Activation and Termination of Peptidoglycan Synthesis in Escherichia Coli
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Bohrhunter, Jessica L.
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CitationBohrhunter, Jessica L. 2019. Activation and Termination of Peptidoglycan Synthesis in Escherichia Coli. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractThe peptidoglycan cell wall (PG) is a contiguous matrix formed from long glycan strands crosslinked by short peptides. It is essential for bacterial survival and morphology and is an important antibiotic target. Construction of PG is accomplished through the concerted activities of peptidoglycan synthases, which have glycosyltransferase activity to polymerize glycan strands and transpeptidase activity to form peptide crosslinks, and PG cleaving enzymes. Although numerous PG synthases and PG cleaving enzymes have been identified, little is known about how their activities are controlled or what their physiological roles are.
MltG is a lytic transglycosylase, which cleaves glycan strands, that has been previously characterized as a glycan strand terminase, due to its unique inner membrane localization and the finding that ∆mltG increased average glycan strand length. However, further study of this enzyme was hampered by lack of a ∆mltG phenotype. We identified mltG mutations as suppressors of lethality under multiple conditions where peptidoglycan synthesis is impaired. These phenotypes allowed us to perform domain function analysis on MltG. We found MltG requires its membrane proximal LysM domain and inner membrane localization for full activity, suggesting the primary substrate of MltG is nascent glycan strands. Combined with the phenotypic results, these findings are consistent with a model of MltG as a glycan strand terminase. Additionally, we identified genetic and bacterial two-hybrid data which support an association between MltG and the Rod system.
PBP1a is a PG synthase and requires activation by the outer membrane lipoprotein LpoA for in vivo activity. However, it was not known how LpoA achieved activation of PBP1a. We identified PBP1a variants that bypass the requirement for LpoA activation in vivo. The amino acid substitutions in these variants cluster in the linker region between the two catalytic domains, suggesting a role for this region in modulating PBP1a activity. We further found that these variants have increased glycosyltransferase activity, suggesting that LpoA may play a role in stimulating glycan synthesis in vivo, despite primarily affecting the crosslinking activity of PBP1a in vitro.
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