Search

The low-molecular-weight (LMW) penicillin-binding protein, PBP 5, plays a dominant role in determining the uniform cell shape of Escherichia coli. However, the physiological functions of six other LMW PBPs are unknown, even though the existence and enzymatic activities of four of these were established three decades ago. By applying fluorescence-activated cell sorting (FACS) to quantify the cellular dimensions of multiple PBP mutants, we found that the endopeptidases PBP 4 and PBP 7 also influence cell shape in concert with PBP 5. This is the first reported biological function for these two proteins. In addition, the combined loss of three DD-carboxypeptidases, PBPs 5 and 6 and DacD, also impaired cell shape. In contrast to previous reports based on visual inspection alone, FACS analysis revealed aberrant morphology in a mutant lacking only PBP 5, a phenotype not shared by any other strain lacking a single LMW PBP. PBP 5 removes the terminal D-alanine from pentapeptide side chains of muropeptide subunits, and pentapeptides act as donors for cross-linking adjacent side chains. As endopeptidases, PBPs 4 and 7 cleave cross-links in the cell wall. Therefore, overall cell shape may be determined by the existence or location of a specific type of peptide cross-link, with PBP 5 activity influencing how many cross-links are made and PBPs 4 and 7 acting as editing enzymes to remove inappropriate cross-links.

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.