Murein synthesis and beta-lactam antibiotic susceptibility during rod-to-sphere transition in a pbpA(Ts) mutant of Escherichia coli

Abstract

The conditional morphology mutant of Escherichia coli SP45 grows as a rod at 30°C and assumes a spherical shape after 90 min of incubation at 42°C. The rod-to-sphere morphological transition has been found to be associated with the disappearance of penicillin-binding protein 2 (PBP-2), the progressive reduction (as much as 50%) of murein synthesis, as measured both in intact cells and ether-permeabilized bacteria, and alterations in the structure of the cell envelope, including detachment of the outer membrane from the underlying structures. The detachment was initially localized at the poles of the cells and then spread over the entire surface. Shape transition was also linked to increased susceptibility to beta-lactam antibiotics which preferentially bound to PBP-1A (cephalothin, cephaloridine) or to PBP-3 (furazlocillin, piperacillin). Treatment with beta-lactams possessing a high affinity for PBP-1A, although inducing a low degree of peptidoglycan synthesis inhibition (5 to 10%), was associated with a marked loss of cell viability and massive lysis. On the other hand, the simultaneous absence of PBP-2 and inhibition of PBP-3 caused a significant reduction of peptidoglycan synthesis, yet only slightly affected cell viability. Whereas PBP-1A inhibition during shape transition had no effect on morphology, addition of antibiotics binding to PBP-3 30 min after the temperature shift-up caused formation of elongated cells with a centrally located bulge, not observed in similarly treated cells grown at 30°C. Inhibition of PBP-3 in round cells 90 min after temperature shift caused formation of giant cells, indicating complete loss of elongation ability. The different effects of the simultaneous inhibition of two PBPs, combining mutational loss with specific binding in vivo of another PBP by beta-lactams, proved new insight into the role of these proteins and the killing mechanisms of this class of antibiotics.