Analysis of Teg41 and PSMα promoter activity using a divergent fluorescent reporter plasmid

<i>Staphylococcus aureus</i> is a common commensal of human skin and anterior nares but also a notable pathogen, causing a range of diseases from minor skin infections to serious, often fatal conditions. The global health community considers <i>S. aureus</i> a serious threat due to the rise of multidrug-resistant strains, including community-acquired methicillin-resistant <i>S. aureus</i>. Effective host tissue invasion by <i>S. aureus</i> requires precise spatiotemporal regulation of virulence factor expression, which can be controlled by small RNAs (sRNAs). Our lab has identified over 300 potential sRNAs in <i>S. aureus</i> USA300, highlighting their regulatory significance. Recently, our group characterized an sRNA named Teg41, which positively influences transcription from the PSMα promoter, enhancing toxin production and virulence. Teg41, located 203 bp downstream of the <i>psmα</i> transcription start site, exhibits relatively stable growth phase expression, a first for sRNAs in <i>S. aureus</i>. We demonstrate that the Teg41 and <i>psmα</i> promoters respond to different stimuli, and the Teg41 promoter has distinct regulatory regions. Single-cell analysis reveals non-homogeneous Teg41 expression within the bacterial population, with a subset of cells showing high expression. This study introduces new tools for studying divergent promoters and elucidates the Teg41-<i>psmα</i> expression relationship.IMPORTANCE<i>Staphylococcus aureus</i> is a leading cause of human infections and a major public health concern due to rising antibiotic resistance. Understanding how this pathogen regulates its virulence is critical to developing new therapeutic strategies. sRNAs are key regulators of bacterial gene expression, yet most remain uncharacterized. Here, we investigate Teg41, an sRNA that activates expression of the potent PSMα toxins. Using a novel dual-fluorescence reporter system and single-cell analysis, we uncover that Teg41 and its target promoter are regulated independently and heterogeneously across the bacterial population. These findings reveal new insights into sRNA-mediated regulation of virulence and provide innovative tools to dissect complex gene regulatory networks in <i>S. aureus</i>.