ABSTRACTPeptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are tightly regulated under harsh environmental stresses, few related mechanisms have been investigated. In this study, we aimed to investigate the coordinated and distinct roles of the PGdd-carboxypeptidases (DD-CPases) DacC and DacA in cell growth under alkaline and salt stresses and shape maintenance inEscherichia coli. We found that DacC is an alkaline DD-CPase, the enzyme activity and protein stability of which are significantly enhanced under alkaline stress. Both DacC and DacA were required for bacterial growth under alkaline stress, whereas only DacA was required for growth under salt stress. Under normal growth conditions, only DacA was necessary for cell shape maintenance, while under alkaline stress conditions, both DacA and DacC were necessary for cell shape maintenance, but their roles were distinct. Notably, all of these roles of DacC and DacA were independent ofld-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and the outer membrane lipoprotein Lpp. Instead, DacC and DacA interacted with penicillin-binding proteins (PBPs)—dd-transpeptidases—mostly in a C-terminal domain-dependent manner, and these interactions were necessary for most of their roles. Collectively, our results demonstrate the coordinated and distinct novel roles of DD-CPases in bacterial growth and shape maintenance under stress conditions and provide novel insights into the cellular functions of DD-CPases associated with PBPs.IMPORTANCEMost bacteria have a peptidoglycan architecture for cell shape maintenance and protection against osmotic challenges. Peptidoglycandd-carboxypeptidases control the amount of pentapeptide substrates, which are used in the formation of 4-3 cross-links by the peptidoglycan syntheticdd-transpeptidases, penicillin-binding proteins (PBPs). Sevendd-carboxypeptidases exist inEscherichia coli, but the physiological significance of their redundancy and their roles in peptidoglycan synthesis are poorly understood. Here, we showed that DacC is an alkalinedd-carboxypeptidase for which both protein stability and enzyme activity are significantly enhanced at high pH. Strikingly,dd-carboxypeptidases DacC and DacA physically interacted with PBPs, and these interactions were necessary for cell shape maintenance as well as growth under alkaline and salt stresses. Thus, cooperation betweendd-carboxypeptidases and PBPs may allowE. colito overcome various stresses and to maintain cell shape.