Using Whole Genome Sequencing to Identify Mutations in E. coli Leading to Cefsulodin Resistance. Escherichia coli K-12 strain:MG1655

Peptidoglycan (PG) is the key component of the cell wall and a hallmark of bacteria. It forms a net-like structure that surrounds the bacterial cell, giving the organism its characteristic shape and protecting it from lysis due to turgor pressure. PG is synthesized by Class A and Class B Penicillin-Binding Protein (aPBP, bPBP). Cefsulodin is a beta-lactam antibiotic that inactivates aPBPs. Even though the bPBPs are still active, they are not sufficient to support proper peptidoglycan synthesis, so E. coli growing in the presence of cefsulodin will lyse and die. We identified cefsulodin resistant mutants by plating E. coli onto media containing cefsulodin and waiting for (rare) colonies to grow. Characterization of seven CefR mutants confirmed increased resistance to cefsulodin. Whole genome sequencing revealed one CefR mutant had a mutation in ihf-alpha (a DNA binding protein), and another had a mutation in yidC (inserts proteins into the membrane). The remaining five CefR mutants had point mutations in mrcB, which encodes the class A PBP called PBP1b, a target of cefsulodin. Biochemical assays confirmed two PBP1b mutants had reduced binding to cefsulodin. Mapping the mutations onto a published crystal structure of PBP1b revealed the mutations are near the active site. Thus, selection for antibiotic resistance identified amino acids that affect binding of cefsulodin to its target PBP1b. This information may help in developing new antibiotics to avoid resistance.