Abstract
Cytokinesis in gram-negative bacteria requires the constriction of all three cell envelope layers: the inner membrane (IM), the peptidoglycan (PG) cell wall and the outer membrane (OM). In order to avoid potentially lethal breaches in cell integrity, this dramatic reshaping of the cell surface requires tight coordination of the different envelope remodeling activities of the cytokinetic ring. However, the mechanisms responsible for this coordination remain poorly defined. One of the few characterized regulatory points in the envelope remodeling process is the activation of cell wall hydrolytic enzymes called amidases. These enzymes split cell wall material shared by developing daughter cells to facilitate their eventual separation. In Escherichia coli, amidase activity requires stimulation by one of two partially redundant activators: EnvC, which is associated with the IM, and NlpD, a lipoprotein anchored in the OM. Here, we investigate the regulation of amidase activation by NlpD. Structure-function analysis revealed that the OM localization of NlpD is critical for regulating its amidase activation activity. To identify additional factors involved in the NlpD cell separation pathway, we also developed a genetic screen using a flow cytometry-based enrichment procedure. This strategy allowed us to isolate mutants that form long chains of unseparated cells specifically when the redundant EnvC pathway is inactivated. The screen implicated the Tol-Pal system and YraP in NlpD activation. The Tol-Pal system is thought to promote OM invagination at the division site. YraP is a conserved protein of unknown function that we have identified as a new OM-localized component of the cytokinetic ring. Overall, our results support a model in which OM and PG remodeling events at the division site are coordinated in part through the coupling of NlpD activation with OM invagination.
Highlights
Gram-negative bacterial cells are surrounded by a complex envelope consisting of an inner membrane (IM), a polysaccharide matrix called the peptidoglycan (PG) cell wall, and an outer membrane (OM) [1]
Gram-negative bacterial cells are surrounded by a complex cell envelope consisting of two membranes, the inner and outer membranes, with a layer of cell wall matrix sandwiched between them
Coordinating cell envelope constriction during bacterial cytokinesis the basic mechanisms required for envelope assembly promises to teach us how to defeat this barrier and make gram-negative infections easier to treat
Summary
Gram-negative bacterial cells are surrounded by a complex envelope consisting of an inner (cytoplasmic) membrane (IM), a polysaccharide matrix called the peptidoglycan (PG) cell wall, and an outer membrane (OM) [1]. This triple-layered surface structure is essential for cell integrity and serves as a formidable barrier hindering the penetration of drug molecules into cells. A greater understanding of envelope assembly and remodeling promises to identify better ways of getting drugs into this class of bacteria for the development of effective anti-gram-negative therapeutics In this regard, cell division is an attractive area of study because the process involves a substantial remodeling of the envelope where all three layers are constricted in concert. Most of the components of the divisome have been identified, relatively little is known about how it orchestrates envelope constriction to ensure that cell wall synthesis and remodeling are coordinated with the invagination of the two membrane layers
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