The complete genomes of Staphylococcus aureus bacteriophages 80 and 80α--implications for the specificity of SaPI mobilization

Abstract

Staphylococcus aureus pathogenicity islands (SaPIs) are mobile elements that are induced by a helper bacteriophage to excise and replicate and to be encapsidated in phage-like particles smaller than those of the helper, leading to high-frequency transfer. SaPI mobilization is helper phage specific; only certain SaPIs can be mobilized by a particular helper phage. Staphylococcal phage 80α can mobilize every SaPI tested thus far, including SaPI1, SaPI2 and SaPIbov1. Phage 80, on the other hand, cannot mobilize SaPI1, and ϕ11 mobilizes only SaPIbov1. In order to better understand the relationship between SaPIs and their helper phages, the genomes of phages 80 and 80α were sequenced, compared with other staphylococcal phage genomes, and analyzed for unique features that may be involved in SaPI mobilization.

Figure 1. Genomic maps of phages 80α and 80

Predicted open reading frames of at least 50 codons are indicated. Open reading frames shown in black are those for which gene products have been identified in phage virions by mass spectrometry. Genes encoding proteins with at least 50% amino acid identity are indicated by shaded regions between the two genomes; darker shading indicates amino acid identity of at least 75%.

Figure 2. Mobilization of SaPI1 by phage 53

RN10822, a SaPI1-containing derivative of RN4220, was infected with either 80α or 53 as described in Materials and Methods. Standard minilysates were prepared at indicated times (minutes) after infection, separated on agarose, and probed for SaPI1 DNA by Southern blotting. The upper ‘bulk’ DNA band includes chromosomal DNA, replicating and linear phage-sized DNA, and replicating SaPI1 DNA; the lower band is SaPI1 linear monomers released from phage heads.

Figure 3. Identification of 80 virion proteins

Numbers indicate bands excised from the Coomassie blue-stained 10% SDS polyacrylamide gel; size markers are on the left. The protein(s) present in each band were identified by MS/MS and are indicated in the table.

Figure 4. 80α integrase and the 80α attachment site

(A) 80α int is closely related to ccrB, and not to the tyrosine integrase of 80 and others commonly found in staphylococcal Siphoviridae. (B) Map illustrating the location of attB in an intergenic region between the coding regions of rmpF and sirH. (C) Sequences of the phage (attP) and bacterial (attB) attachment sites, as well as the left (attL) and right (attR) prophage junctions. Nucleotides corresponding to the bacterial sequence are shown in bold letters. Conserved nucleotides in the short, imperfect core sequence are indicated by asterisks. Broken arrows above the sequence mark the imperfect inverse repeat flanking the crossover site. The location of the crossover, which is inferred from the phage-bacterial junction revealed by alignment of the prophage att sites with attB and attP, is shown by a bold arrow.

Fig. 5. Specificity of SaPI mobilization by different helper phages

RN4220 and derivatives RN10822, RN10823 and JP45, carrying the indicated SaPIs, were infected with each of the indicated phages as described in Materials and Methods. Standard minilysates were prepared 60 minutes after infection, separated on agarose, and probed for SaPI DNA by Southern blotting with a ter probe, which is common to all three SaPIs. The upper ‘bulk’ DNA band includes chromosomal DNA, replicating and linear phage-sized DNA, and replicating SaPI1 DNA; the lower band is SaPI1 linear monomers released from phage heads.