ABSTRACT Bacterial infectious disease is a global threat to human health and there is an urgent need to develop new antimicrobials that limit the impact of life-threatening pathogens. These pathogens include the major causative agents of nosocomial infections, e.g., Acinetobacter baumannii and Staphylococcus aureus, and a major respiratory pathogen, Streptococcus pneumoniae. In this renewal application, we seek continuation of our innovative, strongly integrated and topical research program positioned at an intersection of inorganic chemistry and microbial physiology, designed to tackle significant gaps in our knowledge in bacterial transition metal homeostasis (metallostasis) and hydrogen sulfide homeostasis. My group has long-standing interests in the transcriptional repressor proteins (metallosensors) and metallochaperones that allow a bacterium to respond to host efforts to restrict transition metal availability or induce metal toxicity. Our subsequent discovery of transcriptional regulators that “sense” downstream more oxidized forms of hydrogen sulfide, collectively termed reactive sulfur species (RSS), is foundational to our understanding of hydrogen sulfide signaling via protein persulfidation (S-sulfuration). Indeed, an emerging consensus holds that the biogenesis of hydrogen sulfide and RSS provides protection against host weapons reactive oxygen and reactive nitrogen species, and antibiotics, where they function as antioxidants and signaling molecules. Future research will be carried out in four general areas: 1) Investigating allostery in transcriptional regulation, where we extend our comprehensive physical description of metallosensors as dynamically-anchored “allosteric inorganic switches” to RSS sensors, using state-of-the-art methyl-specific NMR relaxation experiments and a novel mass spectrometry-based kinetic profiling method used to elucidate the broad principles of RSS specificity in diverse structural classes of regulators; 2) critically evaluate the RSS signaling hypothesis in A. baumannii, which posits that persulfidation is a regulatory modification, completely unexplored in bacteria; 3) deduce the global impact of host transition metal (zinc, iron) starvation (nutritional immunity) using complementary proteomics and metalloproteomics workflows to define changes in the metalloproteome while identifying metallochaperone targets, in A. baumannii; and 4) elucidate a poorly understood, infection-relevant iron-catecholate acquisition and detoxification pathway in S. pneumoniae. Our multidisciplinary approach, which seamlessly spans biophysical, bioinorganic and analytical chemistries to microbial physiology, will transform our understanding of foundational principles of pathogen metallostasis and hydrogen sulfide/RSS biogenesis in an effort to discover and characterize new players and biological processes that can be targeted by novel antibacterial strategies.