Deficiency in peptidoglycan recycling promotes β-lactam sensitivity in Caulobacter crescentus

Abstract

Peptidoglycan (PG)-modifying enzymes play a crucial role in cell wall remodeling, essential for growth and division. Cell wall degradation products are transported to the cytoplasm and recycled back in most gram-negative bacteria, and PG recycling is also linked to β-lactam resistance in many bacteria. Caulobacter crescentus is intrinsically resistant to β-lactams. Recently, it was shown that a soluble lytic transglycosylase, SdpA, is essential for β-lactam resistance. However, the precise role of SdpA in β-lactam resistance is unknown. This study investigated the PG recycling pathway and its role in antibiotic resistance in C. crescentus. Anhydromuropeptides generated by the action of lytic transglycosylases (LTs) are transported to the cytoplasm by the permease AmpG. C. crescentus encodes an ampG homolog, and deletion mutants of sdpA and ampG are sensitive to β-lactams. The ampG deletion mutant displays a significant accumulation of anhydromuropeptides in the periplasm of C. crescentus, demonstrating its essential role in PG recycling. While single knockout mutants of sdpA and ampG exhibit no growth defects, double-deletion mutants (∆sdpA∆ampG) exhibit severe growth and morphological defects. These double mutants also show enhanced sensitivity to β-lactams. Analysis of soluble muropeptides in wild-type (WT), ∆sdpA, and ∆ampG mutants revealed reduced levels of PG precursors (UDP-GlcNAc, UDP-MurNAc, and UDP-MurNAc-P5), suggesting that PG recycling products contribute toward de novo PG biosynthesis. Furthermore, supplementing the growth media with GlcNAc sugar enhanced the fitness of ∆sdpA and ∆ampG mutants under β-lactam stress. In conclusion, our study indicates that defects in PG recycling compromise cell wall biogenesis, leading to antibiotic sensitivity in C. crescentus.