Non-canonical stringent response signaling mediates antimicrobial fatty acid sensitivity in <i>Staphylococcus aureus</i>

<i>Staphylococcus aureus</i> is a leading cause of skin and soft tissue infections, yet colonization of healthy skin is limited by multiple defenses, including antimicrobial fatty acids (AFAs) produced by host cells and the resident microbiota. The mechanisms by which <i>S. aureus</i> overcomes these lipid-based defenses remain incompletely understood. Here, we show that mutations truncating the essential stringent response regulator Rel confer broad tolerance to both host- and microbially-derived AFAs in diverse <i>S. aureus</i> strains. Unlike classical stringent response activation, C-terminal Rel truncations do not induce typical (p)ppGpp-dependent transcriptional changes but instead enhance activity of the alternative sigma factor SigB and the staphylococcal accessory regulator SarA. This SigB-SarA regulatory cascade promotes transcriptional remodeling, including upregulation of pyrimidine biosynthesis genes, and coincides with alterations to cell envelope structure. Moreover, in the absence of SigB or SarA, tolerance can be restored through mutations in the serine/threonine phosphatase Stp1, highlighting additional pathways that modulate cell envelope-mediated resistance to AFAs. These findings identify a previously unrecognized consequence of Rel mutation in <i>S. aureus</i>; whereby small truncations may promote survival in AFA-rich host environments facilitating skin colonization and infection.