The light-organ symbiont Vibrio fischeri releases N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramylalanyl-␥-glutamyldiaminopimelylalanine, a disaccharide-tetrapeptide component of peptidoglycan that is referred to here as "PG monomer." In contrast, most gram-negative bacteria recycle PG monomer efficiently, and it does not accumulate extracellularly. PG monomer can stimulate normal light-organ morphogenesis in the host squid Euprymna scolopes, resulting in regression of ciliated appendages similar to that triggered by infection with V. fischeri. We examined whether the net release of PG monomers by V. fischeri resulted from lytic transglycosylase activity or from defects in AmpG, the permease through which PG monomers enter the cytoplasm for recycling. An ampG mutant displayed a 100-fold increase in net PG monomer release, indicating that AmpG is functional. The ampG mutation also conferred the uncharacteristic ability to induce light-organ morphogenesis even when placed in a nonmotile flaJ mutant that cannot infect the light-organ crypts. We targeted five potential lytic transglycosylase genes singly and in specific combinations to assess their role in PG monomer release. Combinations of mutations in ltgA, ltgD, and ltgY decreased net PG monomer release, and a triple mutant lacking all three of these genes had little to no accumulation of PG monomers in culture supernatants. This mutant colonized the host as well as the wild type did; however, the mutant-infected squid were more prone to later superinfection by a second V. fischeri strain. We propose that the lack of PG monomer release by this mutant results in less regression of the infection-promoting ciliated appendages, leading to this propensity for superinfection.Microbe-associated molecular patterns (MAMPs) are recognized by hosts in a variety of pathogenic and symbiotic relationships. MAMP is an umbrella term for a variety of semiconserved bacterial molecules, including lipopolysaccharide, lipoproteins, flagella, and peptidoglycan (PG), that are sensed by conserved host surveillance mechanisms (e.g., the innate immune system), triggering context-dependent reactions to bacterial colonization. Mounting evidence shows that PG-derived MAMPs play important and previously underappreciated roles in host-bacterium interactions (11).The PG layer of gram-negative bacteria is a rigid network in the periplasm that protects against osmotic lysis and helps to determine cell size and shape while still allowing diffusion of molecules into the cell (14). In PG, repeated subunits of Nacetylglucosamine and N-acetylmuramic acid are connected to a short pentapeptide side chain of L-alanyl-D-␥-glutamyl-mesodiaminopimelyl-D-alanyl-D-alanine (Ala-Glu-DAP-Ala-Ala). Adjacent peptides are cross-linked through Ala-DAP or DAP-DAP peptide bonds, and side chains are converted to tetra-, tri-, and dipeptides through the action of carboxypeptidases in the periplasm (19,49).Despite its mechanical stability, PG is a dynamic structure that undergoes remodeling and recycling. Murein hydrola...