Mechanisms and dynamics of superinfection exclusion in microviral prophage populations PROJECT SUMMARY The Microviridae, small, single-stranded DNA (ssDNA) viruses that infect bacteria are among the most abundant and stable colonizers of the human gut. Therefore, competition for host cells between lysogenic microviruses is expected to be fierce. Hypervariable regions (HVRs) mediating prophage defense against other viruses (superinfection exclusion, SiEx) as well as overcoming these defenses have evolved numerous times concurrently with the evolution of lysogeny in different microviral lineages. We thus hypothesize that microviruses have also evolved one or more entirely novel ways by which they prevent viral superinfections, and that these mechanisms have profound influence on their genome dynamics. In addition, there is evidence that these mechanisms might have been adapted by bacteria to become anti-phage defense systems. Our project’s objectives are thus to understand a) the origin(s) of microviral SiEx and b) the influence of HVR diversity on SiEx dynamics. To this end, we will compare the basic mechanisms of SiEx in independently evolved microvirus prophages, a putative defense system, and potential SiEx-like mechanisms in lytic microviruses that might have served as exaptations in prophage SiEx. Secondly, we will experimentally test the effect of HVRs on SiEx, in particular whether specific mutations allow the avoidance of SiEx and how different HVR types interact with each other. This will be done through synthesis of variants of the model microvirus Enterogokushovirus carrying all known HVR types. Pairwise infection assays will then determine which HVRs can block or overcome SiEx of other HVRs. This study will provide considerable insights into the biology of the Microviridae and also insights into the influence of SiEx on the population dynamics of prophages in general. Additionally, it will contribute to our growing knowledge of the arsenal of phage defense mechanisms in bacteria.