The work presented herein details post-translational modifications (PTMs) in Staphylococcus aureus that are involved in mediating the stress response and normal cellular processes. The first PTM that was investigated is regulated intramembrane proteolysis (RIP) for the activation of the ECF sigma factor σS. We achieved this by analyzing the role of the site-1 protease, which we termed “putative regulator of sigmaS” (PrsS), as it is predicted to be the first enzyme in the RIP cascade, leading to the activation of σS. It was determined that the putative site-1 protease, prsS, mimics transcriptional profiles of sigS; with expression low in all strains examined other than in the highly mutagenic strain RN4220. Moreover, up-regulation of the protease was observed in response to cell wall-targeting antibiotics, DNA-damaging agents, and during infection in human serum and RAW 264.7 cells, similar to that previously demonstrated for sigS. It was further determined that prsS mutants, like sigS mutants, are more sensitive to cell wall-targeting antibiotics and DNA-damaging agents, which is explained, in part, by alterations in altered abundance of proteins in the prsS mutant that mediate antibiotic resistance (Pbp2a, FemB, and HmrA) and the response to DNA damage (BmrA, Hpt, and Tag). Importantly, transcriptional analyses of proteins affected in the protease mutant, revealed that their expression is decreased in both prsS and sigS mutants, suggesting that this is a result of sigS-mediated regulation. Lastly, it was determined that PrsS, similar to σS, is required for infection in whole human blood and murine models of virulence. Next, since the abundance of a stress response protease, HtrA1, was altered in prsS mutants, we aimed to assess the roles of this enzyme, and its homolog HtrA2 in S. aureus. Interestingly, we first determined that unlike that previously described for the HtrA enzymes, these proteases do not have a role in Agr-mediated virulence regulation. We attribute this finding to unintended mutations likely introduced during strain construction, which is common for S. aureus strains. We next used transcription profiling of the htrA genes in order to understand their role in the cell, and found that they are moderately expressed under standard conditions, and are up-regulated in response to both in vitro and ex vivo stressors that lead to cell protein, DNA, and cell envelope damage. Further to this, the protease mutants are more sensitive to numerous conditions that affect macromolecular stability, including elevated temperature, alterations in pH, reactive oxygen species, DNA damage, and antimicrobial stress. In order to further explore these sensitivities and gain insight into putative substrates, we employed a yeast-2 hybrid screen, and identified numerous proteins that interact with HtrA1 and HtrA2, including those that mediate the response to stress and normal cellular homeostasis. Taken together, we provide evidence to suggest the HtrA proteases in S. aureus are required both during standard conditions and in stress-inducing environments to mediate protein folding and proteolysis of a broad range of substrates. Finally, we performed the first examination of prenylation in a bacterial organism. Prenylation is a well-studied post-translational modification (PTM) in eukaryotes, wherein a prenyl group is added to a metabolite or the C-terminal “CAAX” motif of a protein. Interestingly, the machinery exists for this PTM in a wide variety of prokaryotic species, thus we set out to investigate its impact in S. aureus. To achieve this, we disrupted prenyl group synthesis by inactivating ispA, the gene encoding a prenyl synthetase. The abrogation of prenylation ensued in striking alterations in the cell, including lack of pigmentation and smaller colony size, similar to small-colony variants (SCVs) of S. aureus. In addition to this, the ispA mutant displayed a growth defect, as a result of lower ATP levels. Moreover, the prenylation mutant displayed alterations in resistance to antibiotics, including increased resistance to aminoglycosides and antimicrobial peptides (AMPs), yet elevated sensitivity to cell wall-targeting antibiotics. These differences in susceptibility to cell envelope targeting antibiotics are a result of alterations in cell envelope architecture, including variations in fatty acid composition and increased membrane fluidity. Collectively, the pleotropic consequences of the disruption of prenylation indicate that this process is key to maintaining cellular homeostasis in S. aureus, and perhaps other bacterial species.