The gentamicin 2'-N-acetyltransferase [EC 184.108.40.206; AAC(2')-Ia] of Providencia stuartii was shown to contribute to the O-acetylation of peptidoglycan and mutants that either under- or overexpress the aac(2')-Ia gene was characterized phenotypically to possess either lower or higher levels of peptidoglycan O-acetylation, respectively, compared to the wild-type. These mutants were subjected to scanning electron microscopy. P. stuartii PR100, with 42-44% peptidoglycan O-acetylation compared to 54% for the wild-type, appeared as irregular rods. In direct contrast, strains PR50.LM3 and PR51, with increased levels of peptidoglycan O-acetylation (63 and 65%, respectively), appeared as coccobacilli or chain formers, respectively. Zymogram analysis of the autolysins produced by another member of the closely related Proteeae group of bacteria, Proteus mirabilis, indicated the presence of three classes of enzymes: one that acts preferentially on native, O-acetylated peptidoglycan, a second that hydrolyses non-O-acetylated peptidoglycan, and a third that is not distinguished by the two forms of substrate. On the basis of the apparent morphological changes directly related to levels of O-acetylation combined with the presence of different classes of autolysins, a model is proposed that invokes the role of this modification in the control of autolysins for the maintenance of the structure of the peptidoglycan sacculus.