Molecular interplay between peptidoglycan integrity and outer membrane asymmetry in maintaining cell envelope homeostasis

The bacterial cell envelope is a critical interface with the environment, particularly in Gram-negative species where the outer membrane and peptidoglycan layers coordinate to maintain structural integrity and resist turgor. Although this coordination is essential for survival, the molecular mechanisms linking outer membrane and peptidoglycan homeostasis remain poorly understood. LD-transpeptidases (LDTs) are enzymes that crosslink peptides in peptidoglycan and incorporate D-amino acids, but their physiological roles are not fully defined. Here, we characterize the activity of the LDT enzyme LdtJ in Acinetobacter baumannii and investigate the consequences of its deletion. Loss of LdtJ disrupts cell morphology, downregulates peptidoglycan precursor genes (e.g., dadA , alr ), and activates the stringent response, including elevated ppGpp levels and dksA upregulation. These defects are fully suppressed in a Δ ldtJ Δ mla double mutant, implicating the outer membrane lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the Δ ldtJ mutant are reversed in the double mutant, highlighting a functional interplay between peptidoglycan remodeling and outer membrane lipid asymmetry. Our findings suggest that LdtJ contributes to envelope integrity not only through peptidoglycan modification but also by influencing broader regulatory and metabolic networks.