corresponding bands shown in panel A. While bands 2 to 4 could be assigned collectively to ORFs 59, 61, and 62, specific assignment of each of these three bands to the gene encoding it was not possible, since the three proteins were not resolved from each other. This uncertainty is indicated by the brackets in Fig. 2A and B.

FIG. 3.

Locations of genes encoding the proteins identified in this study. The late region of the 80α prophage genomic map is shown, from nucleotide 16407 to the end. Arrows indicate predicted open reading frames encoding proteins of at least 50 amino acids, as annotated in the database entry (accession no. DQ517338). Genes for which functional assignments have been made based on sequence similarity are indicated above the map. Shaded arrows represent genes for which the assigned function is supported by the protein analysis reported here, the black arrows indicate genes encoding proteins identified by SDS-PAGE and mass spectrometry, and the gray arrows indicate genes encoding proteins identified only in the mass spectrometry analysis of complete virions. The ORF number for each gene encoding an identified virion protein is indicated.