ABSTRACTStreptococcus pyogenescan cause a wide variety of acute infections throughout the body of its human host. An underlying transcriptional regulatory network (TRN) is responsible for altering the physiological state of the bacterium to adapt to each unique host environment. Consequently, an in-depth understanding of the comprehensive dynamics of theS. pyogenesTRN could inform new therapeutic strategies. Here, we compiled 116 existing high-quality RNA sequencing data sets of invasiveS. pyogenesserotype M1 and estimated the TRN structure in a top-down fashion by performing independent component analysis (ICA). The algorithm computed 42 independently modulated sets of genes (iModulons). Four iModulons contained thenga-ifs-slovirulence-related operon, which allowed us to identify carbon sources that control its expression. In particular, dextrin utilization upregulated thenga-ifs-slooperon by activation of two-component regulatory system CovRS-related iModulons, altering bacterial hemolytic activity compared to glucose or maltose utilization. Finally, we show that the iModulon-based TRN structure can be used to simplify the interpretation of noisy bacterial transcriptome data at the infection site.IMPORTANCES. pyogenesis a pre-eminent human bacterial pathogen that causes a wide variety of acute infections throughout the body of its host. Understanding the comprehensive dynamics of its TRN could inform new therapeutic strategies. Since at least 43S.pyogenestranscriptional regulators are known, it is often difficult to interpret transcriptomic data from regulon annotations. This study shows the novel ICA-based framework to elucidate the underlying regulatory structure ofS. pyogenesallows us to interpret the transcriptome profile using data-driven regulons (iModulons). Additionally, the observations of the iModulon architecture lead us to identify the multiple regulatory inputs governing the expression of a virulence-related operon. The iModulons identified in this study serve as a powerful guidepost to further our understanding ofS. pyogenesTRN structure and dynamics.