POSSIBLE ROLE OF ESCHERICHIA-COLI PENICILLIN-BINDING PROTEIN-6 IN STABILIZATION OF STATIONARY-PHASE PEPTIDOGLYCAN

Plasmids for high-level expression of penicillin-binding protein 6 (PBP6) were constructed, giving rise to overproduction of PBP6 under the control of the lambda p(R) promoter in either the periplasmic or the cytoplasmic space. In contrast to penicillin-binding protein 5 (PBP5), the presence of high amounts of PBP6 in the periplasm as well as in the cytoplasm did not result in growth as spherical cells or in lysis. Deletion of the C-terminal membrane anchor of PBP6 resulted in a soluble form of the protein (PBP6s350). Electron micrographs of thin sections of cells overexpressing both native membrane-bound and soluble PBP6 in the periplasm revealed a polar retraction of the cytoplasmic membrane. Cytoplasmic over-expression of native PBP6 gave wise to the formation of membrane vesicles, whereas the soluble PBP6 formed inclusion bodies in the cytoplasm. Both the membrane-bound and the soluble forms of PBP6 were purified to homogeneity by using the immobilized dye Procion rubine MX-B. Purified preparations of PBP6 and PBP6s350 formed a 14[C]penicillin-protein complex at a 1:1 stoichiometry. The half-lives of the complexes were 8.5 and 6 min, respectively. In contrast to PBP5, no DD-carboxypeptidase activity could be detected for PBP6 by using bisacetyl-L-Lys-D-Ala-D-Ala and several other substrates. These findings led us to conclude that PBP6 has a biological function clearly distinct from that of PBP5 and to suggest a role for PBP6 in the stabilization of the peptidoglycan during stationary phase.