Deficiency in peptidoglycan recycling promotes β-lactam sensitivity in <i>Caulobacter crescentus</i>

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. <i>Caulobacter crescentus</i> 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 <i>C. crescentus</i>. Anhydromuropeptides generated by the action of lytic transglycosylases (LTs) are transported to the cytoplasm by the permease AmpG. <i>C. crescentus</i> encodes an <i>ampG</i> homolog, and deletion mutants of <i>sdpA</i> and <i>ampG</i> are sensitive to β-lactams. The <i>ampG</i> deletion mutant displays a significant accumulation of anhydromuropeptides in the periplasm of <i>C. crescentus,</i> demonstrating its essential role in PG recycling. While single knockout mutants of <i>sdpA</i> and <i>ampG</i> exhibit no growth defects, double-deletion mutants (∆<i>sdpA</i>∆<i>ampG</i>) exhibit severe growth and morphological defects. These double mutants also show enhanced sensitivity to β-lactams. Analysis of soluble muropeptides in wild-type (WT), ∆<i>sdpA</i>, and ∆<i>ampG</i> mutants revealed reduced levels of PG precursors (UDP-GlcNAc, UDP-MurNAc, and UDP-MurNAc-P5), suggesting that PG recycling products contribute toward <i>de novo</i> PG biosynthesis. Furthermore, supplementing the growth media with GlcNAc sugar enhanced the fitness of ∆<i>sdpA</i> and ∆<i>ampG</i> mutants under β-lactam stress. In conclusion, our study indicates that defects in PG recycling compromise cell wall biogenesis, leading to antibiotic sensitivity in <i>C. crescentus</i>.<b>IMPORTANCE</b>β-lactam antibiotics target the peptidoglycan cell wall biosynthetic pathway in bacteria. In response to antibiotic pressures, bacteria have developed various resistance mechanisms. In many gram-negative species, cell wall degradation products are transported into the cytoplasm and induce the expression of β-lactamase enzymes. In this study, we investigated the cell wall recycling pathway and its role in antibiotic resistance in <i>Caulobacter crescentus</i>. Based on our data and prior studies, we propose that cell wall degradation products are utilized for the synthesis of peptidoglycan precursors in the cytoplasm. A deficiency in cell wall recycling leads to cell wall defects and increased antibiotic sensitivity in <i>C. crescentus</i>. These findings are crucial for understanding antibiotic resistance mechanisms in bacteria.