A B C D Alpha toxin PSMs 35kDa <10kDa PSMs <10kDa

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A B C D Alpha toxin PSMs 35kDa <10kDa PSMs <10kDa MW MupS MupR MupS MupR Alpha toxin 35kDa PSMs <10kDa C SH1000 p(pmtC) Uninduced SH1000 p(pmtC) Induced D SH1000 Dagr SH1000 MupS MupR PSMs <10kDa Fig. 2: The mupR mutation reduces toxin production by the SH1000 strain. A: a representative image of western blot on TCA precipitated bacterial supernatant using anti-alpha toxin antibodies. On average we found the wild type mupS strain produced 2-fold more alpha toxin compared to the mupR strain. B: Coomassie stained SDS-PAGE gel with butanol extractions of bacterial supernatant, containing the PSMs. On average we found the wild type mupS strain produced 3.4-fold more alpha toxin compared to the mupR strain. C: Over-expression of the pmtC gene, which encodes one of the ATP binding proteins of the PSM secretory system, Pmt, in the wild type SH1000 strain causes a reduction in the abundance of the PSM in the S. aureus supernatant. An Agr mutant has been provide as a control. D: The intra- and extra-cellular levels of PSMs were pooled to compare the overall levels of PSM production where on average the wild type strain produced 3.2 fold more PSMs. A full length SDS-PAGE gel has been provided in Supplementary material (Supp. Fig. 3), to illustrate why we only provide a ‘letter-box’ snap-shot of the PSM gels here.