ENVIRONMENTAL REGULATION OF CELL GROWTH AND DIVISION IN MYCOBACTERIA

The mycobacterial cell wall is profoundly altered in response to environmental conditions; however, cell wallregulatory mechanisms, which cause antibiotic tolerance, are not well understood. Our long-term goal is tobuild a comprehensive molecular model of mycobacterial cell wall regulation in clinically relevant stresses. Theobjective of this proposal is to determine how two essential factors, FtsQ and SepIVA, contribute to theregulation of cell elongation and division during stress in Mycobacterium smegmatis. The central hypothesis ofthis proposal is that FtsQ and SepIVA are cell division regulators, are post-translationally modified in responseto stress, and that these modifications can lead to altered cell division behavior and stress tolerance. Thishypothesis was formulated based on preliminary data showing that FtsQ phosphorylation impairs survivalunder antibiotic stress, and that the arginine methylations of SepIVA promote survival in stationary phase. Therationale behind this research is that knowledge of the signaling events that allow mycobacteria to respond tostress will lead to a better understanding of mycobacterial infection physiology and mechanisms of antibiotictolerance, which could lead to the development of new anti-mycobacterial drugs. The research in this proposalpursues two specific aims: Aim 1) Determine how phosphorylation of FtsQ contributes to the regulationof cell division and cell wall metabolism. The data suggest that phosphorylation of FtsQ alters the activity ofcell wall enzymes under stress, leading to changes in cell division and stress tolerance. This work will: a)assess how FtsQ phosphorylation impacts cell division and survival in stresses and antibiotic treatment, b)identify the kinase of FtsQ and the conditions under which it is phosphorylated, and c) characterize how FtsQphosphorylation affects protein interactions and divisome assembly. Aim 2) Characterize the mechanism ofSepIVA?s function in cell division, and determine how arginine methylation affects this function. Thedata suggest that SepIVA regulates cell wall precursor enzymes required for cell division, that this regulationcontributes to survival under stress, and that arginine methylations on SepIVA help modulate its function. Thiswork will: a) Identify the stage of cell wall synthesis that SepIVA regulates and determine how SepIVA affectsthe localization of other cell division factors, b) assess how the post-translational regulation of SepIVA alters itslocalization, survival under stress, and role in cell division, and c) identify SepIVA interaction partners anddetermine how arginine methylation affects protein interactions. This work takes an innovative approach to thestudy of cell division regulation by focusing on post-translational modifications of core septal factors - includingarginine methylations, which have never before been described in bacteria. The results of this work willilluminate cell wall regulatory mechanisms that are likely to be important for infection and antibiotic tolerance inpathogenic mycobacteria.