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To characterize the consequences of eliminating essential functions needed for peptidoglycan synthesis, we generated deletion mutations of Acinetobacter baylyi by natural transformation and visualized the resulting microcolonies of dead cells. We found that loss of genes required for peptidoglycan precursor synthesis or polymerization led to the formation of polymorphic giant cells with diameters that could exceed ten times normal. Treatment with antibiotics targeting early or late steps of peptidoglycan synthesis also produced giant cells. The giant cells eventually lysed, although they were partially stabilized by osmotic protection. Genome-scale transposon mutant screening (Tn-seq) identified mutations that blocked or accelerated giant cell formation. Among the mutations that blocked the process were those inactivating a function predicted to cleave murein glycan chains (the MltD murein lytic transglycosylase), suggesting that giant cell formation requires MltD hydrolysis of existing peptidoglycan. Among the mutations that accelerated giant cell formation after ß-lactam treatment were those inactivating an enzyme that produces unusual 3->3 peptide cross-links in peptidoglycan (the LdtG L,D-transpeptidase). The mutations may weaken the sacculus and make it more vulnerable to further disruption. Although the study focused on A. baylyi, we found that a pathogenic relative (A. baumannii) also produced giant cells with genetic dependencies overlapping those of A. baylyi. Overall, the analysis defines genetic pathway for giant cell formation conserved in Acinetobacter species in which independent initiating branches converge to create the unusual cells. [ABSTRACT FROM AUTHOR]