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 (OM) and peptidoglycan (PG) layers coordinate to maintain structural integrity and resist turgor. Although this coordination is essential for survival, the molecular mechanisms linking OM and PG homeostasis remain poorly understood. LD-transpeptidases (LDTs) are enzymes that crosslink peptides in PG and incorporate d-amino acids, but their physiological roles are not fully defined. Here, we characterize the activity of the LDT enzyme LdtJ in <i>Acinetobacter baumannii</i> and investigate the consequences of its deletion. Loss of LdtJ disrupts cell morphology, downregulates PG precursor genes (e.g., <i>dadA</i> and <i>alr</i>), and activates the stringent response, including elevated ppGpp levels and <i>dksA</i> upregulation. These defects are fully suppressed in a ∆<i>ldtJ</i> ∆<i>mla</i> double mutant, implicating the OM lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the ∆<i>ldtJ</i> mutant are reversed in the double mutant, highlighting a functional interplay between PG remodeling and OM lipid asymmetry. Our findings suggest that LdtJ contributes to envelope integrity not only through PG modification but also by influencing broader regulatory and metabolic networks.IMPORTANCE<i>Acinetobacter baumannii</i> is a leading cause of hospital-acquired infections and is highly resistant to antibiotics. Its survival relies on the integrity of the cell envelope, composed of the peptidoglycan (PG) layer and outer membrane (OM). While LD-transpeptidases are traditionally known for reinforcing PG structure through non-canonical crosslinking, our findings reveal that the LdtJ enzyme also plays a critical role in regulating cellular metabolism and stress responses. Deletion of <i>ldtJ</i> results in pronounced growth defects and abnormal cell morphology-phenotypes that are fully suppressed by disrupting the OM lipid asymmetry transport system, Mla. This genetic interaction uncovers a previously unrecognized link between PG remodeling and OM lipid homeostasis. These insights deepen our understanding of envelope coordination in Gram-negative bacteria.