Peptidoglycan recycling is critical for cell division, cell wall integrity and β-lactam resistance in Caulobacter crescentus

Reviewer #1 (Public review):

Summary:

In their manuscript, Richter and colleagues comprehensively investigate the cell wall recycling pathway in the model alphaproteobacterium Caulobacter crescentus using biochemical, imaging, and genetic approaches. They clearly demonstrate that this organism encodes a functional peptidoglycan recycling pathway and demonstrate the activities of many enzymes and transporters within this pathway. They leverage imaging and growth assays to demonstrate that mutants in peptidoglycan recycling have varying degrees of beta-lactam sensitivity as well as morphological and cell division defects. They propose that, rather than impacting the levels or activity of the major beta-lactamase, BlaA, defects in PG recycling lead to beta-lactam sensitivity by limiting the availability of new cell wall precursors. The findings will be of interest to those in the field of bacterial cell wall biochemistry, antibiotics and antibiotic resistance, and bacterial morphogenesis.

Strengths:

Overall, the manuscript is laid out logically, and the data are comprehensive, quantitative, and rigorous. The mutants and their phenotypes will be a valuable resource for Caulobacter researchers.

Weaknesses:

The only major missing piece is the complementation of mutants to demonstrate that loss of the targeted gene is responsible for the observed phenotypes.

Reviewer #2 (Public review):

Summary:

Pia Richter et al. investigated the peptidoglycan (PG) recycling metabolism in the alpha-proteobacterium Caulobacter crescentus. The authors first identified a functional recycling pathway in this organism, which is similar to the Pseudomonas route, and they characterized two key enzymes (NagZ, AmiR) of this pathway, showing that AmiR differs in specificity from the AmpD counterpart of E. coli. Further, they studied the effects of deletions within the PG recycling pathway (ampG, amiR, nagZ, sdpA, blaA, nagA1, nagA2, amgK, nagK mutants), showing filamentation and cell widening, thereby revealing a link between PG recycling and cell division. Finally, they provide a link between PG recycling and beta-lactam sensitivity in C. crescents that is not caused by activation of a beta-lactamase, but rather is a result of reduced supply of PG building blocks increasing the sensitivity of penicillin-binding proteins.

Strengths:

This work adds to the understanding of the role of PG recycling in alpha-proteobacteria, which significantly differ in their mode of cell wall growth from the better studied gamma-proteobacteria.

Weaknesses:

The findings are not entirely novel as recent studies by Modi et al. 2025 mBio (studying C. crescentus) and Gilmore & Cava 2022 Nat. Commun. (studying Agrobacterium tumefaciens) came to similar conclusions.