Skills to Develop
- State the three parts of a peptidoglycan monomer and state the function of peptidoglycan in bacteria.
- Briefly describe how bacteria synthesize peptidoglycan, indicating the roles of autolysins, bactoprenols, transglycosylases, and transpeptidases.
- Briefly describe how antibiotics such as penicillins, cephalosporins, and vancomycin affect bacteria and relate this to their cell wall synthesis.
- State what color Gram-positive bacteria stain after Gram staining.
- State what color Gram-negative bacteria stain after Gram staining.
- State what color acid-fast bacteria stain after acid-fast staining.
The mycoplasmas are the only bacteria that naturally lack a cell wall. Mycoplasmas maintain a nearly even pressure between the outside environment and the cytoplasm by actively pumping out sodium ions. Their cytoplasmic membranes also contain sterols that most likely provide added strength. The remaining bacteria in the domain Bacteria, with the exception of a few bacteria such as the Chlamydias, have a semirigid cell wall containing peptidoglycan. (While bacteria belonging to the domain Archaea also have a semirigid cell wall, it is composed of chemicals distinct from peptidoglycan such as protein or pseudomurein. We will not take up the Archaea here.)
Function of Peptidoglycan
Peptidoglycan prevents osmotic lysis. As seen earlier under the cytoplasmic membrane, bacteria concentrate dissolved nutrients (solute) through active transport. As a result, the bacterium's cytoplasm is usually hypertonic to its surrounding environment and the net flow of free water is into the bacterium. Without a strong cell wall, the bacterium would burst from the osmotic pressure of the water flowing into the cell.
Structure and Composition of Peptidoglycan
With the exceptions above, members of the domain Bacteria have a cell wall containing a semirigid, tight knit molecular complex called peptidoglycan. Peptidoglycan, also called murein, is a vast polymer consisting of interlocking chains of identical peptidoglycan monomers (Figure 1). A peptidoglycan monomer consists of two joined amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with a pentapeptide coming off of the NAM (Figure 2). The types and the order of amino acids in the pentapeptide, while almost identical in gram-positive and gram-negative bacteria, show some slight variation among the domain Bacteria.
Figure 1: Peptidoglycan is composed of cross-linked chains of peptidoglycan monomers (NAG-NAM-pentapeptide). Transglycosylase enzymes join these monomers join together to form chains. Transpeptidase enzymes then cross-link the chains to provide strength to the cell wall and enable the bacterium to resist osmotic lysis. (left) In a peptidoglycan monomer of S. aureus, the pentapeptide coming off the NAM is composed of the amino acids L-alanine, D-glutamine, L-lysine, and two D-alanines. The peptide cross-link forms by formation of a short peptide interbridge consisting of 5 glycines. In the process the terminal D-alanine is cleaved from the pentapeptide to form a tetrapeptide in the peptidogycan. (right) In a peptidoglycan monomer of E. coli, the pentapeptide coming off the NAM is composed of the amino acids L-alanine, D-glutamic acid, meso-diaminopimelic acid, and two D-alanines. The peptide cross-link forms between the diaminopimelic acid of one peptide chain with the D-alanine of another and in the process the terminal D-alanine is cleaved from the pentapeptide to form a tetrapeptide in the peptidogycan.
The peptidoglycan monomers are synthesized in the cytosol of the bacterium where they attach to a membrane carrier molecule called bactoprenol. 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.
Figure 2: (left) A peptidoglycan monomer consists of two joined amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with a pentapeptide coming off of the NAM. In E. coli, the pentapeptide consists of the amino acids L-alanine, D-glutamic acid, meso diaminopimelic acid, and two D-alanines. (right) A peptidoglycan monomer consists of two joined amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with a pentapeptide coming off of the NAM. In S. aureus, the pentapeptide consists of the amino acids L-alanine, D-glutamine, L-lysine, and two D-alanines.
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. By linking the rows and layers of sugars together in this manner, the peptide cross-links provide tremendous strength to the cell wall, enabling it to function similar to a molecular chain link fence around the bacterium (see Figure 1).
Synthesis of Peptidoglycan
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:
a) Break the glycosidic bonds between the peptidoglycan monomers at the point of growth along the existing peptidoglycan (see Fig. 3, steps 1-3); and
b) Break the peptide cross-bridges that link the rows of sugars together (see Fig. 3, steps 1-3).
Step 2. The peptidoglycan monomers are synthesized in the cytosol (see Fig. 4, step-1 and Fig. 4, step-2) and bind to bactoprenol. The bactoprenols transport the peptidoglycan monomers across the cytoplasmic membrane and interacts with transglycosidases to insert the monomers into existing peptidoglycan (see Fig. 4, step-3, Fig. 4, step-4, Fig. 4, step-5, and Fig. 4, step-6)
In Escherichia coli, the terminal D-alanine is cleaved from the pentapeptides to form a tetrapeptides. This provides the energy to bond the D-alanine of one tetrapeptide to the diaminopimelic acid of another tetrapeptide (see Fig. 1B). In the case of Staphylococcus aureus, the terminal D-alanine is cleaved from the pentapeptides to form a tetrapeptides. This provides the energy to bond a pentaglycine bridge (5 molecules of the amino acid glycine) from the D-alanine of one tetrapeptide to the L-lysine of another (see Fig. 1A).
Exercise: Think-Pair-Share Questions
- As we will see in Unit 2, the antibiotic bacitracin binds to bactoprenol after it inserts a peptidoglycan monomer into the growing bacterial cell wall.
Explain how this can lead to the death of that bacterium.
- As we will see in Unit 2, the penicillin antibiotics binds to the bacterial enzyme transpeptidase.
- Explain how this can lead to the death of that bacterium.
- Could this antibiotic be used to treat protozoan infections such as giardiasis and toxoplasmosis?
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. These proteins form the cell division apparatus known as the divisome and are directly involved in bacterial cell division by binary fission (see Fig. 1 and Fig. 2).
The divisome is responsible for directing the synthesis of new cytoplasmic membrane and new peptidoglycan to form the division septum.
Antimicrobial Agents that Inhibit Peptidoglycan Synthesis Causing Bacterial Lysis
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.), the cephalosporins (cephalothin, cefazolin, cefoxitin, cefotaxime, cefaclor, cefoperazone, cefixime, ceftriaxone, cefuroxime, etc.), the carbapenems (imipenem, metropenem), the monobactems (aztreonem), the carbacephems (loracarbef), and the glycopeptides (vancomycin, teichoplanin).
- For example, penicillins and cephalosporins bind to the transpeptidase enzymes (also called penicillin-binding proteins) responsible for resealing the cell wall as new peptidoglycan monomers are added during bacterial cell growth. This blocks the transpeptidase enzymes from cross-linking the sugar chains and results in a weak cell wall and subsequent osmotic lysis of the bacterium (see Fig. 8).
Gram-Positive, Gram-Negative, and Acid-Fast Bacteria
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.
- Gram-positive Bacteria: These retain the initial dye crystal violet during the Gram stain procedure and appear purple when observed through the microscope. Common Gram-positive bacteria of medical importance include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, and Clostridium species.
(left) Gram Stain of Staphylococcus aureus which are gram-positive (purple) cocci in clusters. (right) Gram Stain of Escherichia coli which are Gram-negative (pink) bacilli.
- Gram-negative Bacteria: These decolorize during the Gram stain procedure, pick up the counterstain safranin, and appear pink when observed through the microscope. Common Gram-negative bacteria of medical importance include Salmonella species, Shigella species, Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Proteus species, and Pseudomonas aeruginosa. Also see gram stain of a mixture of gram-positive and gram-negative bacteria.
A Gram Stain of a Mixture of Gram-Positive and Gram-Negative Bacteria. Note Gram-negative (pink) bacilli and Gram-positive (purple) cocci.
- acid-fast Bacteria: These resist decolorization with an acid-alcohol mixture during the acid-fast stain procedure, retain the initial dye carbolfuchsin and appear red when observed through the microscope. Common acid-fast bacteria of medical importance include Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium-intracellulare complex.
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 16. We will now look at each of these three bacterial cell wall types.
1. 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 2-8 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:
- S-Layer Proteins, the Structural Biology Homepage at Karl-Franzens University in Austria.
- Characteristic Properties of S-layer Proteins, at Foresight Nanotech Institute in Austria.
2. Functions and Significance to Bacteria Causing Infections
The S-layer has been associated with a number of possible functions. These include the following:
a. The S-layer may protect bacteria from harmful enzymes, from changes in pH, from the predatory bacterium Bdellovibrio, a parasitic bacterium that actually uses its motility to penetrate other bacteria and replicate within their cytoplasm, and from bacteriophages.
b. The S-layer can function as an adhesin, enabling the bacterium to adhere to host cells and environmental surfaces, colonize, and resist flushing.
c. The S-layer may contribute to virulence by protecting the bacterium against complement attack and phagocytosis.
d. The S-layer may act as a as a coarse molecular sieve.
- The vast majority of the domain Bacteria have a rigid cell wall composed of peptidoglycan.
- The peptidoglycan cell wall surrounds the cytoplasmic membrane and prevents osmotic lysis.
- Peptidoglycan is composed of interlocking chains of building blocks called peptidoglycan monomers.
- In order to grow following binary fission, bacteria have to synthesize new peptidoglycan monomers in the cytoplasm, transport those monomers across the cytoplasmic membrane, put breaks in the existing cell wall so the monomers can be inserted, connect the monomers to the existing peptidoglycan, and cross-link the rows and layers of peptidoglycan.
- Many antibiotics inhibit peptidoglycan synthesis in bacteria and lead to osmotic lysis of the bacteria.
- 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. These staining reactions are due to fundamental differences in the bacterial cell wall.
- Gram-positive bacteria stain purple after Gram staining while Gram-negative bacteria stain pink.
- Acid-fast bacteria stain red after acid-fast staining.
Study the material in this section and then write out the answers to these questions. Do not just click on the answers and write them out. This will not test your understanding of this tutorial.
- A monomer of peptidoglycan consists of _____________, _____________, and _______________. (ans)
- State the function of peptidoglycan in bacteria. (ans)
- State the role of the following enzymes in peptidoglycan synthesis:
- autolysins (ans)
- bactoprenols (ans)
- transpeptidases (ans)
- transglycosylase (ans)
- A penicillin is used to treat a bacterial infection. Describe the mechanism by which this antibiotic eventually kills the bacteria. (ans)
- Gram-positive bacteria stain ____________ (ans) after Gram staining while Gram-negative bacteria stain _____________ (ans).
- Bacteria normally live in a hypotonic environment. Since water flows into a cell in an environment that is hypotonic, why don't the bacteria burst from osmotic pressure? (ans)
- Multiple Choice (ans)
Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)