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

Unlabelled: 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 and investigate the consequences of its deletion. Loss of LdtJ disrupts cell morphology, downregulates peptidoglycan precursor genes (e.g., , ), and activates the stringent response, including elevated ppGpp levels and upregulation. These defects are fully suppressed in a Δ Δ double mutant, implicating the outer membrane lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the Δ 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.

Importance: 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 layer and outer membrane. While LD-transpeptidases (LDTs) are traditionally known for reinforcing peptidoglycan 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 results in pronounced growth defects and abnormal cell morphology - phenotypes that are fully suppressed by disrupting the outer membrane lipid asymmetry transport system, Mla. This genetic interaction uncovers a previously unrecognized link between peptidoglycan remodeling and outer membrane lipid homeostasis. These insights deepen our understanding of envelope coordination in Gram-negative bacteria and suggest that targeting interconnected stress response pathways could offer novel strategies to undermine bacterial resilience.