Project Summary/Abstract Human monocytic ehrlichiosis caused by Ehrlichia chaffeensis and human granulocytic anaplasmosis resulting from Anaplasma phagocytophilum are responsible for significant morbidity and are also responsible for mortalities in a subset of high-risk populations. Despite vertebrates and ticks having sophisticated systems of defense, rickettsial pathogens have evolved strategies to evade host immunity and cause persistent infections. Mutational studies to be performed with ease in all members of the rickettsiales, including for Anaplasmataceae and Rickettsiaceae family pathogens, remain a major limiting factor. Similarly, the inability to grow the organisms in the absence of host cell support (axenic media growth) is another major hurdle in research progress on all important rickettsial pathogens. The ability to grow obligate intracellular bacteria under axenic conditions can be a major advancement enabling new paths of investigation, such as manipulating the pathogenic organisms with ease, allowing rapid clonal purification of bacterial mutants, permitting detailed biochemical characterization and genetic studies. Our published research progress on mutagenesis studies and axenic media development forms the basis for the current proposed research goals. We propose to address these gaps through the following three focused specific aims: 1) optimize Himar1 mutagenesis supporting the development of a mutational library spanning a greater portion of E. chaffeensis genome; 2) expand the targeted mutation capability in E. chaffeensis and A. phagocytophilum valuable in diverse research applications, and 3) optimize axenic media conditions aiding the continued replication of E. chaffeensis and A. phagocytophilum. Our prior research progress in creating mutations and similarly the preliminary research advances on the axenic media development in Ehrlichia and Anaplasma species highlights the importance of the planned research and substantiates our expertise that the proposed research goals will be accomplished in a timely manner. At the conclusion of this research, we anticipate filling in major research gaps that will aid in extending investigations on several important tick-borne rickettsial pathogens.