A Mycobacterial Enzyme Essential for Cell Division Synergizes with Resuscitation-Promoting Factor

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Abstract
The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein–protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall–targeting β-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein–protein interaction as one way of regulating its activity. Mycobacteria, like all peptidoglycan-containing bacteria, must extend and cleave the surrounding structurally rigid layer of peptidoglycan to grow and divide. The peptidoglycan hydrolases responsible for this cleavage often have redundant functions, both revealing their importance and making them difficult to study. Furthermore, such hydrolases must be tightly regulated, due to their potentially lytic nature. We recently demonstrated the interaction between a lytic transglycosylase (Rpf) and an endopeptidase (RipA) at the septum of dividing bacteria. To investigate the role of these two hydrolases, we generated conditional mutants of each and were surprised to find that depletion of ripA resulted in long chains of cells. This phenotype was reversed upon induction of ripA, indicating that cell wall expansion and septum formation can be decoupled from the process of septum resolution. In addition, we present data showing that the combination of Rpf and RipA results in enhanced hydrolysis of peptidoglycan in an in vitro assay, suggesting protein–protein interactions as one potential mechanism of regulation.