Abstract Bacteria, from commensals to pathogens, utilize stress responses to maintain cellular homeostasis in fluctuating environments, enabling cells to survive nutrient deprivation, antibiotic treatments and host defenses. A well conserved key component of the bacterial stress response is the small signaling molecule (p)ppGpp (guanosine tetraphosphate and pentaphosphate). (p)ppGpp can be produced in almost all bacteria to mediate stress resistance and adaptation. (p)ppGpp is also critical for the generation of persister cells that are metabolically dormant and refractory to killing by antibiotics. However, (p)ppGpp regulation in Gram-positive bacteria, including many human pathogens, is poorly understood. The overall rationale of the proposed work is to unravel (p)ppGpp regulation in Gram-positive bacteria, which will provide important insights into the bacterial stress response, antibiotic tolerance, and interactions between microbes and hosts.