SUMMARY The enterococci are ancient microbes that appear to have co-evolved along with the spread of animals on land. They possess an intrinsic ruggedness that allows them to survive unusually harsh conditions, including starvation and desiccation, yet efficiently recolonize the gut of a new host. These traits distinguish them from their ancestors, which led to the identification of an initial set of enterococcal-specific genes and phenotypes. Functional analysis by Tn-seq recently implicated a subset of those genes is important for growth under non-challenged conditions, with still others assuming importance upon antibiotic challenge. This subproject proposes to take advantage of the power of high throughput single cell imaging to more rigorously identify genes critical to the hardiness of enterococci, and associate them with precisely defined growth defects. Moreover it expands this to include genes of importance to various probes of the cell wall, including innate defense molecules, and in the process it will generate an ordered array of known transposon insertion mutations in these genes of importance. As most of the genes that encode the distinctive ruggedness of enterococci encode proteins of unknown function, these mutants will be of considerable value for discovering the underlying mechanisms. Genes found to be important are bioinformatically organized into putative functional networks, which create models that can be tested experimentally. One such network emerged from preliminary studies, and incorporates functions of three critical enterococcal genes with limited distribution outside of the species, that encode genes of unknown function. Bioinformatic analysis suggests that these genes play important roles in regulation of cell wall homeostasis, and appear to be influenced by a quorum sensing mechanism. From functional analysis, this network appears to be highly sensitive to the influence of rifampicin, supporting the role of several key inferred transcription factors in the network. This network will be defined by creating tightly controlled gene silencing constructs and identifying elements of the network through comparative transcriptomics and proteomics. Because of the apparent involvement of three unique enterococcal genes in this network, it appears to be a promising target for limiting the growth and survival of enterococci in patients or in the hospital environment. It is expected that new insights gleaned from the applying Mother Machine imaging technology will add new components to these networks with rigorously defined contributions to enterococcal cell fitness.