Date of Award

12-26-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Kevin D. Young

Abstract

In the final stages of the bacterial growth cycle, daughter cells must be released from one another by cleaving the shared peptidoglycan cell wall that separates them. Although enzymes capable of cleaving bonds in the murein sacculus are ubiquitous among the bacteria, the physiological roles of only a few of these enzymes are clear. The peptidoglycan-specific amidases play an important role in cleaving the septum to release daughter cells after cell division in Escherichia coli. E. coli has three amidases dedicated to this task: AmiA, AmiB and AmiC. These enzymes remove side chains from the peptidoglycan by hydrolyzing the bond between the peptide and N-acetylmuramic acid and amidase mutants grow as long chains of unseparated cells. E. coli contains seven low molecular weight (LMW) penicillin binding proteins (PBPs) and, with the exception of PBP 5, the physiological roles of most are still obscure. In this work I examined whether any of the PBPs played a role in cell separation. One class of PBPs, the endopeptidases PBPs 4 and 7, cleave the peptide crosslinks between two peptidoglycan monomers. Thus, PBPs 4 and 7 could possibly play a role in septum cleavage either by directly cutting the peptide side chains or by modifying the peptidoglycan to provide alternate substrates for the amidases. To examine the above possibilities mutants lacking multiple LMW PBPs as well as AmiC were created. The results show here that deleting PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the amidases during cell separation. The second endopeptidase, PBP 7, plays a secondary role. In addition, we find that the amidases may have different substrate preferences. The results suggest that AmiC probably prefers to cleave tetrapeptides, while AmiA and AmiB are more prone to cleave pentapeptides. In addition, deletion of LMW PBPs not only exacerbates cell separation defects but also alters the chain morphology. Amidase mutants lacking PBP 5 grow as long twisted chains instead of as straight filaments, indicating that the overall septal morphology is changed. Finally amidase mutants exhibit altered septa, suggesting that in addition to their roles during cell separation, amidases appear to be required for proper orientation of the septum.

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