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The Role of PBP1B and ppGpp in ß-Lactam Resistance and Characterization of Mecillinam Heteroresistance in Escherichia coli MG1655
Mack, Isabella
Mack, Isabella
Abstract
β-lactams are the most widely prescribed class of antibiotics. Overproduction of the alarmone ppGpp in Escherichia coli confers resistance to the β-lactams mecillinam, meropenem, and doripenem, which target penicillin-binding protein 2 (PBP2) and disrupt cell wall synthesis. This resistance requires PBP1B, a related cell wall synthase, and DksA, a transcription factor that binds ppGpp. We sought to uncover the mechanism by which ppGpp and PBP1B cause resistance to these β-lactams. Measuring minimum inhibitory concentrations (MICs) of PBP1B separation-of-function mutants revealed that full enzymatic activity of PBP1B is essential for resistance. Prior work showed that overproduction of ppGpp upregulates expression of the PBP1B activator lpoB. An LpoB-bypass PBP1B mutant revealed that the LpoB-PBP1B interaction is partially required for resistance. Separately, we also report the first evidence of mecillinam heteroresistance (HR), a subpopulation-driven resistance phenotype. HR was detected in E. coli by Etests and measured with population analysis profiling (PAP). The HR observed was unstable, exhibiting a decrease in antibiotic resistance in the absence of selective pressure. Mutants lacking ppGpp, DksA, and PBP1B exhibited reduced HR. HR was not dependent on the recombinase protein RecA, which has previously been implicated in HR to other antibiotics. qPCR analysis of select mecillinam resistance genes revealed unstably increased copy number of ftsZ, dksA, and mrcB (encodes PBP1B) in the resistant subpopulation. These findings highlight the roles of ppGpp, DksA, and PBP1B in β-lactam resistance and uncover HR as an additional survival pathway during mecillinam exposure.
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2025-12-09
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2027-12-09
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Biology