T HE BACTERIAL CELL WALL

The integrity of the bacterial cell wall is of indispensable importance to cell viability. Its rigidity provides mechanical support to the cell and allows the bacterium to maintain its characteristic shape. By stabilizing the semipermeable, remarkably fragile plasma membrane, the primary function of the cell wall is to protect the underlying protoplast against environmental stresses from the outside and to prevent rupture or lysis caused by osmotic force from the inside. Since procaryotes usually live in hypotonic habitats with a lower solute concentration outside the cell, bacteria evolved to form such a bag-shaped sacculus.

Therefore, bacteria are able to accumulate high concentrations of nutrients inside the cell accompanied by the development of an exceedingly high internal osmotic pressure of about 2 to 5 atmospheres in Gram-negative bacteria and up to 50 atmospheres in Gram-positive bacteria (Archibald et al., 1993; Seltmann and Holst, 2002). Accordingly, the cell wall is made of a rigid, sufficiently porous material that has considerable tensile strength, thus

permitting the diffusion of metabolites to the plasma membrane as well as reversible cell expansion or shrinkage to tolerate changes in osmolarity of the environment. Peptidoglycan, also known as murein, is such a material, the major structural constituent of bacterial cell walls. Peptidoglycan is a heteropolymer that consists of long glycan strands of repeating disaccharide residues, cross-linked by short peptide bridges to form one large molecule of strong but elastic nature (Höltje, 1998; Park, 1996; Weidel and Pelzer, 1964). It is present in all bacteria with the exception of Mycoplasma, Planctomyces and a few other bacterial species that lack a cell wall (Moulder, 1993; Seltmann and Holst, 2002).

While in both Gram-negative and Gram-positive bacteria the chemical composition of peptidoglycan is very similar, its arrangement in the murein sacculus is different (Fig. 1). In Gram-negative bacteria, a thin peptidoglycan layer of a few nanometers, which accounts for less than 5% of the cell mass, is located in the periplasm between the inner and outer membrane (Fig. 2A). The peptidoglycan layer is covalently attached to the outer membrane by the murein lipoprotein called Lpp or Braun´s lipoprotein (Braun, 1975). About two thirds of the peptidoglycan in the Gram-negative bacterium E. coli is composed of only one layer, whereas the remainder has three layers (Labischinski et al., 1991). Unlike Gram-negative bacteria, peptidoglycan is highly abundant in Gram-positives, in which it makes up more than 20% of the cell mass.

Fig. 1. Most bacteria are protected against environmental stress by an exoskeleton-like structure called the bacterial cell wall. While the cell wall of Gram-positive bacteria is mainly comprised of a thick layer of peptidoglycan with covalently bound anionic polymers, Gram-negative bacteria possess a predominant monolayer of peptidoglycan within the periplasm, which is covered by an outer membrane. CAP, covalently attached protein; IMP, integral membrane protein; LP, lipoprotein; LPS, lipopolysaccharide; LTA, lipoteichoic acid; OMP, outer membrane protein; WTA, wall teichoic acid. Figure was adapted from (Silhavy et al., 2010).

Since Gram-positive bacteria typically lack an outer membrane, they are surrounded by multiple layers of peptidoglycan which can be up to 20-fold thicker than that of Gram-negative bacteria (Shockman and Barrett, 1983). Most Gram-positive bacteria protect the peptidoglycan sacculus, which is exposed to the external environment, by covering the surface with structures such as layers of proteins (S-layer) or polysaccharides (capsules) (Navarre and Schneewind, 1999). They may function, among others, as protective barriers or are involved in cell adhesion. In addition to peptidoglycan, teichoic acids are embedded in an approximately equal proportion within the Gram-positive cell wall (Foster and Popham, 2002). These anionic polymers consist of repeating residues of glycerol phosphate or ribitol phosphate and occur in two distinct forms. While wall teichoic acids are covalently attached to the peptidoglycan, the second major type that is anchored to the cytoplasmic membrane includes the lipoteichoic acids. Under phosphate-limiting growth conditions teichoic acids are almost entirely replaced by teichuronic acids, which are devoid of phosphate residues.

Glycopolymers like teichoic acids are not found within the cell wall of Gram-negative bacteria.

Fig. 2. Cryo-transmission electron microscopies of bacterial cell walls from negative and Gram-positive bacteria revealed distinct architectural differences. (A) The Gram-negative cell wall of E. coli includes an outer membrane (OM) that surrounds a thin peptidoglycan layer (PG) embedded in a periplasmic space between the plasma membrane (PM) and the outer membrane. Scale bar, 200 nm. (B) In contrast, Gram-positive bacteria like B. subtilis lack an outer membrane but possess a thick cell wall with varying mass distribution. The plasma membrane (PM) is enclosed by a low-density inner wall zone (IWZ), representing the Gram-positive periplasmic space, which is surrounded by a high-density outer wall zone (OWZ). Scale bare, 50 nm. Figure (A) was taken from (Matias et al., 2003) and (B) from (Matias and Beveridge, 2005).

Remarkably, a two-layered organization of the cell wall has been detected in Gram-positive bacteria using cryo-transmission electron microscopy (Fig. 2B). These studies revealed that Bacillus subtilis as well as Staphylococcus aureus possess a low-density inner cell wall zone overlying the outer surface of the plasma membrane, followed by an outer wall zone of higher density peptidoglycan with covalently linked teichoic acid (Matias and Beveridge, 2005, 2006). This inner cell wall zone contains soluble components but, in contrast to the outer wall zone, it is rid of the previously mentioned polymeric network. It is, therefore, suggested that the inner cell wall zone represents a periplasmic space analogous to that found in Gram-negative bacteria, as part of the Gram-positive cell wall (Matias and Beveridge, 2006).

The current knowledge of the cell wall peptidoglycan regarding structure, pathways and models presented in this thesis will focus predominantly on the rod-shaped Gram-negative bacterium E. coli and its Gram-positive counterpart B. subtilis, respectively.