Bacterial cell wall synthesis: new insights from localization studies

Abstract

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.

FIG. 1.

Incorporation of new cell wall in differently shaped bacteria. Rod-shaped bacteria such as B. subtilis or E. coli have two modes of cell wall synthesis: new peptidoglycan is inserted along a helical path (A), leading to elongation of the lateral wall, and is inserted in a closing ring around the future division site, leading to the formation of the division septum (B). S. pneumoniae cells have the shape of a rugby ball and elongate by inserting new cell wall material at the so called equatorial rings (A), which correspond to an outgrowth of the cell wall that encircles the cell. An initial ring is duplicated, and the two resultant rings are progressively separated, marking the future division sites of the daughter cells. The division septum is then synthesized in the middle of the cell (B). Round cells such as S. aureus do not seem to have an elongation mode of cell wall synthesis. Instead, new peptidoglycan is inserted only at the division septum (B). Elongation-associated growth is indicated in red, and division-associated growth is indicated in green.

FIG. 2.

Building blocks and synthesis reactions of the peptidoglycan. (A) The basic unit of the peptidoglycan is a disaccharide-pentapeptide composed of the amino sugars N-acetylglucosamine and N-acetylmuramic acid, which are linked together by β-1,4 glycosidic bonds. The pentapeptide is covalently linked to the lactyl group of the muramic acid, and its composition can vary between different bacteria. In both E. coli and B. subtilis, the dibasic amino acid of the stem peptide, which allows the formation of the peptide cross bridge, is meso-diamoinopimelic acid (m-A₂pm), while in S. aureus it is l-lysine (l-Lys), to which a pentaglycine cross bridge is bound. (B) Peptidoglycan chains are synthesized by transglycosylation and transpeptidation reactions which lead to the formation of long glycan chains cross-linked by peptide bridges. In the transglycosylation reaction, the reducing end of the N-acetylmuramic acid (M) of the nascent lipid-linked peptidoglycan strand is likely transferred onto the C-4 carbon of the N-acetylglucosamine (G) residue of the lipid-linked PG precursor, with concomitant release of undecaprenylpyrophosphate (ppB). In the transpeptidation reaction, the d-Ala-d-Ala bond of one stem peptide (donor) is first cleaved by a PBP enzyme, and an enzyme-substrate intermediate is formed, with the concomitant release of the terminal d-Ala. The peptidyl moiety is then transferred to an acceptor, which is the last amino acid of the pentaglycine cross bridge in the depicted case of S. aureus, and the PBP enzyme is released.

FIG. 3.

Proposed mode of insertion of new peptidoglycan in gram-negative bacteria. (A) The three-for-one growth mechanism suggests that three newly synthesized, cross-linked glycan chains in a relaxed state (white circles) are covalently attached to the free amino groups present in the donor peptides of the cross-links on both sides of a strand, called the docking strand (hatched circles), which is substituted by acceptor peptides. Specific cleavage of the preexisting cross-links results in the replacement of the docking strand by the three new, cross-linked glycan chains. (Adapted from reference with permission from Elsevier.) (B) It is proposed that the three new cross-linked glycan strands (shown in gray) are synthesized by a mutienzyme complex, which also attaches these strands to the cross bridges on both sides of a docking strand (single gray strand) and at the same time degrades this strand by the action of a lytic enzymes. The peptidoglycan synthases should be in front of the hydrolases, thereby acting according to the make-before-break strategy. LT, lytic transglycosylase; EP, endopeptidase; TP, transpeptidase; TP/TG, bifunctional transpeptidase-transglycosylase; TG, transglycosylase. (Adapted from reference with permission.)

FIG. 4.

Different methods to visualize the incorporation of PG precursors. (A) Autoradiogram of sacculi prepared from steady-state E. coli cells grown at 28°C in the presence of [³H]diaminopimelic acid. (Adapted from reference with permission of the publisher.) (B) Immunoelectron microscopy of sacculi prepared from d-Cys-labeled E. coli cells chased for one mass doubling and a schematic representation of the result (inset). (Adapted from reference with permission.) (C) Immunoelectron microscopy of sacculi prepared from d-Cys-labeled E. coli cells chased in the presence of aztreonam to a fivefold increase in optical density. Note the accumulation of label at poles and the generation of split poles in between accumulation of label. (Adapted from reference with permission.) (D) Van-FL staining of nascent PG in wild-type B. subtilis during various stages in the cell cycle (i) and in an mbl null strain (ii). Note the absence of the helical staining of the lateral wall in the mbl null strain. (Adapted from reference with permission from Elsevier.) (E) Van-FL labeling of new PG after transient incubation with excess d-serine in S. aureus, showing different stages of septum formation (i), and wheat germ agglutinin-Oregon green labeling of S. aureus cells followed by incubation in absence of the dye (ii). New wall material appears as nonfluorescent regions. Bars, 1 μm. (Adapted from reference with permission of Blackwell Publishing.)

FIG. 5.

Localization of PBPs in different organisms. (A) Three different patterns, i.e., disperse, septal, and spotty, are observed in B. subtilis. Shown are GFP-PbpH (i), GFP-PBP1 (ii), and GFP-PBP3 (iii). Bar, 5 μm. (Adapted from reference with permission of Blackwell Publishing.) (B) Localization of GFP-PBP2 in E. coli. Left, phase-contrast image; right, GFP fluorescence image. Bar, 1 μm. (Adapted from reference with permission of Blackwell Publishing.) (C) Localization of GFP-PBP2 during the S. aureus cell division cycle. GFP-PBP2 localizes as two spots at the start of septum formation (i) and then forms a line indicating localization along the entire closed septum (ii) and remains at the septum site when separation has started (iii). Bar, 2 μm. (Adapted from reference with permission of Blackwell Publishing.) (D) Septal localization of PBP2x (left) and equatorial localization of PBP2b (right) in S. pneumoniae. DNA staining (DAPI [4′,6′-diamidino-2-phenylindol]) (blue), immunofluorescence of FtsZ (red) and PBP2x and PBP2b (green), and an overlay of the FtsZ and PBP patterns (merge), during various stages in the cell cycle are shown. The top and bottom series represent the same localization patterns in a single cell and a diplococcus, respectively. (Adapted from reference with permission of Blackwell Publishing.) (E) Immunostaining of PBP2 in C. crescentus after two-dimensional deconvolution of the fluorescence images. (Adapted from reference with permission of Blackwell Publishing.)

FIG. 6.

Model for PBP2 localization by substrate recognition in S. aureus. In a wild-type cell growing in the absence of inhibitors (upper panels), the substrate of PBP2 is exported from the inside of the cell at the division site, possibly because the translocator of the PBP2 substrate (presumably an FtsW homologue) localizes at the division site by interaction with the divisome. PBP2 recognizes and binds the substrate, therefore localizing at the division site, initially forming a ring around the cell (cell on the upper right). Several events can lead to delocalization of PBP2 (lower panels): depletion of FtsZ leads to delocalization of the substrate translocator and therefore to a nonlocalized export of the PBP substrate; inactivation of the PBP2 binding site by β-lactam antibiotics or alteration or blockage of the substrate (for example, by addition of d-cycloserine or vancomycin, respectively [see text]) prevents enzyme-substrate binding. Both delocalization of the substrate and inability of PBP2 to bind its substrate can cause the loss of the precise localization of PBP2 at the division septum, and the protein becomes dispersed over the entire surface of the cell (cell on the lower right).