A hierarchical regulatory cascade defines the temporal window of natural transformation in Staphylococcus aureus
Natural transformation (NT), a major mechanism of horizontal gene transfer, promotes bacterial genome plasticity and contributes to the dissemination of antibiotic resistance. NT requires the development of competence, a transient and tightly regulated physiological state that is induced by environmental cues. Although competence has recently been demonstrated in the human pathogen Staphylococcus aureus, the regulatory architecture and temporal coordination governing its development remain poorly understood. Using a highly sensitive luciferase-based transcriptional reporter, we demonstrate that NT gene expression in S. aureus is transient, thereby defining a temporal window during which DNA uptake and recombination can occur. We further identify two classes of NT genes with distinct regulatory requirements and activation kinetics: Class I genes require both SigH and ComK1 and are activated approximately 2.5 hours later than Class II genes, which are regulated exclusively by ComK1. Mechanistically, we demonstrate that ComK1 activates sigH transcription, establishing a hierarchical regulatory cascade that explains its essential role in Class I gene expression. Finally, time-resolved transcriptomic analyses identify the global regulator CodY as an early regulator of competence that differentially controls comK1 and sigH transcription, thereby linking metabolic adaptation to competence development. Together, our findings define the regulatory architecture that governs competence development in S. aureus and reveal how a hierarchical cascade integrates environmental and metabolic cues to restrict horizontal gene transfer to a precise developmental window. This work provides a mechanistic framework for understanding how bacteria coordinate physiological adaptation with genome diversification through NT. Author summaryBacteria constantly evolve by acquiring new genetic information from their environment. One important mechanism driving this process is natural transformation, which allows bacterial cells to take up and integrate exogenous DNA into their genomes. However, this process is not continuously active: bacteria must first enter a specialized physiological state called competence. How pathogens control the development of competence remain poorly understood. In this study, we investigate how the human pathogen Staphylococcus aureus regulates natural transformation. Using a highly sensitive reporter system that allows us to monitor gene expression over time, we show that competence is activated only during a limited period, creating a defined window during which bacteria can acquire new DNA sequences. We uncover a hierarchical regulatory cascade involving the transcription factors ComK1 and SigH that controls the sequential activation of natural transformation genes. We further identify the metabolic regulator CodY as an upstream regulator that connects bacterial physiological state with the initiation of competence. Our findings reveal that natural transformation is not a random or continuous process, but a precisely timed developmental program controlled by multiple regulatory layers. By defining how S. aureus coordinates environmental sensing, metabolism, and horizontal gene transfer, this work provides new insight into how bacterial pathogens generate genetic diversity and adapt to changing environments, including conditions that may promote the emergence of antibiotic resistance.