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Schaefer, Kaitlin

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Schaefer

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Kaitlin

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Schaefer, Kaitlin

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2014 - 20183

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Author's Publications: search.filters.isAuthorOfPublication.fef7ed7e-780b-44df-9e80-c7a5415a6aa1

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    Detection of Lipid-Linked Peptidoglycan Precursors by Exploiting an Unexpected Transpeptidase Reaction
    (American Chemical Society, 2014) Qiao, Yuan; Lebar, Matthew D.; Schirner, Kathrin; Schaefer, Kaitlin; Tsukamoto, Hirokazu; Kahne, Daniel; Walker, Suzanne
    Penicillin-binding proteins (PBPs) are involved in the synthesis and remodeling of bacterial peptidoglycan (PG). Staphylococcus aureus expresses four PBPs. Genetic studies in S. aureus have implicated PBP4 in the formation of highly cross-linked PG, but biochemical studies have not reached a consensus on its primary enzymatic activity. Using synthetic Lipid II, we show here that PBP4 preferentially acts as a transpeptidase (TP) in vitro. Moreover, it is the PBP primarily responsible for incorporating exogenous d-amino acids into cellular PG, implying that it also has TP activity in vivo. Notably, PBP4 efficiently exchanges d-amino acids not only into PG polymers but also into the PG monomers Lipid I and Lipid II. This is the first demonstration that any TP domain of a PBP can activate the PG monomer building blocks. Exploiting the promiscuous TP activity of PBP4, we developed a simple, highly sensitive assay to detect cellular pools of lipid-linked PG precursors, which are of notoriously low abundance. This method, which addresses a longstanding problem, is useful for assessing how genetic and pharmacological perturbations affect precursor levels, and may facilitate studies to elucidate antibiotic mechanism of action.
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    Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams
    (2017) Qiao, Yuan; Srisuknimit, Veerasak (Jeep); Rubino, Frederick; Schaefer, Kaitlin; Ruiz, Natividad; Walker, Suzanne; Kahne, Daniel
    Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. Beta-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by beta-lactams has been hampered by lack of access to substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate utility by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2, PBP2, which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different beta-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors.
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    MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis
    (eLife Sciences Publications, Ltd, 2018) Hussain, Saman; Wivagg, Carl N; Szwedziak, Piotr; Wong, Felix; Schaefer, Kaitlin; Izoré, Thierry; Renner, Lars D; Holmes, Matthew; Sun, Yingjie; Bisson-Filho, Alexandre W; Walker, Suzanne; Amir, Ariel; Löwe, Jan; Garner, Ethan
    MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.