Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteinsto maintenance of cell shape in Escherichia coli

Escherichia coli has 12 recognized penicillin binding proteins (PBPs), four of which (PBPs 4, 5, and 6 and DacD) have DD-carboxypepidase activity. Alt hough the enzymology of the DD-carboxypeptidases has been studied extensive ly, the in vivo functions of these proteins are poorly understood. To expla in why E. coli maintains four independent loci encoding enzymes of consider able sequence identity and comparable in vitro activity, it has been propos ed that the DD-carboxypeptidases may substitute for one another in vivo. We tested the validity of this equivalent substitution hypothesis by investig ating the effects of these proteins on the aberrant morphology of Delta dac 4 mutants, which produce no PBP 5, Although cloned PBP 5 complemented the m orphological phenotype of a Delta dacA mutant lacking a total of seven PBPs , controlled expression of PBP 4, PBP 6, or DacD did not. Also, a truncated PBP 5 protein lacking its amphipathic C-terminal membrane binding sequence did not reverse the morphological defects and was lethal at low levels of expression, implying that membrane anchoring is essential for the proper fu nctioning of PBP 5. By examining a set of mutants from which multiple PBP g enes were deleted, we found that significant morphological aberrations requ ired the absence of at least three different PBPs. The greatest defects wer e observed in cells lacking, at minimum, PBPs 5 and 6 and one of the endope ptidases (either PBP 4 or PBP 7). The results further differentiate the rol es of the low-molecular-weight PBPs, suggest a functional significance for the amphipathic membrane anchor of PBP 5 and, when combined with the recent ly determined crystal structure of PBP 5, suggest possible mech anisms by w hich these PBPs may contribute to maintenance of a uniform cell shape in E. coli.