Mechanisms Controlling the Cell Envelope Remodeling Activities of the Escherichia Coli Cytokinetic Ring
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CitationTsang Mui Ching, Mary-Jane. 2016. Mechanisms Controlling the Cell Envelope Remodeling Activities of the Escherichia Coli Cytokinetic Ring. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractIn Escherichia coli, cytokinesis requires the constriction of all the cell envelope layers: the inner membrane, the peptidoglycan (PG) cell wall and the outer membrane (OM). Such dramatic cell envelope remodeling requires tight coordination of the different activities of the division complex or cytokinetic ring in order to avoid potentially lethal breaches in cell envelope integrity. However, the mechanisms coordinating these cell wall and membrane remodeling events remain poorly defined.
To better understand the constriction process, I studied an allele of the essential division gene ftsL with an unusual lytic phenotype and showed that it accelerated the division process by prematurely initiating cytokinesis. Additionally, this mutant bypassed the requirement for other essential division proteins. These results suggest a role for FtsL as part of a mechanism that senses the assembly status of the cytokinetic apparatus and triggers constriction when assembly is complete.
Cell wall remodeling during cytokinesis requires the hydrolytic enzymes called amidases and their activators. One of the activators is the OM lipoprotein NlpD, but how amidase activation by NlpD is controlled is unknown. To gain insight into its activity and regulation, I performed a structure-function analysis. Sub-domains required for recruitment to the division site or proper regulation of PG hydrolysis were identified. Interestingly, I found that aberrant amidase activation by a subset of NlpD truncations disrupted cytokinetic ring assembly and inhibited cell division. These findings support a model in which cell wall processing is communicated to cytoskeletal polymers in the cytoplasm to ensure continued cell constriction at the chosen division site.
Next, I investigated the regulation of NlpD using a flow cytometry-based enrichment strategy to isolate mutants with a cell chaining phenotype similar to that of NlpD-defective cells. I identified the Tol-Pal system, which has been implicated in OM constriction during cytokinesis, and an OM lipoprotein of unknown function called YraP as possible control factors for NlpD. My genetic analysis suggests a possible coupling between PG processing and OM constriction during cell division. Overall, these studies reveal a central role for cell wall remodeling in the coordinated constriction of the cell envelope layers.
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