Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli

Abstract

The penicillin-binding proteins (PBPs) are a set of enzymes responsible for the terminal stages of peptidoglycan biosynthesis, where they carry out transpeptidation, transglycosylation, endopeptidase, or carboxypeptidase functions. It is the transpeptidation, endopeptidase, and carboxypeptidase functions that are inhibited when these proteins are acylated by a ␤-lactam antibiotic (10). In all cases where this reaction has been investigated biochemically, the ␤-lactam ring opens and forms a covalent bond with an active-site serine residue on the PBP (10, 11). Among the most extensively investigated PBPs are those of the gram-negative bacillus Escherichia coli, and these have been the subject of numerous publications and reviews. It is for E. coli that we have perhaps the clearest understanding of the roles that the individual PBPs perform in peptidoglycan synthesis and cell morphogenesis (7,19,21). At the same time, however, much remains to be understood concerning the role of the PBPs in cell wall growth and their participation in cell division.More than 18 years ago, Spratt published an estimate of the numbers of individual PBPs in an average E. coli cell (25). In this seminal work, the PBPs of isolated cell membranes were labeled with [ 14 C]penicillin and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After electrophoresis, the radiolabeled PBPs were detected by fluorography. The numbers of PBPs per cell were calculated by scanning fluorographs, obtaining the areas under the curves and calculating a percentage distribution for each of the individual PBPs. This relative distribution was then combined with a literature value for the total amount of [ 14 C]cefoxitin bound per milligram (dry weight) and a literature value for cell number per milligram (dry weight) to arrive at absolute values of individual PBP numbers per cell (20,25). Although this was clearly meant as a rough estimate, this set of numbers has been cited repeatedly in numerous publications and several textbooks since that time (1,7,19,21,26).Because of the importance of these proteins in ␤-lactam antibiotic action, cell wall assembly, and cell division, we decided to measure the numbers of the individual PBPs of E. coli by a direct method. In the present study, the numbers of molecules per cell of the individual PBPs were calculated directly by measuring both the cell numbers present and the radioactivity in the individual PBPs from a precise quantity of cells. Care was taken to ensure that the individual PBPs were saturated with [ 3 H]penicillin, and a sufficiently large number of samples were taken in separate experiments to allow statistical assessment of the data sets. The results obtained in the present study are in some cases significantly different from the previously published values. In addition, measurements were performed with both rapidly growing cells in Luria broth (LB) medium as well as with cells growing in M9 minimal medium. The absolute numbers of PBPs were reduced in the cells growing at a lower ...

A dynamically assembled cell wall synthesis machinery buffers cell growth

“…Transient interactions between MreB and PBP2 may explain how E. coli maintains robust cell growth despite only expressing ∼100 PBP2 molecules per cell (21). If PBP2 were part of a stable multienzyme complex, then previous calculations have indicated that ∼100 PBP2 molecules would be required to maintain a 20-min doubling time (3), and reductions in PBP2 levels would lead to a decrease in growth rate.…”

“…In fact, transient associations are beneficial for buffering growth against fluctuations in enzyme abundance. At a PBP2 abundance of ∼100 enzymes per cell (21), one would expect 1/√N ∼ 10% fluctuations, yet growth rate remains consistent through cell division (24). In addition, transient association loosens the requirement for an MreB complex to spatially and temporally order the steps of cell wall synthesis.…”

“…Previous studies have estimated that there are ∼100 PBP2 molecules per E. coli cell (21). To estimate whether each PBP2 could participate in five crosslinking events per second, we assume that cell wall material is synthesized in a spatially uniform fashion across the cylindrical portion of the cell, which has an average length of 3 μm and an average surface area of ∼9 μm 2 .…”

Localization of the Escherichia coli cell division protein FtsI (PBP3) to the division site and cell pole

“…As there are only about 100 molecules of FtsI per cell (Spratt, 1977;Dougherty et al, 1996), we used immunofluorescence microscopy (IFM) rather than immunoelectron microscopy (IEM) to visualize FtsI. IFM is more sensitive than IEM, in part because in IFM one views whole cells and can, in principle, detect all of the molecules of a given protein within a cell.…”

Comparative analysis of Pseudomonas aeruginosa penicillin-binding protein 7 in the context of its membership in the family of low-molecular-mass PBPs

“…In E. coli the DD-endopeptidase activity appears to be due to redundant catalytic activities of DacB and PbpG, while DD-carboxypeptidase activity appears to be due to the redundant catalytic activities of E. coli DacA, DacC and DacD. In E. coli PbpG has been shown to constitute up to 30% of the total penicillin-binding proteins (Dougherty et al, 1996).…”