Peptidoglycan DD-peptidases have distinct activities that impact fitness of <i>Acinetobacter baumannii</i>

The gram-negative cell envelope is a critical interface between the bacterium and its environment, serving as a selective barrier for nutrient uptake and defense against harmful agents. It also facilitates environmental sensing and adaptive responses. Structurally, it comprises the outer membrane, inner membrane, and periplasmic space, which contains the peptidoglycan layer-a conserved polymer that maintains cell shape and withstands internal turgor pressure. Peptidoglycan consists of glycan strands connected by short peptides, forming a mesh-like structure. In gram-negative bacteria, most peptidoglycan subunits contain tetrapeptides, generated by DD-carboxypeptidases (DD-CPases) that cleave the terminal D-alanine from pentapeptides. Although gram-negative bacteria encode multiple DD-CPases, their precise roles in maintaining cell shape and structural integrity remain poorly understood. The nosocomial pathogen <i>Acinetobacter baumannii</i> encodes three putative DD-CPases. To investigate their functions, we generated single and double mutants in <i>dacC</i>, <i>dacD</i>, and <i>pbpG</i>, which encode homologs of <i>Escherichia coli</i> DD-CPases PBP5 and PBP6a, PBP6b, and the endopeptidase PBP7, respectively. We assessed the mutants for changes in cell morphology, growth dynamics, and pH-dependent stress tolerance. Additionally, we analyzed their peptidoglycan composition to determine the biochemical consequences of enzyme inactivation. Each mutant showed distinct alterations in coccobacillary morphology and growth. Peptidoglycan analysis showed DD-CPase activity, with PBP6b also exhibiting endopeptidase activity. Together, our results demonstrate that each peptidoglycan-modifying enzyme contributes uniquely to cell growth, morphology, and pH tolerance, underscoring their non-redundant functions.IMPORTANCEDD-peptidases, including carboxypeptidases and endopeptidases, are crucial for maintaining cell envelope homeostasis, with distinct roles for each enzyme in cell wall biogenesis and structural integrity. The enzymatic characterization presented in this study not only advances our understanding of fundamental <i>Acinetobacter baumannii</i> biology but also highlights these enzymatic activities as targets for the development of innovative therapeutic strategies to combat infections caused by this multidrug-resistant microbe.