Lecture 3

BACTERIAL STRUCTURE AND CELL FUNCTION

 Contents
• Bacterial Structure
– Bacterial Ultastructures
– Bacterial Capsules
– Lipopolysaccharide
– Peptidoglycan
• Bacterial Cell Function
– Growth, Culturability and Viability
– Bacterial Energy Metabolism
 Bacterial Structure
• Bacteria are small, prokaryotic cells, generally of
the size of mitochondria.
• Their length varies from about 0.2 to more than
10 μm, and their width from about 0.2 to 1.5 μm.
• A variety of bacterial shapes can be observed
under the light microscope, including cocci, rods,
spiral, and even cubes!
• These shapes can also be observed by scanning
electron microscopy, which provides both a
three-dimensional view of cellular structures and
information about their surface topography.
Scanning electron micrograph of rod-shaped cells of Clostridium difficile
adhering to the villi of human gut epithelium
Generalized diagram of the main features of a
bacterial cell
 Flagella

• Flagella are key structures concerned with bacterial

motility.
• Nevertheless, bacteria that lack flagella may still be
motile.
• A type of gliding motility can be achieved by the
flexible movement of the whole cell.
• Flagella can be located singly at one cell pole
(monotrichous flagella), at both poles
(amphitrichous flagella), in large numbers along the
length of the cell (peritrichous flagella), or as a tuft
of flagella at a polar end (lophotrichous flagella).
 Pili and Fimbriae
• Pilus’ (plural pili), are involved in
conjugation, that is the transfer of genetic
material.
• ‘Fimbria’ (plural fimbriae) are structures
concerned with the adhesion of bacteria to
various surfaces, including cell surfaces, and
to each other in coaggregation.
 Glycocalyx
• The glycocalyx is a polysaccharide and
protein film that surrounds bacterial cells.
• It may be found surrounding individual
cells or colonies, and it forms part of the
complex organization of bacterial biofilms.
• The glycocalyx can be divided into two
main types: capsules and S-layers
 Capsules
• Capsules are well-defined outer layers of uniform diameter on
bacterial surfaces. This is in contrast to slime layers, which
extend outwards from bacterial surfaces in a loosely organized
network of material.
• Capsular layers are mainly composed of a material made up of
an arrangement of polysaccharide chains that form a hydrated
‘polysaccharide gel’ on the cell surface.
• The thickness of this material depends upon genetic and
environmental factors.
• The capsule allows for the diffusion of small molecules, but can
exclude bacteriophages, protect against phagocytosis or
binding of host-mediated antibodies, and in some strains
encapsulation may be associated with virulence.
Pathogenic bacteria with capsules
 CHEMISTRY OF CAPSULES
Primary Structures
• Except for the PGA capsule of B. anthracis, the aetiologic agent of anthrax, other known capsules are all
composed of polysaccharides.
• There is great biochemical and structural diversity in capsule polysaccharides of both Gram-negative and -
positive bacteria.
• However, the CPSs of most Gram-negative (Neisseria, Haemophilus and Klebsiella) and Gram-positive
bacteria (Streptococcus and Staphylococcus) appear as a more or less homogeneous group of acidic
polysaccharides with comparable chemistry and biosynthesis.
• The CPSs are acidic polysaccharide polymers made up of repeating oligosaccharide units that may be
either linear or branched.
• The primary structures can be formulated by the respective repeating unit and the joining linkages.
• The negative charges are due to acidic sugar components or substitution with charged noncarbohydrate
moieties.
• The CPSs are sometimes substituted with O-formyl or O-acetylgroups (in ester linkage) or with pyruvate
(in ketosidic linkage).
• Amino acids are found linked either to the hydroxyl groups of sugar rings (esters) or to the carboxyl
group of hexuronic acids (amides).
 CHEMISTRY OF CAPSULES
Secondary Structures
• The secondary structures of bacterial
capsules are poorly characterized.
• The ploysia (K1 antigen) of E. coli and CPS
of N. meningitidis group B appear to be
helical, either throughout the
polysaccharide chain or as helical regions
of the polysaccharides.
 S-layers
• S-layers are found in many types of bacteria, including Gram-
positive and Gram-negative bacteria, cyanobacteria and the
Archaea.
• They are composed of crystalline arrays of protein or
glycoprotein subunits.
• In Gram-positive organisms the S-layer lies on the outer surface
of the peptidoglycan layer, while in Gram-negative bacteria it
is present on the outer surface of the outer membrane.
• It is probable that the main functions of the S-layer are to
protect the bacterial cell from hostile external environments,
and to act as a permeability barrier to macromolecules.
• To achieve the selective permeability of the S-layer, pores are
present within its protein subunits to facilitate the movement
of various molecules
 CELL WALL
• Beneath the S-layer is the cell wall proper, (approx. 2,040 nm thick).
• It is important for maintaining the bacterial shape.
• It also has to withstand an internal osmotic pressure of 520 atmospheres. This
is made possible by the presence of a strong, flexible peptidoglycan layer.
• The cell wall allows for the passage of macromolecules from the external
environment to the cytoplasmic membrane, and thence into the cytoplasm. If
the cell wall is ruptured, the cytoplasmic contents may be lost and the bacterial
cell undergoes lysis.
• By light microscopy, based on the application of the Gram stain, bacteria can
be divided into twogroups: Gram-negative and Gram-positive. The basis of the
different staining reaction of the two groups has been established by means of
electron microscopy.
• In the case of Gram-positive cells the cell wall peptidoglycan is not removed
by the decolourization step of the Gram-staining procedure, so the cytoplasm
retains the crystal violet stain.
• In the case of Gram-negative organisms the outer membrane is lost and the
cytoplasmic membrane becomes permeable, allowing the loss of stain from the
cytoplasm. Counter-staining with carbol fuchsin is necessary to observe such
Gram-negative bacteria under the light microscope
Gram-negative bacterial cell wall

Gram-positive bacterial cell wall

 Lipopolysaccharide
• Lipopolysaccharide (LPS) is a surface molecule unique to Gram-
negative bacteria and an integral component of the outer
leaflet of the outer membrane.
• Some examples of the Gram-negative bacteria with LPS are
Escherichia coli, Salmonellae, other Enterobacteriaceae like
Yersiniae or Shigellae, Enterobacter, Proteus, and pathogens like
Vibrio cholerae, Yersinia pestis, Brucella abortus
• LPS consists of three structural moieties:
– Lipid A: the hydrophobic domain, which is an endotoxin and the
main virulence factor.
– O-antigen, the repeating hydrophilic distal oligosaccharide.
– The hydrophilic core polysaccharide

While lipid A-core is universally present, not all bacteria produce OAg.
 Structural details of
lipopolysaccharide from a
Gram-negative bacterium.

LPS provides structural and

functional integrity to outer
membrane of Gram-negative
bacteria.
LPS is an amphipathic molecule
with a general structure consisting
of three different regions:
hydrophobic lipid A, core
polysaccharide, and O-antigen
(repeats of polysaccharide chain,
where n can be up to 40 repeats).
Lipid A consist of
bisphosphorylated diglucosamine
backbone substituted with six acyl
chains that are attached by ester
or amide linkage.
 Reading assignment
• BIOSYNTHESIS AND ASSEMBLY OF THE
CORE OLIGOSACCHARIDE
• BIOSYNTHESIS AND ASSEMBLY OF OAG
(a) Wzy-dependent pathway;
(b) ABC-dependent pathway;
(c) Synthase-dependent pathway.
The review summarizes the LPS biosynthesis and transport pathways and discuss efforts to find small molecule
inhibitors against targets within these pathways.
 Peptidoglycan
• Peptidoglycan is a large polymer that forms a mesh-like
scaffold around the bacterial cytoplasmic membrane.
 BASIC CHEMICAL STRUCTURE OF PEPTIDOGLYCAN

• Peptidoglycan is built from two monosaccharides (sugars);

– N-Acetylmuramic acid (NAM) and N-acetylglucosamine (NAG)
 Structure of the bacterial
peptidoglycan unit.

• Peptidoglycan consists of
repeating disaccharyl units (N-
acetylglucosamine-
Nacetylmuramic acid), to which
a pentapeptide is linked each N-
acetylmuramic acid.
• Crosslinking between adjacent
pentapeptides provides rigidity
to the bacterial cell
 BASIC CHEMICAL STRUCTURE OF PEPTIDOGLYCAN

• Peptidoglycan is generally composed of

linear glycan strands that are cross-linked by
short peptides.
• The glycan strands are made of alternating,
β-1-4 connected N-acetylglucosamine
(GlcNAc) and N-acetylmuramic acid
(MurNAc) residues.
• MurNAc is the typical sugar present in
peptidoglycan and represents GlcNAc with a
D-lactoyl group, which is linked to its C3
position and functions as an attachment site
for the stem peptide
 Contents

• Bacterial Structure
– Bacterial Ultrastructures
– Bacterial Capsules
– Lipopolysaccharide
– Peptidoglycan
• Bacterial Cell Function
– Growth, Culturability and Viability
– Bacterial Energy Metabolism
 Bacterial Growth
• Growth involves the accumulation of biomass
and may include genomic replication, cell
division and an increase in the number of
propagules of the organism concerned.

Phases of the Bacterial Growth Curve

• Upon inoculation into a new nutrient
medium, the bacteria shows four distinct
phases of growth. Let us dive into each of the
phases in detail –
Bacterial Growth
Lag Phase
• The bacteria upon introduction into the nutrient medium take some
time to adapt to the new environment. In this phase, the bacteria
does not reproduce but prepares itself for reproduction. The cells are
active metabolically and keep increasing in size. The cells synthesise
RNA, growth factors and other molecules required for cell division.
Log Phase (exponential phase)
• This phase is marked by the doubling of the bacterial cells. The cell
number increases in a logarithmic fashion such that the cell constituent
is maintained.
• The log phase continues until there is depletion of nutrients in the
setup. The stage also comes to a stop if toxic substances start to
accumulate, resulting in a slower growth rate.
• Plotting this phase on the bacterial growth curve gives a straight line.
Upon calculation of the slope of this line, the specific growth rate of
the organism is obtained.
• It is the measure of divisions per cell per unit of time.
 Bacterial Growth
Stationary Phase
• In the stationary phase, the rate of growth of the cells becomes equal to its
rate of death. The rate of growth of the bacterial cells is limited by the
accumulation of toxic compounds and also depletion of nutrients in the
media. The cell population remains constant at this stage. Plotting this phase
on the graph gives a smooth horizontal linear line.

Death Phase
• This is the last phase of the bacterial growth. At this stage, the rate of death is
greater than the rate of formation of new cells. Lack of nutrients, physical
conditions or other injuries to the cell leads to death of the cells.
 Bacterial Culturability
• Isolation of single bacterial strains in pure culture
has provided the foundation stone of medical
microbiology.
• Strategies for enrichment and selective isolation
perhaps represent the best-developed aspects of
the subject throughout the 20th century and they
will not be reviewed here, particularly since the
molecular requirements of isolation procedures are
not defined.
• It is self evident that isolation and culture have
provided abundant and relevant information that
gives us insights into infection.
Some Implications of Different Growth Phases of Bacteria for Medicine
 Viability

• Viability determinations have played a central

role in bacteriology.
• In medical microbiology we are primarily
concerned with measuring the effects of
antimicrobial agents, sterilization and disinfection
regimens and immune effector mechanisms.
• In addition, only viable bacteria are considered
capable of promulgating infection.
A Functional classification of indirect tests that have been applied to
assess the viability of bacteria
As Yet Uncultured’ (AYU) Bacteria
Using methods based on direct recovery of 16S
rRNA subunit genes from samples and in situ
hybridization to ribosomes at the single cell level it
has been possible to specifically recognize and
classify many bacteria that have not been recovered
or characterized in laboratory cultures

Diagram outlining the recognition and

characterization of ‘new’ organisms by
microscopy and rRNA gene recovery.
 Definitions of Key
Terms Relating to
Growth, Culturability
and Viability of
Bacteria
Reading Assignment