Binding of bicyclomycin to inner membrane proteins of E. coli.

Abstract

[14C]Bicyclomycin was observed to bind to several inner membrane proteins of E. coli, but not to outer membrane proteins. In SDS polyacrylamide (12.5%) slab gel electrophoresis, 4 major bands and 3 minor bands of binding proteins were demonstrated. Benzylpenicillin showed no competition with bicyclomycin for bicyclomycin-binding proteins (BBPs), and bicyclomycin no competition with penicillin for penicillin-binding proteins (PBPs). The absence of competition and the difference of mobilities of BBPs and PBPs suggested that BBPs differ from PBPs. The molecular weights of BBPs were estimated in comparison with PBPs on slab gel electrophoresis: BBP-1 ca. 93, 000, BBP-2 72, 000, BBP-3 53, 000, BBP-4 46, 000, BBP-5 41, 000, BBP-6 30, 000, and BBP-7 27, 000. The binding to all the proteins seemed to be irreversible in that the antibiotic was not released from the proteins during a 3-hour incubation. From the kinetics of binding it is likely that the binding is a simple bimolecular irreversible reaction. At saturation, 8 pmoles of [14C]bicyclomycin were bound to 1 mg (dry weight) of E. coli, i.e. 2, 400 molecules per cell. An estimate of the number of molecules of each BBP per cell was calculated from measurements of the amount of bicyclomycin bound per cell and the relative proportions of the antibiotic bound to each protein. The results indicated that, besides PBPs, there exist(s) inner membrane protein(s) participating in cell division.