Peptidoglycan permease AmpG1 couples lytic transglycosylase activity to muropeptide import in Acinetobacter baumannii

Peptidoglycan (PG) recycling contributes to envelope integrity and antibiotic resistance in gram-negative bacteria. Many species import PG turnover products through multiple systems, including AmpG, OppBCDF, MurP, and NagE. Acinetobacter baumannii, however, lacks OppBCDF, MurP, and NagE but encodes three ampG homologs, suggesting a particular reliance on AmpG-mediated recycling. Bioinformatic analysis identified one homolog, AmpG1, which harbors a distinctive periplasmic domain conserved within the Acinetobacter genus. Because PG turnover occurs in the periplasm through the action of PG hydrolases, we used AlphaFold 3-based prediction to identify potential interacting partners of AmpG1 and found the soluble lytic transglycosylase (Slt) as a top candidate. Microscale thermophoresis confirmed direct binding between Slt and the periplasmic domain of AmpG1. LC-MS/MS analysis of cellular extracts revealed that ΔampG1 and ΔampG1Δslt mutant cells accumulated GlcNAc-1,6-anhydroMurNAc-tri/tetrapeptides and exhibited reduced levels of UDP-MurNAc-pentapeptide, changes not observed in Δslt mutant cells. Moreover, ΔampG1 mutant cells showed aberrant morphology and increased β-lactam sensitivity. This sensitivity results from disrupted PG metabolism due to loss of AmpG1-dependent muropeptide import, rather than from altered β-lactamase activity or membrane permeability. Together, these results support a model in which the periplasmic domain of AmpG1 directly interacts with Slt to couple glycan cleavage with import, thereby promoting efficient uptake of anhydromuropeptides and sustaining precursor supply. These findings delineate a key step in the PG recycling pathway of A. baumannii.