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

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.

Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression

“…The essential cell wall hydrolase is believed to work in concert with its interacting partner, RpfB, and promote the reactivation process wherein the M. tuberculosis pathogen exits from a non-replicating state to resume active multiplication (36,48). Thus, the ability of MtrB to affect ripA expression via MtrA could be one way of achieving regulated cell wall metabolism.…”

GENES DIFFERENTIALLY EXPRESSED BY Mycobacterium tuberculosis AFTER EXPOSURE TO RUTHENIUM PHOSPHINIC COMPOUND AND ISONIAZID

“…Haiser et al [23] described a related hydrolase of cell wall biosynthesis in Streptomyces coelicolor, indicating the possibility of this class of enzyme be present in Mycobacteriaceae, as observed in our study. Although little is known about the hydrolases in Mycobacteriaceae some authors [24,25] have shown that they may play a role in the late stages of cell division, possibly during re-growth from a stressed state. Additionally, Chauhan et al [24] had proposed previously that the exposure of M. tuberculosis to a variety of DNA damaging agents or certain substances leads to cell division blockage and up regulation of a protein that exhibits cell wall hydrolysis activity, which target the nascent petidoglican synthesis or it may be part of the SOS response in the DNA repair system.…”

Mycobacterium tuberculosis RpfE crystal structure reveals a positively charged catalytic cleft

“…Moreover, Rpf proteins may be regulated by association with distinct partners. Rubin and coworkers 10,11 reported the interaction between RpfB/E and RipA, a peptidase involved in PG remodeling and cell division. The nature of the interaction is uncertain, however, because the structure of RipA 19 revealed that most of the putative Rpf-interacting segment identified using a yeast two-hybrid assay forms a buried bstrand.…”

“…8,9 During growth, the catalytic activity of Rpfs is crucial for cell elongation and division. 10,11 Mtb encodes five Rpf proteins (Rv0867c, Rv1009, Rv1884c, Rv2389c, and Rv2450c named RpfA-E). 12 They are generally predicted to be secreted (RpfA,B,E) or anchored to the membrane (RpfD).…”