BIOL 157: BIOLOGICAL

CHEMISTRY
Lecture 16:
BACTERIAL CELL WALL AND CELL WALLS OF
HIGHER PLANTS

Lecturer:
Amma Larbi, PhD
Introduction

• In contrast to animal cells, most prokaryotic cells are

surrounded by a rather complex and rigid cell wall which
allows bacteria to survive in a hypotonic environment
without bursting.
• Inside bacterial cells are considerable amounts of
dissolved substances (ions, metabolites, etc) which
together exert a high osmotic pressure of up to 20 bars.
• These cell contents are separated from the external
medium by the plasma membrane, and since the external
medium is usually hypotonic relative to the cell interior,
there is a high tendency for the cell to take up water
osmotically.
• Without restriction on this process, the cell is bound to
swell and burst.
• To avoid this, bacteria have a cell wall external to the cell
membrane which is capable of withstanding these osmotic
forces.
• This protection offered by the bacterial cell wall against
osmotic shock is the main function of the wall.
• Apart from this, the bacterial cell wall gives shape to the
cell. Each bacterial cell has a characteristic shape; this
could be cylindrical or rod-like (bacilli), spherical (cocci)
and helical (spirilla) shapes.
• On the surface of the wall are antigenic determinants that
enable the bacteria to evade the immunological
surveillance mounted in vertebrates.
Relevance
• The study of the bacterial cell wall is of relevance due to
the following reasons:
• The cell was bears the antigenic determinants which
enable mammals to recognize them as ‘foreign’, and so
are able to develop antibodies to counteract them;

• The fact that part of the cell wall is subject to breakdown

by specific enzymes throws light on both the chemical
components of the cell wall, and the mechanism of
action of the lytic enzymes (important for drug
development).

• The synthesis of the cell wall is the target for the action
of some antibiotics.
Contributing Studies

• The use of the Gram stain

• Use of light and electron microscopes

• The use of lysozyme and penicillin

Gram Staining
• The primary staining is with crystal violet which stains all
bacteria cells blue purple.
• Then iodine is added as a mordant to fix the primary dye.
It increases the affinity of the primary stain for the
bacterial cells.
• Then follows the rinsing of the cells with an acetone-
alcohol mixture as decolourising agent. This is to leach or
wash out the primary stain. The stain in the gram negative
bacteria would be washed away, while the blue purple
stain on the gram positive bacteria remains.
• To be able to observe the two classes of bacteria, a
counter stain, safranin could be applied in what is called
differential staining. This is able to stain the bacteria that
were decolourised by the acetone-alcohol mixture.
• The micrographs from electron microscopy show an electron-
dense layer of the cell wall outside the plasma membrane.
• There is a difference in the wall thickness and complexity
between the gram positive and gram negative bacteria.
• The gram positive is surrounded by a single electron dense
layer, whereas the material covering the gram negative
bacteria is thinner and more complex.
• The bacterial cell wall can be treated with enzymes such as
lysozymes which are able to break down the cell wall
material.
• Another approach is to inhibit the synthesis of the cell wall,
leading to the accumulation of the precursors of the cell wall
components which can be isolated and identified.
Lysozymes

• The gram positive bacteria cell wall, in particular can be

fragmented using lysozyme, an enzyme found in tears,
saliva and egg white.
• The enzyme hydrolyses the glycan part of peptidoglycan
which is the most abundant material in the cell wall of
gram positive bacteria.
• When the lysosomal action on the bacteria cell wall is
carried out in an isotonic sucrose solution, the ‘naked
cells’ usually assume a spherical shape.
Use of Penicillin
• Where penicillin is used, it inhibits the cell wall synthesis.
• Therefore, in the presence of penicillin, under isotonic
conditions, growing bacterial cells accumulate high
concentrations of cell wall precursors which cannot be
incorporated to form a functional cell wall.
• Such cells may also assume spherical shapes but they
usually retain fragments of the old cell wall materials.
• Such cell wall-fragment-containing spheres are called
spheroplasts. There can be differential centrifugation to
obtain a supernatant from which the cell wall precursors
can be extracted for identification.
• General structure of Penicillins
Chemical composition of bacterial cell wall
• Both Gram positive (40-90%) and negative (1%) bacteria have
peptidoglycan or murein or muropeptide in their cell walls.
• There are two parts of peptidoglycan,
• the peptide portion made up of a short chain of amino acid residues (L-
Ala, D-isoglutamine, L-lysine and D-alanine.)
• the glycan part.

• The glycan component is a heteroglycan which is made up of a

repeating heterodisaccharide, also composed of
• N-acetyl-glucosamine (NAG) and
• N-acetylmuramic acid (NAMA) linked by β(1→4) glycosidic bonds.
• NAMA is N-acetylglucosamine in which the C-3 OH forms an
ether linkage with the OH of lactic acid. The glycan component
forms the backbone of the peptidoglycan.
• The following unique features should be noted
about the tetrapeptide (L-Ala, D-isoglutamine, L-
lysine and D-alanine.).
• The presence of both D- and L-forms of amino acids
(Be reminded that in proteins the amino acids are in the
L-form).
• There is the alternation of the L-and D-amino acids
making them resistant to peptidases. Most of such
peptidases specifically hydrolyse peptides with L- amino
acids due to their stereospecificity.
• The presence of isoglutamine instead of glutamine.
• Lysine occurs in Gram positive bacteria, but in Gram
negative bacteria, its place taken by diaminopimelic
acid, a diamino and dicarboxylic acid.
 Some modifications
• In Staphylococcus
aureus, the peptide
bridge is a pentapeptide
chain from the terminal
COOH of D-ala residue
of a tetrapeptide to the
epsilon amino group of
L-lys in another
tetrapeptide.
• Not all the tetrapeptides
are cross-linked. The
linking determines the
rigidity of the cell wall.
The cross-linking of the
tetrapeptide is called
transpeptidation.
Penicillin Resistance
• The penicillins are a group of compounds, characterized
by the presence of a four-membered amide ring called β-
lactam ring.
• Bacteria develop resistance to penicillin by secreting the
enzyme, β-lactamase which hydrolyses penicillin before it
blocks transpeptidation. Some drugs have been
developed to inhibit the β-lactamase.
Other cell components
• Apart from peptidoglycan, gram positive bacteria cell walls
contain teichoic or teichuronic acids which are acidic
polysaccharides.
• Teichoic or teichuronic acids are also covalently linked to
NAMA in peptidoglycan.
• The teichoic acids are made up of either ribitol or glycerol
connected by internal phosphate diesters. There is a direct
linkage between teichoic acid and C-6-OH of NAMA.
• The transpeptidation of peptidoglycan layer provides
structural rigidity to the cell wall.
• Teichoic or teichuronic acids contribute to the negative charge
associated with the surface of gram positive cell walls. Both
polymers can bind cations such as Mg 2+, which allow the wall
to become more compact, but does not affect the porosity of
the wall material.
• Teichoic and teichuronic acids are also involved in the
control of autolysins (housekeeping enzymes in bacteria).
• These are enzymes that carry out controlled hydrolysis of
some specific bonds in the peptidoglycan in the
restructuring or reshaping of the cell wall.
• Such a controlled hydrolysis of the cell wall components is
necessary in the normal growth cycle as old cell wall must
be cleaved to allow the insertion of new cell wall
components.
• Autolysins may be completely destructive of the cell,
particularly when a cell ages.
• The teichoic acids can bind protons, thereby maintaining
low pH which controls the action of the autolysins.
Lipopolysaccharides
• Two distinct structures make up the Gram negative wall;
• a peptidoglycan layer and
• an outer membrane called the lipopolysaccharide.
• The peptidoglycan layer of the gram negative bacteria is
thinner (about 1%) and it lacks teichoic acid.
• Between the lipopolysaccharide (LPS) and peptidoglycan
layer is an area called the periplasmic space.
• The LPS restricts the movement of certain enzymes which
are thought to be involved in the active transport of
molecules into the cell by binding nutrients before they are
moved into the cell.
• The LPS is a complex molecule composed of three
distinct regions:
• lipid A,
• core polysaccharide and
• O-polysaccharide.
• The innermost portion of the LPS is the lipid A. This lipid
portion anchors the LPS to the hydrophobic portion of the
outer membrane.
• Lipid A is a made up of N-acetylglucosamine
disaccharides (diglucosylamine phosphate) linked through
ester or amide bonds to unusual fatty acids such as β-
hydroxymyristic, caproic and lauric acids.
• The aliphatic side chains of the fatty acids are saturated;
this confers to the lipid A portion the property of low
fluidity.
• The second region, next to the lipid A is the core
polysaccharide.
• This is composed of sugars such as glucose, galactose,
N-acetylglucosamine, and unusual sugars like tin eight-
carbon sugar, ketodeoxyoctulosonic acid (KDO) and
heptoses like glyceromannoheptose.
• The O-polysaccharide or the O-specific chain typically
contains glucose, galatose, rhamnose, mannose and
several dideoxy sugars such as abequose, colitose,
pavanose and tyvelose.
• The O-antigens vary widely in composition in different
bacterial strain, so provides many specific O-antigen
determinants.
• Infection of host by a particular strains of bacteria leads to
an immune response to that microbe. The large variety of
O-antigens, even within single species of bacteria is to
enable the bacteria evade detection by the immune
system.
Capsules
• In addition to the cell wall, some prokaryotic cells have an
additional layer of material called capsule.
• A capsule may be composed of complex carbohydrates
(e.g. dextrins and hyaluronic acid) or proteins.
• Capsules are slimy or sticky substances which can be
observed by the light microscopes, using special staining
techniques.
• Encapsulated Staphylococcus pneumoniae may be able
to resist phagocytosis by certain leucocytes.
• When a few of such encapsulated cells are injected into
an experimental animal like mouse, they will cause the
death of the animal.
• However, the injection of many thousands of non-
encapsulated streptococci will not cause any harm. The
degree to which these bacteria cause harm is determined
by the presence or absence of the capsule.
• The encapsulated form is more resistant to phagocytosis,
more virulent, and able to multiply in the host to cause
havoc. On the contrary, the absence of a capsule renders
the bacteria less virulent and rather, more susceptible to
phagocytosis.
Plant cell walls
• In mature higher plants, a typical cell wall is made up of
three distinct parts: the primary cell wall, an intercellular
substance or middle lamella, and the secondary cell wall.
• Young growing cells, cells of storage tissues,
parenchymatous cells as well as photosynthesizing cells
of leaves contain only a primary cells wall.
• Characteristically, the primary cell wall is thin and it is
formed while the plant cell is undergoing growth.
• This cell wall covers the protoplast, composed of the
plasma membrane and its content.
• In between the primary walls of adjoining cells is the
middle lamella or intercellular substance.
• At maturity, many plant cells, particularly, those of xylem
tissues that have completed their growth, have laid down
a secondary cell wall between the primary wall and the
cell membrane.
• The formation of the primary cell wall is begun in cells
which are completing cell division.
• A cell plate is formed where a new cell wall divides two
daughter cells. It is essentially, the components of the cell
plate which becomes the middle lamella which has a high
content of pectin.
• Compared to the secondary wall, the primary wall is
thinner. It contains about 9-25% cellulose which form long
cylindrical microfibrils. These microfibrils are embedded in
a matrix of hemicellulose and pectins.
• The hemicelluloses form 25 - 50%, and could be xylans,
mannans, glycans, galactomannans, etc.
• Also present in the primary walls are proteins, which could
be involved in cell growth and recognition of foreign
molecules.
• The primary wall is adapted to growth. For those plant
tissues which provide support at maturity, or for tissues
like the xylem which conduct fluid under tension, their
protoplasts secrete a secondary wall alter the cells have
ceased growth. After the secretion of the secondary wall,
the protoplasts die, resulting in the loss of the contents of
the protoplasts, so that only the cell wall remains.
• Secondary walls are thicker than primary walls. They
contain about 41-45% cellulose, 30% hemicellulose and
22-28% lignin.
• Lignin occurs in largest amount in wood, in which it
accumulates in the middle lamella, primary and secondary
walls of the xylem elements.
• It usually occurs between the cellulose microfibrils, and it
is the combination of lignin and cellulose which is
responsible for the strength of wood, enabling wood to
resist compression.
• Besides the strengthening function, lignin offers protection
against attack by pathogens and consumption by
herbivores.
• Generally, lignins contain three types of alcoholic
compounds: coniferyl, sinapyl and p-coumaryl alcohols.
Cuticle and suberin

• Herbaceous plants have their entire shoot system covered

by cuticle.
• This cover slows down water loss from the leaves, stems,
flowers and fruits.
• Cuticle also serves to protect the shoot system from
microbial attack and mechanical damage.
• The cuticle is made up predominantly of a heterogenous
mixture of compounds collectively called cutin, with a
relatively small portion made up of waxes and pectin.
• Cutin is composed of varying combinations of two fatty
acids, a 16-carbon and an 18-carbon fatty acids containing
two or more hydroxyl groups.
• The polyhydroxy nature of these fatty acids enable them
form polymeric esters as a result of the reactions between
the hydroxy groups and the COOH groups of the fatty
acids.
• Also found in cutin are small amounts of phenolic
compounds.
• The waxes in cutin include a long chain monohydric
alcohols and long chain fatty acids.
• The syntheses of the cutins and waxes take place in the
epidermal cells, before being secreted onto the surface of
the shoot system.
• Suberin, on the other hand, is a protective coating over
underground plant parts.
• Its role is to cover cells formed in the tree bark by the
crushing effect of secondary growth.
• It can be formed by cells of scar tissue, following the
wounding of a plant.
• Suberin is also a waxy lipid layer, having a complex mixture
of long chain fatty acids, hydroxylated fatty acids,
dicarboxylic-acids, together with long chain alcohols (chains
often contain more than 16 carbon atoms).
• Suberin also contain phenolic compound swhich are
supposed to secure the binding of lipids to the cell wall.
• The phenolic content of suberin is higher than that of cutin.