Best description of Bacterial anatomy, physiology, growth, nutrition, metabolism, toxin and bacteriocin

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
2
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
3

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

5. Santosh Yadav
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
6
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…

10. Santosh Yadav
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…
11

12. Santosh Yadav
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.
13
Peptidoglycan of Gram negative
bacteria

14. Santosh Yadav
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
15
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).

17. Santosh Yadav
Bacteria with atypical cell wall
17
 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.

18. Santosh Yadav
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

20. Santosh Yadav
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.

21. Santosh Yadav
Cytoplasmic membrane( plasma
membrane)
21
 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.

22. Santosh Yadav
Contd…

23. Santosh Yadav
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.

24. Santosh Yadav
Movement of Molecules through
Cytoplasmic Membrane
24

25. Santosh Yadav
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.

26. Santosh Yadav
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

27. Santosh Yadav
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

28. Santosh Yadav
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…

30. Santosh Yadav
30
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
31
 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)

32. Santosh Yadav
Nucleoid (nuclear material)
32
 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…
33
 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
35
 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.

36. Santosh Yadav
36 Ref:- Brock biology of microorganisms.

37. Santosh Yadav
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

38. Santosh Yadav
38
Contd…

39. Santosh Yadav
Functions
39
 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.

41. Santosh Yadav
41
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
42
 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…
43
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.

44. Santosh Yadav
44
Sporulation

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Contd…
45
 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.

46. Santosh Yadav
Spore germination
46

47. Santosh Yadav
Contd…
47
 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

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

49. Santosh Yadav
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.
49

50. Santosh Yadav
50
Fig- spore staining

51. Santosh Yadav
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.

54. Santosh Yadav
Method of demonstration
54
1) Negative staining.
2) Special capsule
staining using CuSO4
as mordant.
3) Quellung reaction

55. Santosh Yadav
Contd…
55

56. Santosh Yadav
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.

63. Santosh Yadav
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
64
 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
65
 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).

66. Santosh Yadav
 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. 67
GROWTH AND
NUTRITION OF
BACTERIA

68. Santosh Yadav
Nutrition requirements
68
 Bacteria have same general chemical pattern
as the cells of other organism.
 Principal constituent of the cell is water, proteins ,
polysaccharides , lipids, nucleic acids,
mucopeptides and other low molecular weigth
compounds.
 For growth and muliplication of bacteria,the
minimum requirements are
 Water,
 Source of carbon and energy,
 Source of nitrogen,and
 Some inorganic salts.

69. Santosh Yadav
Macronutrients
69
• Microorganisms require some elements in large
quantities, because they are used to construct
carbohydrates, lipids, proteins, and nucleic acids.
• 95% or more of cell dry weight is made up of a few
major elements: C ,O , H, N , S and P.
• Required in large amount.

70. Santosh Yadav
Trace Elements
70
 Microbes require very small amounts of other
mineral elements, such as Fe , Cu, Mo , Zn,
etc .
 Most are essential for activity of certain
enzymes, usually as cofactors.
 Eg- Mg²⁺ for Hexokinase , Ni⁴⁺ for urease,
Fe²⁺ for cytochrome oxidase , etc…

71. Santosh Yadav
71 Ref:-Microbiology by Anderson and

72. Santosh Yadav
Growth Factors
72
 Organic compounds required in minute quantities
and are not synthesized by bacteria, also called
bacterial vitamins.
 Eg…Neisseria spp require at least 40 additional
ingredients, including 7 vitamins and all of the 20
amino acids.

73. Santosh Yadav
73
Carbon and Energy source

74. Santosh Yadav
Bactrial Growth
74
 It is an increase in number of population rather
than in size.
 Bacteria divide by binary fission.
 The number of cell arising from a single cell is
2n after n generations.

75. Santosh Yadav
Binary fission
75
Ref:-Microbiology, Nester

76. Santosh Yadav
Generation time
76
 Time required for a
bacterium to give rise to
2 daughter cells under
optimum conditions
 Also called population
doubling time.
 Escherichia coli – 20 mins.
 Mycobacterium
tuberculosis – 20 hrs.
 M.leprae – 20 days.

77. Santosh Yadav
Bacterial Growth Curve
77 Ref:-Microbiology, Nester

78. Santosh Yadav
Lag phase
78
 Phase of adaptation in new medium.
 Necessary enzymes and metabolites
are built up.
 Bacterial cell attain maximum size and is
critical stage for multiplication.
 Length depends on generation time of
bacteria, size of inoculum, quantity of
culture medium, environmental
factors, etc…
 Average time:- 2-6 hours.

79. Santosh Yadav
Log or exponential phase
79
 Cell division occurs at maximum rate and number
increases exponentially with time.
 Generation time is constant for a given strain
under same set of condition.
 Becomes smaller in size.
 Bacteria are most sensitive to antimicrobial
substances.

80. Santosh Yadav
Stationary phase
80
 Growth ceases due to exhaustion of nutrients,
accumulation of of toxic metabolites and lowering of
pH.
 Produce secondary metabolites.
 Frequently gram variable and stain irregularly.
 Sporulation occurs.
 Total count remains same.

81. Santosh Yadav
Death or decline phase
81
 Cells begin to die due to cessation of growth,
accumulation of toxic products, autolytic and
heterolytic enzymes.
 Rapid fall in viable count.
 Some cells remain survive at the expense of
nutrients released from death of others.

82. Santosh Yadav
Phase of prolonged decline ( survival
phase)
82

83. Santosh Yadav
83
Factors affecting bacterial
growth

84. Santosh Yadav
Water
84
 About 80% of bacterial cell is water hence it is
essential requirement.
 Vehicle for entry of nutrients and elimination
of waste products.
 Participate in metabolic reactions and form
integral part of protoplasm.
 Some organism like Treponema pallidum are
highly sensitive while others like Staphylococci
withstand drying for months.

85. Santosh Yadav
Temperature and Bacterial
growth
85
 Major environmental
factor controlling
growth.
 Minimum:- below
which growth ceases.
 Optimum:- at which
maximum growth
occur.
 Maximum:- above
which growth ceases.

86. Santosh Yadav
Classification by tempr. requirement
86
Psychrophiles:-
 best grow at low temprature (-5 -15⁰C)
 Produce enzymes that functions optimally in cold.
 Membrane contain high content of polyunaturated
fatty acids which maintain rigidity at low temp.
 eg. Yersinia enterocolitica , Listeria monocytogens ,
Pseudomonas fluorescens.
 Psychrotrophs grows optimally at 20-30⁰C but can
grow in refrigerator temprature.

87. Santosh Yadav
Mesophiles
87
 Temprature range for growth 25-45⁰C.
 Optimum growth temp 37⁰C.
 Are mostly pathogenic bacteria.
 Eg..Escherichia coli , staphylococcus aureus ,
etc.

88. Santosh Yadav
Thermophiles and
Hyperthermophiles
88
 Range 45-70⁰C
 Contain heat stable enzymes and proteins.
 Membrane contains lipids rich in saturated fatty
acids and stabilizes the membrane at high temp.
 All thermophiles contain reverse gyrase ( a unique
type of DNA 1 topoisomerase) that stabilizes DNA.
 Eg..Bacillus sterothermophilus.
 Hyperthermophiles- grow at temp above 70⁰C.
 Eg…Thermotoga, Methanopyrus.

89. Santosh Yadav
Oxygen and bacterial growth
89
 Different bacteria have different oxygen
requirement.
 When it accepts electron in ETC , it forms H2O2 ,
O2‾ , and OH‾‾ , all of these are toxic unless
broken down.
 Three enzymes namely catalase , peroxidase
and superoxide dismutase(SOD) prevent
bacteria from oxidative damage by breakdown of
these oxygen radicals.

90. Santosh Yadav
Classification based on O2 requirement
90
Obligate aerobes:-
 Grow only in presence of oxygen.
 Have all three enzymes and they have ETC
as final electron acceptor.
Eg.. Pseudomonas , Brucella , Mycobacteria
, Nocardia spp etc.
Facultative anerobes:-
 prefer aerobic condition but can grow without
oxygen.
 Also have all three enzymes.
 Eg..E. coli, S. aureus, etc
Microaerophiles:-
 Are aerobes but require little of oxygen for
growth.
Eg… Campylobacter jejuni, etc.

91. Santosh Yadav
Anaerobes
91
Obligate anaerobes:-
 Grow only in absence of oxygen and oxygen is
lethal for them.
 Lacks enzymes for using oxygen in respiration.
 Enzymes of anaerobes are active only in
reduced state.
Eg…Clostridium sps, Bacteroides sps, etc...
Aerotolerent anaerobes:-
 Do not utilize oxygen , but can tolerate low
amount of oxygen because they have superoxide
dismutase. ( no catalase)
 Eg..Lactobacilli , anaerobic streptococci , etc

92. Santosh Yadav
Contd…
92

93. Santosh Yadav
Carbondioxide( CO2)
93
 Small amount of CO2 is required by all
bacteria.
 Made available endogenously or exogenously.
 Some organisms grow best at higher CO2
tension and are called capnophillic.
 Eg..Brucella sps , gonococcus,
meningococcus.

94. Santosh Yadav
Hydrogen ion
concentration(pH)
94
 Most bacteria grow at neutral or slightly
alkaline pH.
 pH can harm organisms by disrupting the
plasma membrane , inhibiting the activity of
enzymes and membrane proteins and
ionization of nutrient molecules.

95. Santosh Yadav
Classification
95
Acidophiles:-
 grow at acidic pH.
 pH range ( < 5.0).
Eg..Lactobacillus sps , Thiobacillus spp,etc.
Neutrophiles:-
 pH range ( > 5.0 and <8 ).
 Maintains pH by exchanging potassium ion with proton
by antiport transport system.
 Many pathogenic bacteria are neutrophiles.
Alkalophiles:-
 pH range ( > 8 ).
 Maintain pH by exchanging internal sodium ion for
external proton.
 Eg.. Vibrio cholera , Bacillus alkalophilus, etc…

96. Santosh Yadav
Osmotic pressure
96
 Bacteria can withstand wide range of external osmotic
pressure because of protective cellwall.
 0.5% of NaCl is added in most culture medium to
maintain suitable environment.
 Hypertonic solution cause withdrawl of water and
shrinkage of protoplasm (plasmolysis).
 Hypotonic solution cause swelling and hence rupture of
cell (plasmoptysis).

97. Santosh Yadav
contd…
97
 Some bacteria grow at high pressure , i.e. 400-
500 atm (mostly in deep sea) and are called
barophiles.
 They have huge amount of monounsaturated
fatty acids in membrane and OmpH in cellwall
outer membrane.
 Eg..Photobacterium shewanella , Colwella sps,
etc.

98. Santosh Yadav
BACTERIAL METABOLISM
98

99. Santosh Yadav
Metabolism
99
 Sum total of all the chemical reactions
occuring in the cell ( i.e. biosynthetic and
degradative)
 Metabolism in bacteria is essential for their
existance , for environment , and products
are commercially and medically important for
human beings.

100. Santosh Yadav
CATABOLIC AND ANABOLIC
REACTIONS
100
 Reactions that cause
breakdown of complex
molecules into simpler
form with relase of
energy is catabolic
reactions.
 Energy requiring
reactions that build up
complex organic
molecules from
simpler ones is
anabolic reactions.

101. Santosh Yadav
Components of metabolism
101
COMPONENTS FUNCTIONS
Enzymes Biological catalyst, fascilitates each step of metabolic
reaction by lowering the activation energy of reaction.
Adenosine triphosphate
(ATP)
serves as energy currency of cell ,
Energy source Compund that is oxidised to release energy
, also called an electron donor.
Electron carriers carry the electrons that are removed during the oxidation of
energy source (NAD⁺, NADP⁺ , and FAD ( their reduced
form NADH , NADPH , and FADH₂) .
Precursor metabolites Intermediate metabolite that link anabolic and catabolic
pathways, like pyruvate, acetyl-coA, glucose -6-p, etc.

102. Santosh Yadav
Role of ATP
102
 Is energy currency of cell,
serving as ready and
immediate donor of free
energy.
 Energy is releases when
phosphate bond is broken,
hence it is called high
energy phosphate bond.
 Synthesis and breakdown
of ATP continuously occurs
in cell during degradative
and synthetic process.

103. Santosh Yadav
Generation of ATP
103
Bacteria uses three mechanism of
phosphorylation to generate ATP
from ADP.
1)Substrate level
phosphorylation
C-C-C-P + ADP C-C-C +
ATP
In this mechanism , a high energy
phosphate from a phosphorylated
substrate is directly transferred
to to ADP.

104. Santosh Yadav
Contd…
104
2) Oxidative phosphorylation
3)Photophosphorylation
 Occurs in phototrophs.
 Derive ATP using radiant energy of the sun.
 These ATP are then utilized to synthesize mainly
glucose .

105. Santosh Yadav
METABOLIC PATHWAYS OF
ENERGY GENERATION
105

106. Santosh Yadav
GLYCOLYSIS
106
 Embden-Mayerhof Parnas
pathway.
 Stepwise Conversion of glucose to
pyruvate and each step require
specific enzyme.
 Occurs in cytosol.
 Does not require oxygen and hence
occur in both aerobic and
anaerobic bacteria.
 Three phases :- preparatory phase,
splitting phase and energy
generation phase.
 2 molecules of pyruvic acids are
formed from each glucose .
 Net gain of 2 ATP by substrate
level phosphorylation and
formation of 2 reduced substrate
i.e. NADH.

107. Santosh Yadav
Pathways alternative to glycolysis
107
 Many bacteria have another pathway in addition to
glycolysis for degradation of glucose.
1) Pentose phosphate pathway, and
2) Entner Doudoroff pathway.

108. Santosh Yadav
Pentose phosphate pathway
108
 Hexose monophosphate shunt.
 Occurs simultaneously with
glycolysis and provides
breakdown of both pentose
sugar and glucose.
 Important Feature :- intermediate
pentoses are used for nucleic
acid synthesis, amino acid
synthesis and glucose from
CO2 in photosynthetics.
 Important producer of reduced
coenzyme i.e. NADPH , used for
biosynthetic reactions.

109. Santosh Yadav
Entner –Doudoroff pathway
109
 Bacteria having enzyme for
Entner –Doudoroff pathway
can metabolize without glycolysis
or PPP.
 Found in some Gram negative
bacteria like Psedomonas spp,
Rhizobium, Agrobacterium,etc..
and generally not found in Gram
positive bacteria.
 Produces 1 molecule NADH, 1
molecule NADPH and 1
molecule of ATP ( from 1
glucose).

110. Santosh Yadav
Cellular respiration and
fermentation
110
 Pyruvate obatained from glucose breakdown are
channeled either to respiration or to
fermentation.
RESPIRATION:- is ATP generating process in
which molecules are oxidized and the final
electron acceptor is an inorganic molecules.
TYPES OF RESPIRATION :-
Aerobic respiration:- final electron acceptor is
O₂ and occurs in aerobes.
Anaerobic respiration: final electron acceptor is
inorganic molecule other than O₂ .

111. Santosh Yadav
Krebs cycle or TCA cycle
111
 Is second phase of aerobic respiration.
 Cytoplasmic membrane.
 Pyruvate formed enters TCA cycle only after
converted into acetylCoA by decarboxylation rxn (
transition phase).
 Starts with condensaion of acetylCoA and
oxaloacetate.
 AcetylCoA is oxidized and released as CO2 and
oxaloacetate is regenerated.

112. Santosh Yadav
Contd…
112
Cntd…….

113. Santosh Yadav
Contd…
113
 From TCA cycle, oxidation of one acetylCoA
molecule produce 3 NADH , 1 FADH₂ and 2
GTP.
 All the NADH and FADH2 enters into electron
transport chain and finally ATP are generated.

114. Santosh Yadav
Electron transport chain
114
 Last phase of respiration which generates ATP
from reduced substrates.
 Consists of a sequence of carrier molecules
though which electrons passes.
 Occurs in plasma membrane ( eukaryotic cell-
inner mitochondrial membrane).

115. Santosh Yadav
Contd…
115
 Three classes of carrier molecules
1)Flavoprotein :- coenzyme derived from viatmin B2 (riboflavin)
and perform alternating oxidation and reduction reaction.
3)Ubiquinones :- small non protein carriers.
2)Cytochromes :-protein with iron containing group (heme).

116. Santosh Yadav
116
• Electron transport chain is different in different bacteria ,
even a single bacteria have more than one type of ETC
but their target is to derive energy in the form of ATP.
Contd…

117. Santosh Yadav
Contd…
117

118. Santosh Yadav
Anaerobic respiration
118
 Final electron acceptor is NO3⁻ , NO2⁻, N₂O,
SO₄⁻⁻,CO₃⁻⁻, etc. and occurs in strict anaeres
and facultative anaerobes.
 Sometimes Pseudomonas sps and Bacillus sps
can use nitrate as final e⁻ acceptor.
 ATP generation varies from bacteria to bacteria
and always less than aerobic respiration.
 Alternative electron carriers are used in the ETC.

119. Santosh Yadav
Fermentation
119
 Used by organisms that cannot respire because
of either lack of inorganic electron acceptor or
absence of ETS.
 Terminal electron acceptor is always organic
compound.
 End product depends on type of microrganisms.
 Analysis of end product is valuable in identifying
particular bacteria.
 ATP generating pathway is usually glycolysis(
2ATP).

120. Santosh Yadav
Products of fermentation
120

121. Santosh Yadav
Catabolism of other substrates
121

122. Santosh Yadav
BACTERIAL TOXINS
122

123. Santosh Yadav
Toxins
123
 Are virulence factor of most of bacteria and one of
the major cause of tissue damage.
 Two types :- endotoxin and exotoxin.

124. Santosh Yadav
124
Exotoxin Endotoxin
Secreted outside cell by both
Gram positive and Gram
negative bacteria
Relesed after lysis of Gram
negative cellwall
Protein Lipopolysaccharide
Heat labile (except- enterotoxin
of S.aureus )
Heat stable ( upto 250⁰c)
Highly antigenic Less antigenic
Highly toxic in minute dose (
microgram is fatal to animals)
Moderately toxic
Filterable not so(obtained only by cell
lysis )
Can be converted to toxoid cannot
Often enzymatic action no
Properties of bacterial toxin

125. Santosh Yadav
Endotoxin
125
 LPS of Gram negative cell
has three parts:- O-
antigen , core
polysaccharide and
lipid A.
 Released usually when the
cell is lysed but can also be
released during vegetative
growth.
 Has same chemical
composition in almost all
bacteria and has same
toxic effect ( no matter
which bacteria produce it).
 Encoded by chromosomal
gene.

126. Santosh Yadav
Mode of action
126
Endotoxin
( lipid A)
Activates
macrophages
IL-1/IL-6(fever)
TNF(fever and
hypotension) i
NO
(hypotension)
Activates
complement
C3a
(hypotension ,
edema)
C5a
(neutrophil
chemotaxis)
Activates
tissue factor
Coagulation
cascade ( DIC)

127. Santosh Yadav
Exotoxin
127
 Are most powerful and active in small quantities.
 Either secreted by organism or leak into the
surrounding fluid after lysis of bacterial cell.
 Gene for exotoxin may be present on
chromosome or plasmid or bacteriophage
DNA.

128. Santosh Yadav
128

129. Santosh Yadav
A-B toxin
129
 Have two components (A
and B)
 B components binds to
specific cell receptor and
facilitate the internalization
of A.
 Component A is active
(toxic) component.

130. Santosh Yadav
Superantigen
130

131. Santosh Yadav
Contd…
131
 Some bacteria directly
inject exotoxin into
target cell via needle
like projections called
injectosome.
 Also called type III
secretory system.
 Bacteria having type III
secretory system are
more virulent.
Fig:- Injectosome

132. Santosh Yadav
BACTERIOCIN
132

133. Santosh Yadav
BACTERIOCIN
133
 Produced by many bacteria, protein in nature and
have bactericidal activity.
 Have killing action on strains of same or closely
related species.
 First reported by Gratia in 1925, Escherichia coli
producing a substances which is active against other
strains of the same species.
 Colicin – Gratia and Frederique in 1946.
 Bacteriocin- Jacob and Woolman in 1953.

134. Santosh Yadav
contd…
134
 Are named on the basis of their bacterial species of
origin . Some of them are
i) Colicins are bacteriocins of E.coli,
ii) Aeruginocin of P. aeruginosa,
iii) Diphthericins - C. diphtheriae
iv) Cloacin of Enterobacter cloaceae,
v) Pesticin of Y. pestis,
vi) Monocin of Listeria monocytogenes,
vii) Cerecin of Bacillus cereus
viii) Staphylococcin of Staphylococcus aureus,
ix) Warnerin of et S. warneri

135. Santosh Yadav
contd…
135
 Target cells have specific receptor for
attachment of bacteriocin( same as
bacterophage)
 Bacteria producing bacteriocin also carry
gene for immunity to them on chromosome or
plasmid.
 Many have narrow inhibitory spectrum of
activity ( but some have activity on broad
class of bacteria)
 Are plasmid or chromosomal mediated.

136. Santosh Yadav
136
Gram negative bacteriocin

137. Santosh Yadav
Gram positive bacteriocin
137

138. Santosh Yadav
Mode of action
138

139. Santosh Yadav
Importance
139
 Bacteriocins produced by non-pathogenic
bacteria kills other pathogenic
bacteria(Normal flora vs. Pathogens).
 Bacteriocins have also been suggested for
certain cancer treatment.
 Used for food presrvation in food industry (
eg..nisin produced by Lactococcus lactis is
active against many food spoiling bacteria)
 Used for bacteriocin typing of clinical strains
to aid in their identification and
characterization.

140. Santosh Yadav
Bacteriocin typing
140
 Done for identification of isolated strains : as they
are same or different.
 A strain may be typed by:
1) Activity of their bacteriocin against a set of indicator
strains of same or closely related species, or
2) Pattern of their susceptibility to the bacteriocin of a
set of indicator strains.
 If the isolates are same strains, their bacteriocin
production and susceptibility patterns will be
identical.

141. Santosh Yadav
Pyocin typing
141
Gillies and Govan(1966)- cross streaking
method
- The test strains are inoculated across the
surface of BHI agar plates.
- After overnight incubation at 37ᴼC , culture is
exposed to chloroform to kill the test strains and
then the test (producing) strains are scrapped-
off from the plate by using slide.
- The sensitive strains (Indicator strains) are
cross-streaked at right angle to the test strains.
- Incubated at 37ᴼC for 24 hours.
- Observed for inhibition of growth at each side of
the producing strain.

142. Santosh Yadav
Contd…
142
 In pyocin typing technique with cross streaking
method, 105 main types and 25 subtypes can be
identified on the basis of pyocin production by
test strains using 13 indicators(1 to 8 and A to E).
Disadvantages of streak method
 To remove test strains growth before application
of indicator strain.
 Not reliable for mucoid colony of P. aeruginosa.
 48 hour period is needed to obtain result.

143. Santosh Yadav
Bibiliography
143
 Microbiology : A Human Perspective by
Anderson and Nester
 Topley and Wilsons Microbiology and Microbial
infections, vol. 2
 A Handbook of Clinical Microbiology ; Prof. Dr.
Bharat Mani Pokhrel
 Microbiology by Tortora
 Ananthanarayan and Paniker Text Book Of
Microbiology
 Brock Biology of Microorganisms; Medigan and
Martinko.
 Microbiology ; Lansing M. Prescott.

144. Santosh Yadav
THANK YOU
144