The Capsule Regulatory Network of Defined by density-TraDISort.

infections affect infants and the immunocompromised, and the recent emergence of hypervirulent and multidrug-resistant lineages is a critical health care concern. Hypervirulence in is mediated by several factors, including the overproduction of extracellular capsule. However, the full details of how capsule biosynthesis is achieved or regulated are not known. We have developed a robust and sensitive procedure to identify genes influencing capsule production, density-TraDISort, which combines density gradient centrifugation with transposon insertion sequencing. We have used this method to explore capsule regulation in two clinically relevant strains, NTUH-K2044 (capsule type K1) and ATCC 43816 (capsule type K2). We identified multiple genes required for full capsule production in , as well as putative suppressors of capsule in NTUH-K2044, and have validated the results of our screen with targeted knockout mutants. Further investigation of several of the capsule regulators identified-ArgR, MprA/KvrB, SlyA/KvrA, and the Sap ABC transporter-revealed effects on capsule amount and architecture, serum resistance, and virulence. We show that capsule production in is at the center of a complex regulatory network involving multiple global regulators and environmental cues and that the majority of capsule regulatory genes are located in the core genome. Overall, our findings expand our understanding of how capsule is regulated in this medically important pathogen and provide a technology that can be easily implemented to study capsule regulation in other bacterial species. Capsule production is essential for to cause infections, but its regulation and mechanism of synthesis are not fully understood in this organism. We have developed and applied a new method for genome-wide identification of capsule regulators. Using this method, many genes that positively or negatively affect capsule production in were identified, and we use these data to propose an integrated model for capsule regulation in this species. Several of the genes and biological processes identified have not previously been linked to capsule synthesis. We also show that the methods presented here can be applied to other species of capsulated bacteria, providing the opportunity to explore and compare capsule regulatory networks in other bacterial strains and species.