# Enzymatic activity of PBP1B is required for growth rate-independent ppGpp-mediated resistance to PBP2 inhibitors in E. coli

## TL;DR

This study shows how ppGpp and PBP1B work together in E. coli to resist certain antibiotics, even when bacteria are actively growing.

## Contribution

The study reveals a novel mechanism of antibiotic resistance involving ppGpp and PBP1B enzymatic activity in E. coli.

## Key findings

- E. coli cells overproducing ppGpp showed up to 64-fold resistance to PBP2-targeting β-lactams.
- Resistance required the enzymatic activity of PBP1B and the activator LpoB.
- Nutritional conditions may influence antibiotic efficacy through ppGpp levels.

## Abstract

The alarmone (p)ppGpp (ppGpp) accumulates in response to starvation and other stress, leading to inhibition of multiple biosynthetic pathways and, at high concentrations, suppression of bacterial growth. Growth suppression by ppGpp is implicated in the formation of persister cells, which survive antibiotic challenge only to regrow once the drug is removed. However, there is also evidence that low levels of ppGpp contribute to resistance to certain cell wall-active antibiotics in actively growing cells. To characterize ppGpp’s contribution to antibiotic resistance, we measured MICs of a panel of β-lactams in actively growing Escherichia coli cells overexpressing a ppGpp synthase (relA*). Cells engineered to modestly overproduce ppGpp exhibited up to 64-fold increases in resistance to PBP2-targeting β-lactams only, with mecillinam the most dramatically affected. Resistance required the transcription factor DksA and the class A penicillin-binding protein (PBP) PBP1B. PBP1B variants defective for transpeptidase activity, glycosyltransferase activity, or both were incapable of supporting resistance, suggesting the full enzymatic activity of PBP1B is required for resistance. Transcriptomics revealed that ppGpp overproduction leads to increased expression of lpoB, which encodes an activator of PBP1B. LpoB was required for mecillinam resistance, with an lpoB deletion mutant exhibiting a loss of ppGpp-dependent resistance. An lpoB deletion strain expressing an LpoB-bypass variant of PBP1B (mrcB*) exhibited an intermediate level of resistance. Together, these results suggest that ppGpp overproduction and the LpoB-dependent enzymatic activity of PBP1B function synergistically to promote survival in the presence of PBP2 inhibitors.

Antimicrobial resistance is an increasing global health threat, but its underlying molecular mechanisms remain incompletely understood. This work clarifies ppGpp’s role in mediating antibiotic resistance in Escherichia coli. Elevated levels of ppGpp caused resistance to β-lactam antibiotics targeting the cell wall synthesis enzyme PBP2. Resistance required transcriptional regulation by ppGpp and enzymatic activity of the cell wall enzyme PBP1B. ppGpp overproduction was found to increase expression of the PBP1B activator lpoB. Because ppGpp levels are controlled by nutritional conditions, this work suggests that nutritional availability may impact antibiotic efficacy.