The two-competing-sites model for peptidoglycan assembly for bacterial cell shape regulation suggests that in rods, bacterial cell shape depends on the balance between two reactions (sites), one responsible for lateral wall elongation and the other responsible for septum formation. The two reactions compete with each other so that no lateral wall can be formed during septum formation and vice versa. When the site for lateral wall elongation overcomes that for septum formation, long rods or filaments are formed and cell division may be blocked. When the reaction leading to septum formation is hyperactive compared with the other, coccobacilli or cocci are formed. Other bacteria carry only one site for peptidoglycan assembly and can grow only as cocci. The two-competing-sites model predicts that two different types of cocci exist (among both morphology mutants and wild-type strains); one carries only the site for septum formation, whereas the other also carries the site for lateral wall elongation, the former site predominating over the latter. As a consequence of the inhibition (by antibiotics or by mutations) of septum formation in wild-type cocci of various species and in coccoid morphology mutants, some cocci are expected to undergo transition to rod shape and others are not. We have evaluated these predictions and show that they are in agreement. In fact, we found that among wild-type cocci belonging to 13 species, those of 6 species formed rods, whereas the remaining organisms maintained their coccal shape when septa were inhibited by antibiotics. Some coccoid morphology mutants of rod-shaped bacteria underwent coccus-to-rod transition after septum inhibition by antibiotics, whereas others maintained their coccal shape. When a mutation that causes septum inhibition was expressed in a morphology mutant of Klebsielia pneumoniae grown as a coccus, transition to rod shape was observed. A total of 914 mutants unable to form colonies at 42TC were isolated from the coccoid species mentioned above. Between 75 and 95% of the mutants isolated from the species that formed rods when septum formation was inhibited by antibiotics but none of those isolated from the others underwent coccus-to-rod transition upon incubation at the nonpermissive temperature.In previous papers, we proposed a model for shape regulation in bacteria and presented a large body of experimental data in support of it (33,34,37). In this study, we have extensively tested predictions concerning the effects on cell shape of septum inhibition by antibiotics and mutations in various wild-type and mutant coccoid bacteria (see predictions 6 and 7 of Table 1, which have previously been evaluated only preliminarily). We show that all predictions are fulfilled and demonstrate that some coccoid species but not others undergo transition to rod shape when septa are specifically blocked by antibiotics and that mutants undergoing coccus-to-rod transition can be isolated in some coccoid species but not in others.The two-competing-sites (TCS) model for peptidoglycan ...