As discussed below, The bactoprenols transport the peptidoglycan monomers across the cytoplasmic membrane and work with the enzymes discussed below to insert the monomers into existing peptidoglycan enabling bacterial growth following binary fission. Once the new peptidoglycan monomers are inserted, glycosidic bonds then link these monomers into the growing chains of peptidoglycan. These long sugar chains are then joined to one another by means of peptide cross-links between the peptides coming off of the NAMs.
In order for bacteria to increase their size following binary fission, links in the peptidoglycan must be broken, new peptidoglycan monomers must be inserted, and the peptide cross links must be resealed. The following sequence of events occur:. Step 1. Bacterial enzymes called autolysins:. Step 2. Step 3. Step 4. In Escherichia coli , the terminal D-alanine is cleaved from the pentapeptides to form a tetrapeptides.
In the case of Staphylococcus aureus , the terminal D-alanine is cleaved from the pentapeptides to form a tetrapeptides.
In the center of the bacterium, a group of proteins called Fts filamentous temperature sensitive proteins interact to form a ring at the cell division plane. The divisome is responsible for directing the synthesis of new cytoplasmic membrane and new peptidoglycan to form the division septum. Many antibiotics work by inhibiting normal synthesis of peptidoglycan in bacteria causing them to burst as a result of osmotic lysis.
As just mentioned, in order for bacteria to increase their size following binary fission, enzymes called autolysins break the peptide cross links in the peptidoglycan, transglycosylase enzymes then insert and link new peptidoglycan monomers into the breaks in the peptidoglycan, and transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong.
Interference with this process results in a weak cell wall and lysis of the bacterium from osmotic pressure. Examples include the penicillins penicillin G, methicillin, oxacillin, ampicillin, amoxicillin, ticarcillin, etc. Antimicrobial chemotherapy will be discussed in greater detail later in Unit 2 under Control of Bacteria by Using Antibiotics and Disinfectants. Most bacteria can be placed into one of three groups based on their color after specific staining procedures are performed: Gram-positive, Gram-negative, or acid-fast.
Note Gram-negative pink bacilli and Gram-positive purple cocci. Acid-Fast Stain of Mycobacterium tuberculosis in Sputum. Note the reddish acid-fast bacilli among the blue normal flora and white blood cells in the sputum that are not acid-fast. These staining reactions are due to fundamental differences in their cell wall as will be discussed in Lab 6 and Lab We will now look at each of these three bacterial cell wall types.
Structure and Composition. The most common cell wall in species of Archaea is a paracrystalline surface layer S-layer. It consists of a regularly structured layer composed of interlocking glycoprotein or protein molecules. In electron micrographs, has a pattern resembling floor tiles.
Although they vary with the species, S-layers generally have a thickness between 5 and 25 nm and possess identical pores with nm in diameter. Several species of Bacteria have also been found to have S-layers. To view electron micrographs of S-layers see the following:. Functions and Significance to Bacteria Causing Infections. The S-layer has been associated with a number of possible functions. These include the following:. Furthermore, peptidoglycan is a polymer while glycoprotein is not a polymer.
Peptidoglycan is composed of NAG, NAM attached to N-acetylmuramic acid while a glycoprotein is composed of oligosaccharide chains attached to a protein. Hence, this is an important difference between peptidoglycan and glycoprotein. Moreover, the sugars in peptidoglycans are not available elsewhere while the sugars in glycoproteins naturally occur in the other biological systems as well.
Also, proteins do not occur in peptidoglycans while proteins occur in glycoproteins. Another difference between peptidoglycan and glycoprotein is that the peptidoglycans occur in the bacterial cell wall while glycoproteins occur on the eukaryotic cell membrane and in the blood.
Besides, their role is another difference between peptidoglycan and glycoprotein. Peptidoglycans give structural strength to the bacterial cell wall while counteracting osmotic pressure whereas glycoproteins help in cell recognition, cell attachment, signal recognition, etc. It exclusively occurs in the bacterial cell wall, providing structural strength and controlling osmotic pressure.
Moreover, its sugars do not occur elsewhere and it does not contain a defined protein. In contrast, glycoprotein is a protein attached to oligosaccharides. It occurs both on the cell membrane of eukaryotes and in the blood.
It is responsible for cellular recognition, attachment, and signal transduction of chemical signals. Therefore, the main difference between peptidoglycan and glycoprotein is their structure, occurrence, and function. Fluorescence-labelled dextrans of different sizes have been used to determine the diameters of holes in the peptidoglycan network in E. Interestingly, the pores were of similar average sizes in peptidoglycans from Gram-negative and Gram-positive species and they were relatively homogenous in size: the mean radius of the pores was 2.
From these values it was calculated that globular, uncharged proteins with molecular weights of up 22—24 kDa should be able to penetrate the isolated, relaxed peptidoglycan. Globular proteins of up to 50 kDa or more should be able to diffuse through stretched peptidoglycan layer in the cell. Indeed, disruption of the outer membrane of E.
Perhaps this value is determined by the molecular sieving properties of the stretched peptidoglycan layer Vazquez-Laslop et al.
To understand how a biological structure grows, a detailed knowledge of how the individual components are organized and arrayed with respect to each other is of prime relevance. Unravelling the molecular architecture of the bacterial sacculus has been a constant aspiration for many microbiologists, but it is proving to be a frustrating topic. In particular, the architecture of the cell wall of Gram-positive bacteria is far from being understood. Gram-positive species not only have a thick, multi-layered peptidoglycan but other major surface polymers linked to it Vollmer, Many species also have capsular polysaccharides which are often covalently linked to the peptidoglycan.
In addition, there are many surface proteins either linked covalently to peptidoglycan or bound noncovalently to cell wall polymers. To decipher the architecture of this three-dimensional assembly of different polymeric components and its enlargement during bacterial growth will be a major challenge for the future.
With respect to the architecture of the sacculus of Gram-negative bacteria, E. Therefore, the structural aspects of sacculi from this organism will be concentrated on.
The idea of the following comments is not so much to criticize existing models, but to point out aspects of cell wall biology and biochemistry which are overlooked, but must be accounted for, by present day models to help improve future developments. As commented above the sacculus is a covalently closed structure built up from glycan strands that are cross-linked to each other through peptide bridges.
These basic properties, defined long ago, naturally lead to the concept of the sacculus as static, regular and planar net-like polymeric macromolecule, a concept which can still be traced down to present-day textbooks. However, this somewhat simplistic vision seems to be far from reality and the bacterial sacculus is proving itself to be a particularly intractable subject for structural studies. Application of even the more powerful tools in structural analysis, as X-ray diffraction, EM, AFM, low angle neutron scattering, and others have provided only limited information Formanek et al.
From a structural point of view, the basic problem is to define how individual glycan strands are arranged relative to each other and to the cell axes. The interactions among neighbouring glycan strands are in turn conditioned by three parameters; thickness, cross-linkage and length distribution of the glycan strands. These three parameters determine the number of chemical bonds per unit of surface area opposing the turgor, and define the basic constrains in model making.
The extreme thinness 3—4 nm assigned to the E. However, later evidence from different fields suggests a more complex situation. Application of AFM Yao et al. Perhaps more convincing than absolute thickness measurements is the fact that sacculi from another typical Gram-negative organism, P.
As sacculi from both species are made of identical subunits Quintela et al. The sacculus is made up of glycan strands cross-linked to each other through peptide bridges. As the physico chemical properties of the glycan and peptide moieties are very different, in particular the ability of each to change conformation under stress Barnickel et al. The peptide stems are of a fixed length, and in principle distributed regularly along the glycan backbone.
Therefore the number of possible interstrand connections is also a direct function of glycan chain length GCL. For a long time the only way to determine GCL was based on quantification of glycan strand terminal muropeptides Schindler et al. Reported results Pisabarro et al. Altmutter, unpublished data. Application of a new method based on enzymatic clipping of peptide stems with human serum amidase followed by HPLC separation of the resulting linear polysaccharides permitted an accurate analysis of the size distribution of glycan strands Harz et al.
However the method still suffers from a key limitation in that only glycan strands between 1 and 30 disaccharide units can be individually resolved. Longer strands cannot be separated from each other but at least the proportion of muropeptides in strands longer than 30 disaccharides can be evaluated, and an average value can be calculated from the proportion of 1,6-anhydroMur N Ac-containing muropeptides.
Information gathered by this method Harz et al. Therefore, sacculi of E. Cross-linking in Gram-negative species, in particular in E. Sacculi from growing, wild type E. That means that on average every third to second disaccharide in a peptidoglycan strand would be cross-linked to another adjacent strand. Similar values seem to be common among Gram-negative species, although data are still limited Quintela, a; , Costa et al. However the function and distribution of A 2 pm-A 2 pm bridges in the sacculus remains unknown.
Tetramers are barely detectable with reported abundances about 0. Following the same argument as above scarce tetramers could still be structurally significant Glauner et al.
Because disaccharide subunits in peptidoglycan strands are rotated with respect to each other due to the influence of the lactyl group in Mur N Ac, consecutive peptide stems point out in different directions Labischinski et al.
The periodicity of peptidoglycan conditions the cross-linking between adjacent strands, as only those peptide stems with the correct relative orientation can be proficient substrates for transpeptidation.
An immediate consequence of these facts is that adjacent peptidoglycan strands are unlikely to be cross-linked to each other by consecutive muropeptides Koch, a , b. The more controversial aspect related to the structure of the sacculus is the orientation and distribution of the glycan strands Formanek et al. Most available evidence favoured models postulating glycan strands oriented parallel to the cell surface, and in most cases with the glycan backbones transversal to the cell longitudinal axis.
More recently an alternative model based on glycan strands oriented perpendicular to the surface of the sacculus has been proposed Dmitriev et al. As indicated above, it is not intended here to enter into a discussion of models so far proposed, but rather point out some aspects, in the authors' opinion, overlooked in those models.
A good starting point is to refer to a recent comment Young, on the same topic which emphasizes what a weak point for most models is: the requirement for rather restrictive structural and morphological parameters. As commented in the preceding sections the main structural parameters in the sacculus are subjected to drastic changes on the course of normal growth. This calls for dynamic rather than static models because the sacculus as such is in a continuous state of change. Furthermore, any credible model should have an intrinsic ability to accommodate the size and shape changes in particular in cell diameter, but also more dramatic ones as round and branched shapes that cells can exhibit under specific conditions.
Sacculi are made of glycan strands with a very wide distribution of sizes, with a substantial proportion of total peptidoglycan in strands too short to be connected to nearby strands by more than two cross-links. Such strands could structurally be assimilated to a long range cross-link, connecting longer and relatively distant strands Costa et al.
Even if it is generally assumed, there is no evidence at all that cross-linkage happens regularly alongside the glycan strands. Indeed, the tendency of glycan strand termini to be highly cross-linked means that cross-linkage in internal positions is lower than the mean value for total peptidoglycan. Therefore, it is likely that long glycan strands might have relatively long uncross-linked stretches. A final comment on the organization of glycan strands comes from the very existence of cross-linked trimers and tetramers.
The interesting point about these two families of muropeptides is that they represent connecting hubs for several crossing glycan strands.
An attractive idea is that trimers and tetramers represent linking points of short-to-long glycan strands. A normal cross-link bridging two nearby, long glycan strands could act as the attachment point for a short glycan strand acting as a long range connection to another relatively remote long glycan strand.
That is, glycan strand termini seem to have a high tendency to cross-link to a dimeric muropeptide to form a trimer. Whether this tendency is more marked in short or long glycan strands cannot be decided at present, and of course trimers could show no preference at all relative to glycan strand length. As for the remaining, major fraction of cross-linked trimers and tetramers geometrical arguments require that three, or four, peptidoglycan strands cross over Glauner et al.
There are no data available on the angles between multiple glycan strands linked together at these positions. In the extreme cases, they could run parallel, antiparallel, or perpendicular to each other. Regions of multiple glycan strand crossings could be related with the postulated regions of multilayered peptidoglycan. Whether or not simple crossing of strands could be enough to explain the neutron diffraction results Labischinski et al.
Finally, another aspect models should be able to account for is the ability of E. Under normal growth conditions results suggest that E. It is interesting to note that recent measurements of cell wall thickness in E. If indeed E. Many data on the chemical structure and the biophysics of peptidoglycan have been gathered over the last decades. However, knowledge of this fascinating molecule is still very limited. For example, the analysis of peptidoglycan composition with high-resolution techniques has been performed so far only for a few bacterial species.
A significantly enlarged data set on peptidoglycan fine structures will be of interest for different research fields including bacterial taxonomy, physiology, and pathogenesis. Moreover, such research can lead to the discovery of peptidoglycans whose structure diverge from the overall structure defined at the beginning of this review. A recent example is represented by T.
It is likely that such an unusual motif, which coexists with the conventional l -Lys-containing motif, is at the origin of a particular type of cross-link. Another example is that of Chlamydiae , for which no muramic acid-containing peptidoglycan was detected to date.
It is difficult to imagine that these enzymes do not participate in the synthesis of a specific macromolecule, the structure of which presumably greatly differs from that of usual peptidoglycan. In the last two decades, the improvement of analytical methods HPLC, MS has allowed to show that, within a particular species, variations in peptidoglycan fine structure occur as a function of aging, medium composition, pathogenesis, or presence of antibiotics.
This type of research has implications not only in the field of bacterial physiology, but also in those of innate immunity, pathogenicity, and antibacterial therapy. One major task for the future is to determine the molecular architecture of peptidoglycan in Gram-positive and Gram-negative species, which is not possible with today's techniques.
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