Detection of penicillin-binding proteins in Bacillus cereus by using biotinylated β-lactams

Abstract

A new method for detection of penicillin-binding proteins from bacterial membranes has been developed in this study. The method, that employed biotin-ampicillin conjugate (Bio-PCA), is very rapid and can be a significant alternative of hazardous and time consumimng conventional radiometric method for detection of PBPs in cell membranes. PBPs from B. cereus were examined by this technique. In vegetative and sporulating cells, 8 major PBPs were detected. Comparing with standard marker proteins, these PBPs were estimated for their molecular weight as 106, 83, 75, 72, 63, 46, 40, and 32 kDa. Affinity of cephalexin, cefoxitin, and cefotaxime to PBPs was measured indirectly by competition for subsequent binding of Bio-PCA. PBPs 2, 3, 4, and 7 were decreased or disappeared in the electrophoregram by prebinding with these β-lactams. Two PBPs, PBP 3 and PBP 4, which were predominant in vegetative and sporulating cells, respectively, showed strong affinity for cephalexin.