A significant amount of global morbidity and mortality is caused by bacterial pathogens, most of which cause disease by destroying host immune cells or employing them as protected intracellular niches for replication. In order to accomplish this, pathogens must secrete toxins into the extracellular milieu or inject effector molecules into host cells. As a result, the investigation of secretion systems and their substrates has been a major focus of bacterial pathogenesis research. One key to export of toxins/effectors is recognition of the substrate by the secretion system; the protein domain responsible for this is commonly referred to as a signal sequence. Gram-negative bacteria employ a variety of specialized secretion systems during pathogenesis and these have been classified as type I-IX, based on the homology of their components. Type IV secretion systems (T4SSs) consist of plasmid transfer systems, often involved in antibiotic resistance, and adapted conjugation systems, which are commonly used by a variety of intracellular pathogens. Legionella pneumophila, a Gram-negative bacterium that causes pneumonia by replicating inside alveolar macrophages, employs a type IV secretion system called Dot/Icm. The L. pneumophila Dot/Icm T4SS is an amazingly robust apparatus as it injects approximately three hundred different effectors, which will be referred to as Legionella Dot/Icm translocated substrates (LDTSs), into the host cell. It has been proposed that the signal sequence for LDTSs consists of the last ~20 amino acids of each protein. However, this has not been experimentally confirmed for the majority of substrates, and the lack of sequence homology between LDTSs in this region called this idea into question. Although several consensus sequences have been proposed, they reflect only a fraction of the total number of LDTSs that have been identified. In addition, it was recently reported that the LDTS SidJ contains both a C-terminal and an internal signal sequence that function redundantly to export the protein, although strikingly only the internal motif is required during infection. This proposal will utilize a novel experimental approach to identify key features of the C- terminal signal sequence (Aim#1), and then discover additional LDTSs that contain an internal signal sequence (Aim#2).