| The peptidoglycan layer is an essential conserved structure that provides bacteria with shape and structural integrity. It is also a dynamic compartment that is continually modified to accommodate growth, division, and adaptation to stressful environments. Peptidoglycan regulation is critical for the survival of the pathogen, Mycobacterium tuberculosis, both in vitro and during host infection. Characterizing how the peptidoglycan structure is created and remodeled provides new avenues for chemotherapy, as well as insights into one of the most basic, yet poorly understood, bacterial regulatory processes. In order to understand how mycobacteria control peptidoglycan remodeling, I studied the regulation of an essential septal endopeptidase, RipA.;Depletion of RipA in mycobacteria causes daughter cells to chain, while dysregulation of the enzyme by overexpression or antibiotic treatment converts RipA into a lethal autolysin. Thus, the cell must tightly control RipA activity during growth. One way that RipA is regulated is through the formation of holoenzyme complexes---dysregulation of RipA interactions in vivo using dominant negative analysis leads to chaining, as well as cell lysis. To identify RipA complex members, I carried out yeast two-hybrid screens. Here, I characterize two interacting partners for RipA---the lysozyme RpfB and the peptidoglycan synthase PBP1. The RipA-RpfB complex synergistically degrades peptidoglycan, while PBP1 antagonizes this synergy, suggesting that RipA can switch partners to fine-tune its hydrolytic capability. In addition to complex formation, RipA in vivo is also regulated through proteolysis. Through western blot analyses, I demonstrate that processing is required for RipA enzymatic activation. This processing relies partly on C terminal protein-protein interactions, which likely target RipA to cognate proteases.;From my results, I propose a model where RipA is localized at the septum through protein-protein interactions. These interactions aid RipA cleavage and activation only when the cell requires peptidoglycan hydrolysis. After processing, downstream RipA interactions may allow additional temporal control of peptidoglycan remodeling. Finally, slow-growing mycobacteria have reduced RipA cleavage, which may represent a way to synchronize peptidoglycan hydrolysis with the slower growth rate of the bacterium. Therefore, dysregulating RipA is highly toxic and may represent a new avenue for developing novel tuberculosis chemotherapeutics. |
