Description of Structure and function of different bacterial cell organelles.

1. Santosh Yadav
BACTERIAL ANATOMY, PHYSIOLOGY,
GROWTH AND NUTRITION
AND
BACTERIAL METABOLISM,
TOXINS AND BACTERIOCINS
Santosh Yadav
M.Sc. Clinical Microbiology
Dept. of Microbiology
Institute of Medicine
Tribhuvan Univarsity Teaching Hospital, Nepal

2. Santosh Yadav
Outline
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Structure and function of different bacterial cell
organelles.
Growth and nutrition of bacteria.
Bacterial metabolism and different pathways.
Bacterial toxins and their clasiification.
Bacteriocins, their classification ,mode of action
and typing.

3. Santosh Yadav
ANATOMY AND PHYSIOLOGY
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4. Santosh Yadav
Bacterial cell structure Cell wall
 Internal to cell wall
(Plasmamembrane,Mesosome,Nucleoid,Plasmid,Ribosome,Inclusi
on body,Endospore)
 External to cell wall(capsule,Flagella,Pili)
3 – 5 um ₓ 0.2 – 1.5 um

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CELL WALL
 Encloses the
protoplast and lies
immediately external
to the cytoplasmic
membrane.
 Relatively rigid with
some elasticity, and
openly porous.
 Freely permeable to
solute molecules
smaller than 10 kDa
in mass and 1 nm in
diameter.
 Thickness depends
on type of bacteria.
Gram positive Gram
negative

6. Santosh Yadav
Comparision of cellwall
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Gram positive Gram negative
Thickness Thicker Thinner
Peptidoglycan 40-65 sheets 1-2 sheets
Varieties of amino
acids
Few Several
Lipids Absent or scant Present
Teichoic acid present Absent
Lipopolysaccharide
s
Absent Present

7. Santosh Yadav
Cell wall of Gram positive bacteria
 Composed of
peptidoglycan and
techoic acid.
 Peptidoglycan comprises
up to about 50% of the cell
wall material.
 Peptidoglycan layer is 15-
50 nm thick.

8. Santosh Yadav
The peptidoglycan layer
 Complex polymer consisting of three parts:
a)Backbone of cellwall, glycan (polymer of alternating
N-acetylglucosamine(NAG) and N-acetylmuramic acid (NAM);
b) tetrapeptide side chains attached to(NAM); and
c) peptide interbridges.

9. Santosh Yadav
Contd…

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Teichoic acid
 Major surface antigen of
Gram positive bacteria.
 Chains of either ribitol-
phosphate or glycerol
phosphate,to which various
sugars and D-alanine are
usually attached.
 Two types:-
 lipotechoic acid (attached to
cytoplasmic membrane) and
 wall techoic acid ( those
attached to NAM portion of
peptidoglycan of cell wall)

11. Santosh Yadav
Contd…
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Cell wall of Gram negative bacteria
• Composed of peptidoglycan and outermembrane.
• Peptidoglycan comprises 5-10% of the wall material
(thickness 2-6 nm).
 Outer membrane contain three components:
lipoprotein ,phospholipid and lipopolysaccharide.

13. Santosh Yadav
Peptidoglycan Cross-Links. (a) E. coli peptidoglycan
with direct cross-linking, typical of many gram-negative bacteria.
(b) Staphylococcus aureus peptidoglycan. S. aureus is a gram-
positive
bacterium. NAM is N-acetylmuramic acid. NAG is N-acetylglucosamine.
Gly is glycine.
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Peptidoglycan of Gram negative
bacteria

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Lipoprotein
• lipoprotein cross-link the outer membrane and
peptidoglycan layers.
• Is peptide, linked to DAP residues of the
peptidoglycan .
• Function is to stabilize the outer membrane and
anchor it to the peptidoglycan layer .
Outermembrane

15. Santosh Yadav
Phospholipid of outer
membrane
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Is distinct from all other biological membranes
 Its outer leaflet contains a
lipopolysaccharides.
 Has special channels, consisting of protein
molecules called porins.

16. Santosh Yadav
Lipopolysaccharide (LPS)
 outermost part of
cellwall of Gram
negative bacteria.
 Consists of
 lipid A,
 core
polysaccharide and
 a terminal series of
repeat units ( O
antigen).

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Bacteria with atypical cell wall
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 Mycobacteria and
Nocardia.
 Contains high
concnentration (
around 60%) of
hydrophobic waxy lipid ,
mycolic acid.
 Mycolic acid prevent the
uptake of dye .
 Mycolic acids are present
outside the thin
peptidoglycan layer linked
by polysaccharides.

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Bacteria without cell wall
 Mycoplasma .
 Plasmamembrane of
Mycoplasma contain
sterol that are tough
to protect from lysis.

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Bacteria with defective cell
wall
 Protoplast
 Spheroplast
 L- forms

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Functions of cell wall
 Provides shape to the bacterium.
 Give rigidity to the organism.
 Protects from environment.
 Contains receptor sites for phages.
 Provides attachment to complement.
 Contains components toxic to host.
 Site of action of colicin.

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Cytoplasmic membrane( plasma
membrane)
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 Lies beneath the cell wall and separating it from the cell
cytoplasm.
 5-10 nm thick, elastic and semipermeable layer and
comprises about 30% of the dry weight of bacterial cell.
 Composed of mainly phospholipid (20-30%) and proteins
(70-80%).
 Phospholipids form bilayered structure in which proteins
are embedded.
 Phospholipid has two parts:
 Hydrophillic head
 Hydrophobic tail
 Two types of proteins are found:
 peripheral protein
 integral protein
 Many enzymes are also present.

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Contd…

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Functions of plasma
membrane
 Regulates the transport of nutrients and
waste products into and out of the cell
 Synthesis of cell wall components
 Assists in DNA replication
 Secretes proteins
 Carries on electron transport system
 Captures energy in the form of ATP, etc.

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Movement of Molecules through
Cytoplasmic Membrane
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Simple or passive diffusion
 Solute molecules cross the membrane as a result of a
difference in concentration of molecules across the
membrane.
 Speed and direction of diffusion depends on the relative
concentration of molecules on each side of the
membrane.
Ref: Microbiology by M.J.

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Facilitated diffusion
 Similar to that of simple diffusion.
 But requires carrier protein called permease located in
the cytoplasmic membrane.
 Entry of glycerol.
Ref: Microbiology by M.J. Pelczar

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Group translocation
• Accumulates the solute inside the cell against concentration gradient
• Solute molecule altered chemically during transport.
• PEP-dependent sugar-phosphotransferase system.
• A heat stable carrier protein (HPr) is first activated by transfer of
phosphate group from PEP inside the cell .
• At the same time sugar combines with enzyme II at the outer
membrane surface and is transported to inner membrane surface .
Here it combines with phosphate group carried by activated HPr.
Ref: Microbiology by M.J.
Pelczar

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Active Transport
 Almost all solutes , including sugars, amino acids, peptides,
nucleosides, and ions are taken up by cells through active transport.
• Entry of solutes occurs in three steps:-
1)Binding of solute to carrier protein.
2)Translocation of the solute- carrier complex, and
3)Coupling of translocation to an energy yielding reaction to lower the
affinity of the carrier protein for the solute at the inner membrane
surface so that the carrier protein will release solute to the cell
interior.
Ref: Microbiology by M.J.

29. Santosh Yadav
Two primary mechanisms of active transports, each utilizing a
different form of energy.
A. Transport system that use proton motive force
Uniporters : (eg. Potassium enters the cell via uniporter)
Antiporters: (eg. Sodium is transported out of the cell as a
proton passes in )
Symporters :(eg. A lactose molecule enters a cell with one
proton)
Contd…

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B.Transport system that
use ATP:-
ABC transport
system: (ABC stands for
ATP Binding Cassette)
The ABC transport
system utilizes a binding
protein that resides
immediately outside of
the cytoplasmic
membrane to deliver a
given molecule to a
specific transport
complex within the
membrane.
Contd…

31. Santosh Yadav
Mesosome
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 Convoluted or multilaminated
membranous bodies.
 Develop by complex
invagination of the
cytoplasmic membrane into the
cytoplasm.
Function
(1)Compartment of DNA at cell
division and at sporulation.
(2)Are principal sites of respiratory
enzymes.(analogous to the
mitochondria of the eukaryotic
cell)

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Nucleoid (nuclear material)
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 Nucleoid contains a
single dsDNA , which
carry genetic
information for cell.
 Circular thread about 1
mm long, being
condensed and looped
into a supercoiled state,
located centrally.
 Nuclear division
preceeding cell division,
two DNA may be
present.

33. Santosh Yadav
Contd…
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 Chromosomes have 2,000 to 4,000 genes.
 Many genes that encodes virulence factors (
adhesins, invasins, exotoxins,etc) are clustered
adjacent to each other on chromosome , called
pathogenicity islands.
 These islands range in size from 10 to 200 kB.
 Can be horizontally transferred between
bacteria, resulting in enhanced virulence in the
recipient.
 made visible under the light microscope by
Feulgen staining ( specific for DNA).

34. Santosh Yadav
Plasmid
 Many bacteria possess
plasmids in addition to
chromosome.
 Are circular dsDNA
molecules .
 size from 1.5 kilobase (kb)
pairs to 120 kb pairs (less
than one tenth the size of
the bacterial chromosome)
 Can exist and replicate
independently of the
chromosome or may be
integrated with it.
 Not required for host
growth
and reproduction.

35. Santosh Yadav
Classification of plasmid
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 Relaxed plasmid : Plasmid occur free in the cytoplasm
and replicate independent of bacterial genome replication
 Stringent plasmid: plasmid that integrate and replicate
along with bacterial chromosome.

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36 Ref:- Brock biology of microorganisms.

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Ribosome
 Is spherical and granular structure with diameter of 100-
200 A.
 Present in cytoplasm and may loosely attached to the
plasma membrane.
 Made up of both protein and ribonucleic acid (RNA).
 70s type.
 Each 70s has two subunit :- larger 50s and smaller 30s.
30S
50
S
50
S
30S

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Contd…

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Functions
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 Site of protein synthesis;
 Matrix ribosomes synthesize proteins to remain
within the cell and Plasma membrane ribosomes
make proteins for transport to the outside.

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Inclusion body
 Are reserve deposits of
bacterial cell.
 Cells accumulate nutrient
when they are plenty and
use when deficient.
Metachromatic
granules:-
 Reserve of polyphosphate
used in synthesis of ATP.
 Characteristics of
Corynebacterium
diphtherie.

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Polysaccharide granules:-
 Mainly consists of glycogen and starch.
Lipid inclusions:-
 Poly – B-hydroxybutyric acid.
 Found in several species of Mycobacteria, Bacillus, Azotobacter, etc
Sulphur granules:- Present in sulphur bacteria .eg. Thiobacillus.
Carboxysomes :- contain enzyme ribulose-1,5-diphosphate
carboxylase ,helps in CO2 fixation in photosynthetic bacteria (
Cyanobacteria and Thiobacillus, etc ).
Gas vacuoles:-
 Hollow cavities found in aquatic procaryotes.
 Consists of gases covered with proteins.
Magnetosomes :-
 Inclusion of iron oxide. (Fe3O4)
 Decompose hydrogen peroxide.
 Found in Magnetospirillum , Magnetotactium, etc.
Contd…

42. Santosh Yadav
Endospore
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 Highly resistant phase of
bacteria, formed in
unfavorable condition.
 Formed internal to
bacterial cell.
 Can survive in extreme
heat, lack of water, many
chemicals, radiation , etc..
STRUCTURE:-
1)Core ,
2)Core wall,
3) Cortex,
4)Coat , and
5)Exosporium .

43. Santosh Yadav
Contd…
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Resistance property of endospore is due to
- impermeability of their cortex and outer coat,
- high content of calcium and dipicolinic acid,
- low content of water (5-20%),
- very low metabolic and enzyme activity,
- DNA-binding proteins saturate spore DNA and protect it
from heat.

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Sporulation

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Contd…
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 Sporulation, occurs when growth ceases due to lack of
nutrients.
Steps:-
• An axial filament of nuclear material forms.
• An inward folding of the cell membrane to enclose part
of the DNA and produce the forespore septum.
• The membrane continues to grow and engulfs the
immature spore in a second membrane.
• Cortex is laid in the space between the two membranes,
and both calcium and dipicolinic acid are accumulated.
• Protein coats then are formed around the cortex.
• Maturation of the spore occurs.
• Lytic enzymes destroy the sporangium releasing the
spore.

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Spore germination
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Contd…
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 It has been estimated that 7500- year old endospore of Thermoactinomyces vulgaris
from the freezing muds of Elk lake in Minnesota have germinated when warmed and
placed in a nutrient medium.
 The transformation of dormant spores into active vegetative cells is complex process.
It occurs in three stages:
 (1) Activation,
 (2) Germination, and
 (3) Outgrowth.
1)An endospore will not germinate successfully, even in a nutrient-rich medium, unless it
has been activated.
 Activation is a reversible process that prepares spores for germination and usually
results from heat treatments.
2)It is followed by germination, the breaking of the spore’s dormant state.
 It is characterized by spore swelling, rupture or absorption of the spore coat,
loss of refractility,
 Release of spore components, and increase in metabolic activity.
 Many normal metabolites or nutrients (e.g., amino acids and sugars) can
trigger germination after activation.
3)Grmination is followed by the outgrowth.
 The spore protoplast makes new components and develops again into an

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 Central (eg. Clostridium bifermentans)
 Subterminal ( eg. Cl. Perfringens)
 Oval and terminal ( eg. Cl. tertium)
 Spherical and terminal (eg.Cl. tetani)

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Demonstration of spore
 Spore stain ( Shaeffer –Fulton and Dorner method)
 Modified Ziehl Neelsen stain ( resist to
decolorisation by 0.25 % H2SO4)
 Evidence of presence of spore can can also be
obtained by Gram staining where spore remains
unstained.
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Fig- spore staining

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Capsule and slime layers
 Hydrophobic gelatinous
material sectreted outside
and lies immediately in
contact with the cell wall.
 Consists largely of water
and small content of
solids (2%).
 In most species, the solid
material is a
complex
polysaccharide, though
in some species
polypeptide or protein.

52. Santosh Yadav
Contd…
 When the material is tightly associated with cell wall it is
called capsule.
 Capsules too thin to seen under the light microscope is
called microcapsule.
 Slime is an amorphous, colloidal material secreted
extracellularly by some non-capsulated bacteria and also
by many capsulated bacteria outside their capsules.
 Glycocalyx is the term used for any carbohydrate
molecule present on surface of cell.

53. Santosh Yadav
Functions of capsule
 Protecting the cell wall against attack by various
kinds of antibacterial agents, e.g. bacteriophages,
colicins, complement, lysozyme and other lytic
enzymes. Thus the capsule is an important
virulence determinant.
 Is usually antigenic and the capsular antigens
play a very important part in determining the
antigenic specificity of bacteria.
 Helps to adhere bacteria to surface.
 Prevents from Phagocytosis.

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Method of demonstration
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1) Negative staining.
2) Special capsule
staining using CuSO4
as mordant.
3) Quellung reaction

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Contd…
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Flagella
 Organ of locomotion.
 Long , thin filaments,
regular.
 15-20 nm thick and several
times the length of the
bacteria cell.
 Originating in the bacterial
protoplasm and extruded
through the cell wall.
 Made up of several
thousand molecules of a
protein subunit called
flagellin.

57. Santosh Yadav
Structure
A flagellum has three basic parts:-
 Outer filament, contains the globular protein flagellin.
 Filament is attached to a slightly wider hook, consisting
of different protein, and
 The basal body, which anchors the flagellum to the cell
wall and plasma membrane.

58. Santosh Yadav
Flagellar arrangements
•Bacteria without flagella are called atrichous.
The arrangement of flagella may be
Polar flagella :- flagella at one or both end.
Monotrichous:- single flagellum at one end (eg. Vibrio cholera)
Amphitrichous:- single flagellum at both ends (eg. Alkaligenes faecalis)
Lophotrichous:- tuft of flagella at one or both end (eg. Helicobacter
pylori)
Peritrichous flagella:- flagella arranged all round the body (eg.
Escherichia coli , Proteus , etc)

59. Santosh Yadav
Flagella and bacterial motility
 The movement of flagella results
from rotation of basal body.
 As the flagella rotates ,they form
a bundle that pushes against the
surrounding liquid and propels
the bacterium and the flagellar
rotation depends on the cells
countinuous generation of
energy.
 Bacterial cells can alter the
speed and direction of rotation of
flagella and thus are capable of
various patterns of motility.
 When a bacterium moves in one
direction for a length of time
,called run or swim.
 Runs are interrupted by random
changes in direction called
tumble,caused by reversal of
flagellar rotation.

60. Santosh Yadav
contd…
 The energy required for rotation of the flagellum comes from the
proton motive force.
 Proton movement across the cytoplasmic membrane through the
Mot complex drives rotation of the flagellum.
 In this model called the proton turbine model, protons flowing
through channels in the Mot proteins exert electrostatic forces on
helically arranged charges on the rotor proteins.
 Attractions between positive and negative charges would then
cause the basal body to rotate as protons flow though the Mot
proteins.

61. Santosh Yadav
Demonstration of motility
 Hanging drop technique,
 Flagella stain,
 Growing in semisolid agar
media,
 Craigie’s tube,
 U –tube.
Craigies tube U-tube
Flagella stain

62. Santosh Yadav
Axial filament or endoflagella
 Present in spirochetes .
 Are bundle of fibrils, arise at the ends of the cell and
spiral around the cell.
 have structure similar to that of flagella.
 The rotation of the filaments produces movement of the
outer sheath that propells the spirochetes in spiral
motion.

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Pili or Fimbriae
 Organ of adhesion.
 Short , hairlike appendages thinner and smaller than
flagella, originated from cellwall.
 About 3 -10 um in length and 0.03-o.2nm in diameter.
 Composed of helically arranged protein subunits , pilin.
 Some fimbriae cause hemagglutination with RBC of
guinea pig, fowl, horses, etc and can be inhibited by 0.1-
0.5% D-mannose (MS).

64. Santosh Yadav
Types of fimbriae
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 6 types of fimbriae.
Type1 : relatively thick and involve in haemagglutination
(MS), (E. coli, Salmonella spp).
Type2 : resembles MS type1 but non-haemagluttinating( S.
gallinarum, S. pullorum).
Type3 : thin and MR and cause indirect haemagglutination
(RBC treated with tannic acid),( Proteus spp).
Type4 : thinner than MR type3 fimbriae and have MR
haemagglutinating activity for fresh RBC.
Type5 : are very long ( some Klebsiella spp)
Type6 : are monopolar and found only in Pseudomonas
spp.

65. Santosh Yadav
Function of pilli
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 Commom pili (fimbriae): Playing a role in the
adherence of symbiotic and pathogenic bacteria to host
cells.
 (Minor proteins termed adhesins are located at the tips
of pili and are responsible for the attachment properties).

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 Sex pili: long and flexible structure,being
responsible for the attachment of donor and
recipient cells in bacterial conjugation.
 Pili also help in formation of pellicle on
surface of stagnant liquid medium.
Contd…

67. Santosh Yadav
THANK YOU
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