Project Summary The emergence of pathogenic bacteria resistant to antibiotics is an urgent public health issue. Resistance typically results from a specific genetic mutation that permanently alters the organism’s sensitivity to a particular antibiotic. However, antibiotic sensitivity can also change transiently as the result of phenotypic antibiotic tolerance, a phenomenon that is thought to underly treatment failures in the case of recurrent bacterial infections. Antibiotic tolerance results from the entry of bacteria into a transient growth-arrested and quiescent state where they are less sensitive to compounds that inhibit processes such as DNA replication or cell wall synthesis and thus can escape killing by antibiotics to which they are normally sensitive. While quiescence is characteristic of most cells in a stationary phase culture, only a small fraction of the cells in a exponentially growing culture are transiently quiescent. It is these cells – “persisters” - that are thought to be responsible for the failure of antibiotic treatment in recurrent infections. How does quiescence occur in only a sub-population? One proposed mechanism involves the nucleotide second messengers (p)ppGpp that inhibit a broad range of physiological processes including transcription and DNA replication leading to growth arrest and quiescence. It has been proposed that variability in the amount of (p)ppGpp in cells could lead to variations in growth state and thereby antibiotic sensitivity. We recently found that ~1% of an exponentially growing population of Bacillus subtilis expresses substantially higher amounts of a (p)ppGpp synthase. The cells in this sub-population exhibit increased antibiotic tolerance as compared to most cells in the population that do not express this protein. In an important extension of this work, we have developed a fluorescent reporter can be used to identify individual cells with elevated levels of (p)ppGpp. We call these cells (p)ppGpphigh and they can be isolated by Fluorescent Activated Cell Sorting (FACS). We propose to investigate (p)ppGpphigh cells in the following Aims. Aim 1 is to characterize the (p)ppGpphigh cells and compare their physiological characteristics including protein synthesis and DNA replication to average cells of the population. Aim 2 is to investigate the cellular mechanisms responsible for the synthesis and degradation of (p)ppGpp in the (p)ppGpphigh cells. The knowledge gained during the course of this project will deepen our understanding of the physiology of persister bacteria and facilitate the identification of metabolic vulnerabilities that could serve as potential targets for anti-persister strategies.