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.
Highlights
Though not formally considered virulence factors, genes required for bacterial cell division clearly are necessary for the growth, and pathogenesis, of bacteria
We recently demonstrated the interaction between a lytic transglycosylase (Rpf) and an endopeptidase (RipA) at the septum of dividing bacteria
We present data showing that the combination of resuscitation-promoting factors (Rpf) and Rpf-interacting protein A (RipA) results in enhanced hydrolysis of peptidoglycan in an in vitro assay, suggesting protein– protein interactions as one potential mechanism of regulation
Summary
Though not formally considered virulence factors, genes required for bacterial cell division clearly are necessary for the growth, and pathogenesis, of bacteria. The distinction between homeostatic and virulence genes is blurred when nonessential genes involved in vegetative cell division become essential under specific stressful conditions encountered inside a host. Such an example is seen with the resuscitation-promoting factors (Rpf) encoded by many different bacteria, including mycobacteria. One single deletion (rpfB) and several of the triple combinations yielded strains unable to grow or divide in stressful conditions in vitro and in vivo [2,3] This suggests that certain potential cell division proteins that appear to play nonessential roles in homeostatic processes can become vital in conditions of stress
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