ABSTRACT Calcium (Ca2+) is a major signaling mechanism in eukaryotic cells, but although Ca2+ signaling has ancient origins, it is not well understood in bacteria. Several bacteria have been shown to maintain a low intracellular Ca2+ concentration, which is the basis for Ca2+ signaling in eukaryotes. In addition, some bacterial Ca2+- binding proteins and putative transporters have been identified, suggesting the presence of a bacterial Ca2+ signaling system analogous to that in eukaryotes. However, many fundamental questions about bacterial Ca2+ signaling remain, from maintenance of Ca2+ gradients to Ca2+ triggers, Ca2+ binding proteins, Ca2+ storage, and finally the effects of changing Ca2+ concentrations on bacterial physiology and pathogenesis. Using a FRET-based Ca2+ reporter system in Mycobacterium tuberculosis (Mtb) that allows for precise, real time measuring of intracellular Ca2+ levels, we will explore these fundamental questions about Ca2+ signaling in Mtb. We will use a novel proteomic approach, thermal protein profiling, to globally detect Ca2+ binding proteins in Mtb. We also identified a novel Ca2+ porin and will use a porin knockout strain to test for the role of Ca2+ in Mtb pathogenesis. Together, we will leverage these new tools to test the hypothesis that Mtb maintains a Ca2+ signaling system analogous to that in many eukaryotes and will begin to dissect the physiologic and pathogenic implications of Ca2+ homeostasis in Mtb.