The presented project is directed at elucidation how, depending on conditions, proteases are structurally and spatially regulated to DEGRADE essential replication proteins. The research will concentrate on bacterial Lon protease and its phosphate containing cofactors, contributing to the protease activities. We will analyse Lon from bacterial species not being investigated so far in this aspect as well as known and newly identified Lon substrates essential for replication of bacterial DNA. By applying in vitro and in vivo experiments, including real-time, single-molecule and single-cell analysis with a variety of Lon proteins, substrates and cofactors, we expect to describe species-specific, substrate-specific and general effects on Lon, and relate them to growth conditions. The cutting-edge AFM and cryo-EM will be utilise for the description of Lon complexes with cofactors and substrates. Our investigations can provide data on Lon as a new target for new antimicrobial strategies.