Bacillus anthracis is a Gram-positive, spore-forming bacillus and causes the disease anthrax. Anthrax is a deadly infection that begins with phagocytosis of a B. anthracis spore by an antigen presenting cell and ends when bacilli-laden blood from the carcass is exposed to oxygen, which initiates sporulation. Each step in the infection process requires access to nutrients, including iron. Iron is required as a protein co-factor for many different cellular processes but is also tightly regulated within organisms, including both the mammalian host and the bacterium. To gather iron during infection, B. anthracis employs a heme acquisition system as well as two siderophores, petrobactin and bacillibactin. Of these three systems, only petrobactin is required for growth in iron-deplete medium, macrophages, and to cause disease in mouse models of infection. Chapter one describes what is understood about petrobactin including biosynthesis by the asb operon, regulation of biosynthesis, how the ferric-petrobactin complex is imported, and relevance to disease. I also highlight remaining questions such as how petrobactin is exported from the cell and its role in spore biology. In chapter two, I describe my work identifying the petrobactin exporter ApeX. Using a bioinformatics-based protocol to identify putative targets for petrobactin export, I generated single deletion mutants. Laser ablation electrospray ionization mass spectroscopy (LAESI-MS) was adapted to screen for deletion mutants that failed to export petrobactin, enabling identification of ApeX as a petrobactin exporter. An apeX deletion mutant unable to export petrobactin, instead accumulated the molecule within the cell pellet and exported components. These petrobactin components are still able to transport iron and cause disease in a mouse model of inhalational anthrax. I also used LAESI-MS in chapter three to detect petrobactin within B. anthracis spores and explore the role of petrobactin in spore biology. Petrobactin is not required for germination from the spore, but is required for rapid sporulation in the iron-rich ModG sporulation medium. Fluorescent reporters show induction of the asb operon during late stage growth and early sporulation. This phenotype is likely relevant to disease transmission since experiments in defibrinated bovine blood demonstrate that petrobactin is the preferred iron acquisition system during growth in blood and is required for sporulation in aerated blood. Chapter four offers hypotheses and suggestions for how to answer remaining questions not addressed by the research done in this work. I also make hypotheses regarding alternative, non-iron-scavenging functions for petrobactin and discuss the future potential for LAESI-MS as a research tool.PHDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144141/1/akhagan_1.pdf