The ABC's of Cell Division: Regulation of Peptidoglycan Amidase Activity during Cytokinesis in Escherichia coli

Abstract

The bacterial cell wall, composed of peptidoglycan (PG), is an essential component of the cell envelope. This macromolecular structure fortifies the cell membrane, determines cell shape, and helps prevent osmotic lysis. The synthesis and remodeling/recycling of this polymer is mediated by PG synthases and hydrolases, respectively. Proper control of the PG hydrolases is particularly important since misregulation of these enzymes can lead to lethal breaches in the cell wall. Surprisingly, however, the precise molecular mechanisms governing the activities of these enzymes remain poorly understood. To help understand how PG hydrolases are regulated, I examined how their activity is controlled during cytokinesis in Escherichia coli. One important class of PG hydrolases necessary for cell division is the LytC-type amidases (AmiA, AmiB and AmiC). These enzymes require activation by the LytM factors EnvC and NlpD. My work focused on elucidating the mechanism by which the LytM factors activate the amidases. Using a genetic enrichment strategy, I isolated amiB misregulation mutants. Interestingly, the mutations mapped to a region of AmiB found only in cell separation amidases. Structural analysis of an AmiB ortholog indicates that this region corresponds to an alpha-helical domain that appears to occlude the active site. Thus, activation of the amidases by the LytM factors likely occurs via a conformational change that displaces the regulatory helix from the active site. In addition to amidase regulation, I also investigated how the LytM activators are recruited to, and regulated at the site of division. Using genetic and biochemical approaches, I showed that EnvC is directly recruited to the division site by FtsEX, an ATP-binding transporter- like complex. Interestingly, ATPase-defective FtsEX derivatives can still recruit EnvC to the divisome, but fail to promote cytokinesis. These results support a model where conformational changes induced by the ATPase activity of FtsE are directly and specifically transmitted to the amidases via FtsX and EnvC. This model is attractive because it provides a mechanism for converting the potentially dangerous activity of septal PG splitting into a discrete process which can be cycled on and off in coordination with the division process.