Figure S1. Phylogenetic analysis of phage S24-1 ORFs in AHJD-like viruses. Phylogenetic trees of major capsid proteins (ORF19) (left), tail lytic protein (ORF11) (middle), and receptor-binding protein (ORF16) (right). The homologous ORFs in AHJD-like viruses were used in the analysis. First, the major capsid protein is typically used for phage phylogenetic analysis. Phages S24-1 and S13′ were considered to be more related to each other compared with the other AHJD-like viruses. Thus, phages S24-1 and S13′ may have evolved locally from the same origin. Next, the genetic distances of the tail lytic proteins and the major capsid proteins appeared to be smaller among the other AHJD-like viruses. By contrast, the putative receptor-binding protein (ORF16) of phage S24-1 was highly diverged from those of the other AHJD-like viruses, including phage S13′. The ORF data were obtained from the genome data: phage ϕ44AHJD (accession number AF513032), phage SAP-2 (accession number EU136189), Bacteriophage 66 (accession number AY954949), and phage ϕP68 (accession number AF513033).

Figure S2. Mass spectrometric analysis of two proteins from phage S24-1. (A) The structural proteins of phage S24-1. The structural proteins of phage S24-1 were separated using SDS-PAGE (7.5% gel) and visualized by CBB staining (see Fig. 2). The protein bands boxed in gray were digested by trypsin and analyzed by mass spectrometry. (B) Mass spectrometric analysis of the digested upper protein band. (C) Mass spectrometric analysis of the digested lower protein band. The mass spectra of the digested protein are shown on the left. The tables on the right show the mass spectrometry results. The peak numbers in the mass spectra correspond to those listed in the table. “Precursor molecular weight” is abbreviated as “Prec. MW.”

Figure S3. Aggregation assay using rORF16-bound beads. (A) Schematic diagram of the aggregation assay system. If the protein had an affinity for the bacteria, aggregation with the bacteria was observed in the mixture (left). If the protein had no affinity for the bacteria, aggregation was not observed in the mixture (right). (B) Establishment of an aggregation assay system using rORF16-bound beads. rORF16-bound beads, BSA-bound beads, and beads only were prepared. Overnight cultures of S. aureus SA14 or Enterococcus faecalis EF24 were prepared. Twofold serial dilutions of the beads and bacteria were prepared using PBS. The beads were mixed with the bacteria and the aggregability was examined. Aggregation was only observed in the mixture of rORF16-bound beads with S. aureus, in a concentration-dependent manner.

Figure S4. Assessment of the rORF16 binding affinity with S. aureus samples treated with heat and various chemicals. After rORF16 was mixed with S. aureus samples treated with heat and various chemicals, the supernatants and bacterial cell pellets were separated by centrifugation. The samples were subjected to rORF16 detection by western blotting. The treatment group number is shown at the top for each result. The types of treatments and their designated numbers are as follows: untreated in No. 1, autoclaved in No. 2, SDS in No. 3, trichloroacetic acid (TCA) in No. 4, phenol– chloroform in No. 5, n-butanol in No. 6, Triton X-100 in No. 7, NaOH in No. 8, and HF in No. 9 “has been replaced with” proteinase K in No. 5, phenol–chloroform in No. 6, n-butanol in No. 7, Triton X-100 in No. 8, NaOH in No. 9, and HF in No. 10. The result with ORF16 only is shown at the top left. Overall, the results agree with the aggregation assay results shown in Figure 5. Unfortunately, S. aureus cells treated with proteinase K did not produce any bands from the pellet or the supernatant (data not shown), probably because proteinase K residuals appeared to digest the treated ORF16, even after thorough washing of the bacterial cells. The NaOH and HF-treated samples (Nos. 8 and 9) had no binding affinity with S. aureus.

Figure S5. Assessment of the binding activity using a recombinant ORF68 of phage K. The binding activity of phage K ORF68 was examined by western blotting using the recombinant proteins KORF68c-his (ORF68 with a 6 His-tag at the C-terminal) and KORF68n-his (ORF68 with a 6 His-tag at the N-terminal). S. aureus SA14 was sensitive to phage K infection. After incubation of the recombinant K_ORF68s with S. aureus strain SA14, the presence of the recombinant ORF68s in the solution or S. aureus cells was examined by western blotting using the anti-6 His antibody. KORF68c-his and KORF68n-his had no binding activity with S. aureus, regardless of the presence of cationic ions such as MgCl2 and CaCl2.

mbo3166-sup-0002-TableS1.pdfapplication/PDF344KTable S1. The phage lysis activities of phages S13′ and S24-1 against 89 S. aureus strains.
mbo3166-sup-0003-TableS2.pdfapplication/PDF218KTable S2. Bacterial strains used in this study and their phage S24-1 infectivity.
mbo3166-sup-0004-TableS3.pdfapplication/PDF150KTable S3. List of primers used in this study.
mbo3166-sup-0005-TableS4.pdfapplication/PDF325KTable S4. PCR products and primer lists.
mbo3166-sup-0006-TableS5.pdfapplication/PDF299KTable S5. Annotation of the phage S13′ genome.
mbo3166-sup-0007-TableS6.pdfapplication/PDF286KTable S6. Annotation of the phage S24-1 genome.

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