Project Summary Mobile genetic elements (MGEs) provide bacterial populations with an accessory genome, the “mobilome,” that helps them survive antibiotics, phage infections, and other stresses. Understanding the core machinery encoded by MGEs can help explain how accessory genes flow within a microbiome and can also reveal interesting new enzymes, often with potential as biotech tools. Furthermore, MGE-encoded enzymes are often simplified or modified variants of chromosomal machinery that, through comparison, can illuminate the evolution of both. Mechanistic studies of MGEs have lagged far behind the bioinformatics. Our work focuses on conserved machinery encoded by two families of staphylococcal MGEs that are of particular importance to human health: SCCs, which are chromosomal islands implicated in the MRSA epidemic, and pathogenicity islands. Our ongoing efforts have defined a set of conserved core SCC genes and assigned biochemical activities to most of them, discovering new enzymes and following the interesting questions they raised along the way. For example, we have discovered a new group of potentially anti-phage DNA glycosylases and a new type of primase that raises the question of how it evolved as well as how DNA polymerases are normally prevented from initiating DNA synthesis de novo. We also found that these MGEs encode helicases with surprising similarity to the eukaryotic and archaeal MCM helicases, but with interestingly different pathways for loading the helicase onto DNA at the origin of replication. In addition to building on the work above, we will broaden our questions to the roles of SCC-encoded machinery in phage defense, horizontal transfer, and copy number expansion. Recent advances in the field combined with our biochemical groundwork position us to mechanistically dissect the horizontal transfer of SCCs to new strains. We will also dissect the mechanism by which antibiotic resistance-carrying SCC elements can form tandem repeats to increase resistance in response to antibiotic treatment.