In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the ␦ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the ␤ and/or ␤= subunits. To identify the impact of the ␦ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the ␦ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection. Bacterial gene transcription is a complex, multifactorial process that involves several key enzymes and regulatory elements. It is driven by the activity of DNA-dependent RNA polymerase (RNAP) and its associated proteins, which form a multisubunit enzyme consisting of one ␤ subunit, one ␤= subunit, two identical ␣ subunits, and one subunit (reviewed in reference 1). Together these form the RNAP apoenzyme, which is able to perform RNA elongation and termination; however, initiation requires the involvement of a factor. Typically, most bacterial species harbor several different factors, a primary one ( A or 70 ) that mediates housekeeping gene transcription and a variety of alternative sigma factors, which aid in the response to unfavorable environmental conditions and stress.In certain Gram-positive species and particularly the Firmicutes (see Fig. S1 in the supplemental material), an additional RNAP subunit is present, termed the ␦ factor, or RpoE (2). In Bacillus subtilis, the 173-amino-acid delta subunit has been shown to reduce nonspecific binding of RNAP to DNA and lead to an elevated preference for DNA regions that include promoter sequences (3, 4). In early experiments, it was shown that RpoE has a role specifically confined to promoter selection and influences the ability of RNAP to form open promoter complexes (5, 6). Fulllength RpoE has also been described as displacing nucleic acids from RNAP-DNA or RNAP-RNA complexes in vitro, which may explain the decreased affinity of RNAP-RpoE for nonpromoter regions. Thi...