Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly resilient, slow growing pathogen. Despite a robust adaptive immune response by the host, Mtb can persist for the lifetime of an infected individual. The identification of Mtb genes necessary for bacterial persistence in vivo provides insight into the biology of the pathogen, as well as the defenses employed by the host. Here, I used an Mtb transposon mutant to study the role of a novel gene in bacterial replication and survival in vitro and pathogenesis in vivo . Rv0955, a predicted integral membrane protein, was previously identified by our lab in a screen for Mtb transposon mutants sensitive to low pH. While further investigation showed the acid susceptibility phenotype to be detergent-dependent, the rv0955 mutant ( rv0855 ::tn) exhibited a dramatic persistence defect in chronic mouse infection, suggesting an important role for this protein in pathogenesis. Rv0855 ::tn was able to replicate in the acute phase of mouse infection, but was attenuated over 300-fold in the chronic phase. Killing of rv0955 ::tn in vivo was interferon-Þ (IFN-Þ)-dependent. Investigation of the immune response to rv0955 ::tn showed increased production of proinflammatory cytokines by rv0955 ::tn-infected macrophages ex vivo ; however, analysis of the immune response in vivo failed to demonstrate differences in cytokine levels or immune recruitment. In vitro, rv0955 ::tn showed no difference in sensitivity to a range of conditions thought to be encountered in the host, but exhibited an increased requirement for magnesium (Mg 2+ ), demonstrating elongation and compromised replication and survival in reduced-Mg 2+ media. Rv0955 ::tn was not impaired for Mg 2+ acquisition, but exhibited increased permeability in reduced Mg 2+ , suggesting that high Mgt 2+ functions to stabilize the mutant cell wall. Transcriptome analysis revealed increased expression of cell division and cell wall biosynthesis genes in rv0955 ::tn grown in reduced Mgt 2+ . The mutant was acutely sensitive to (Ý-lactam antibiotics, including specific inhibitors of division-associated peptidoglycan synthesis protein FtsI. Rv0955 localized to the mid-cell region and poles in Mycobacterium smegmatis . Together, these data implicate Rv0955 in mycobacterial cell division and peptidoglycan biosynthesis, and provide support for the hypothesis that Mgt 2+ is reduced in the phagosome of activated macrophages in vivo .