Bacterial cells are surrounded by multilayered cell envelopes that are required for their growth and play crucial roles in their ability to cause disease. The cytoplasmic membrane forms the foundational layer of the envelope. Integrated within this phospholipid bilayer are the protein machineries that assemble other layers of the surface like the essential peptidoglycan cell wall, which is the target for an enormous number of structurally diverse natural product antibiotics. Clinically used examples include the beta-lactams and vancomycin. With resistance to these antibiotics now widespread, understanding how the cell wall is built has become a high priority.<br/>Enzymology at this interface is inherently challenging because the proteins involved often include integral membrane proteins with many transmembrane segments, and they often only function properly when in larger complexes. Moreover, many membrane-associated enzymes use complicated substrates such as lipid-linked carbohydrates that are not commercially available and must be chemically synthesized, assembled with purified enzymes, or in some cases purified from bacterial cells and then chemically modified to enable reaction monitoring. Some substrates are polymers assembled from lipid-linked precursors, and these are even more difficult to obtain. The challenges of studying cell wall assembly in vitro are compounded by difficulties deconvoluting the cellular roles of many biosynthetic enzymes. Phenotypes of mutants are often masked by a high degree of functional redundancy. Here, we will talk about how we have overcome many of the challenges in studying cell wall assembly by combining chemistry and biochemistry with genetics and functional genomics using small molecule probes to unmask cellular function.

Deconstructing the bacterial cell wall (by putting it back together)

ACS Spring Meeting 2021

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