PLoS Pathogens 9, e1003197 (2013)

Credit: MICHAEL CHAO

Peptidoglycan hydrolases are required to degrade the bacterial cell wall during cell division but must be controlled to avoid excess hydrolysis and loss of cell integrity. Recent work has pointed to functional interactions between peptidoglycan hydrolases and other proteins, and the hydrolase RipA is known to contain a protease-sensitive loop, but the overall regulatory mechanisms remain unclear. Chao et al. now show that RipA from Mycobacterium smegmatis is controlled by two post-translational processing mechanisms. The authors first confirmed that overexpression of RipA is toxic to cells. Inhibition of peptidoglycan formation by addition of the β-lactam meropenem was similarly toxic but not in RipA-depleted cells, strengthening previous evidence of a link between peptidoglycan synthesis inhibition and dysregulation of hydrolase activity. RipA transcription was fairly constant, ruling out this possible regulatory mechanism. Instead, the authors observed cleavage of the protein—most likely within the flexible loop—to form smaller constructs associated with the cell wall. The authors confirmed that proteolysis is necessary for function by showing that an inactivated C408A mutant could compete for the unknown processing enzymes. Similarly, introduction of the homologous M. tuberculosis protein, which is not processed by the M. smegmatis machinery, was nontoxic, whereas a truncated construct was toxic. The data support a zymogen model in which RipA is secreted from cells, binds interaction partners and is cleaved to initiate hydrolase activity.