Pathogenic mycobacteria are a global health problem. We use Mycobacterium marinum as a model system for Mycobacterium tuberculosis, the causative agent of human Tuberculosis. We have developed tools for studying mycobacterial pathogenesis and identifying genes required for virulence. The Esx-1 export system is essential for mycobacterial virulence. We built a saturating transposon library in M. marinum and screened for mutants deficient for Esx-1 export. We identified several Esx-1-deficient strains using these novel assays and characterized them using molecular genetics, biochemistry, and microbiology. In addition to genetic tools, we developed mass spectrometry tools for monitoring Esx-1. We designed and tested a screening method using MALDI-TOF mass spectrometry for high-throughput screening. Western blotting for Esx-1 substrates is the gold standard for monitoring Esx-1 function. However, this is limited by sensitivity and antibody availability. We implemented targeted MRM mass spectrometry to monitor multiple Esx-1 substrates across multiple strains to elucidate distinct genetic requirements for Esx-1 export of several Esx-1 substrates.Using virulence assays, we identified an Esx-1-deficient strain with a nonsense mutation in a known Esx-1-required gene. We isolated a suppressor strain with the nonsense mutation that had restored Esx-1 export and virulence. We characterized both the Esx-1-deficient strain and the suppressor strain using genetics, genomics, biochemistry, and bioinformatics. We found that suppression was linked to a mutation in a gene required to synthesize lipids.Finally, we characterized a novel locus, MMAR_5349-MMAR_5350. We demonstrated that MMAR_5349 is involved in multiple export systems, including three separate Esx systems. We also showed that MMAR_5350 is a novel Gre-like factor that induces a novel RNA cleavage activity by RNA Polymerase and is involved in the temperature sensitivity of M. marinum, which does not typically grow above 35°C. However, we found that deletion of MMAR_5350 led to an increase in growth at 37°C and overexpression of MMAR_5350 completely abrogated growth at 37°C.Through these studies we have characterized several mechanisms by which pathogenic mycobacteria survive in their host environment and cause disease. These studies add to our knowledge base to help develop novel strategies of fighting these pathogens.