the bacterial structures growth & culture of bacteria
TRANSCRIPT
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The Bacterial Structures
Growth & Culture of Bacteria
Di Qu (瞿涤)
MOH&MOE Key Lab of Medical Molecular Virology
School of Basic Medical Sciences
Shanghai Medical College of Fudan University
复旦大学上海医学院分子病毒学
教育部/卫生部重点实验室
Chapter 2, 4, 5
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Key Words
Prokaryotic
Eukaryotic
Eubacteria (Bacteria)
Archaebacteria (Archaea)
Chromosome
Gram staining
Gram negative
Gram positive
Cell wall
Peptidoglycan
(murein, mucopeptide)
Outer membrane (LPS )
Cell membrane
Spheroplast/protoplast
L form
Flagella (Chemotaxis)
Pili (fimbriae)
Capsule (slime layer, glycocalyx)
Spore (resistant)
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Characteristic Prokaryotic Eukaryotic Drug targets?
Size (diameter) 0.1-2.0 mm 10-100 mm homework
Nucleus Nucleoid,
no nucleoli, no membrane
Nucleus
Organelles Absent Present
Glycocalyx Capsule or slime layer In some cell
Cell wall Usually present
-peptidoglycan
Most no
-celluose /chitin
Plasma
membrane
Lack cholesterol cholesterol
Ribosome 70S : 30S (16S rRNA)
50S (5S & 23S rRNA)
80S 40S/60S
70S(mitochondria)
S=sedimentation
coefficient
Chromosome Single circular, Haploid Diploid
Cell division Binary fission Mitosis
Sexual Rec. No,
Horizontal transfer of
DNA
Meiosis
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Size of Bacteria Average bacteria 0.5 - 2.0 um in (- microscope)
-- RBC is 7.5 um in diam.
Surface Area to Volume is 3:1
-- Typical Eukaryote Cell SA vs. Vol is 0.3:1
Nutrition enters through surface area, quickly reaches all
parts of bacteria
-- Eukaroytes need structures & organelles
Light microscope: Bright –field microscope
100xobjective lens Phased contrast microscope
10x ocular lens Dark-field microscope
Fluorescence microscope
Electron microscope
Scan electron microscope
Confocal microscope
Bacteria are transparent
Chapter 2, p. 9
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Spiral:
Spirilla, Spirillum
Rod-shaped:
bacilli, bacillus
Round:
Cocci, coccus
Bacteria are classified by shape into 3 groups:
Shapes of Bacteria
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Shapes of Bacteria
• Coccus
– Chain = Streptoccus
– Cluster = Staphylococcus
– Diplopcoccus
• Bacillus
– Chain = Streptobacillus
• Coccobacillus
• Vibrio = curved
• Spirillum
• Spirochete
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Bacillus
Spiral bacterium
Vibrio Spirillum Helicobaterium
Spirochete
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Bacterial Structures
• Cell Wall
-Lipopolysaccharides
-Teichoic Acids
• Cell Membrane & Cytoplasm
-Inclusions
• Ribosomes
• Nucleoid
-Chromosome & Plasmids
• Capsule
• Flagella
• Pili
• Spores
Chapter 2
Every bacterium has
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The Cell Wall
Gram Positive Gram Negative
1884
Hans Christian Gram
- outside of cell
membrane
- rigid, protecting cell
from osmotic lysis
Gram staining
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Gram’s Serendipitous Stain,
still forms the basis for
identification of bacteria
The Cell Wall
“I am aware that as yet it is very
defective and imperfect”
Hans Christian Gram
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Chapter 2 p. 24Gram stain related with cell wall
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Gram -
Gram +
Cell wall
-peptidoglycan
NucleoidCell membrane
Flagellum
Cell (inner) membrane Outer membraneRibosomes
Granule
Cell wall
-peptidoglycan
Capsule
Pili
The Cell Wall
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G+ Bacteria (~90%) G- Bacteria(~10%)
PeptidoglycanThe Cell Wall
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Peptidoglycan
More than 40 sheets
in Gram positive bacteria
Only 1-2 sheets
In Gram negative bacteria
The Cell Wall
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• Peptidoglycan Polymer (amino acids + sugars)
• Unique to bacteria
• Sugars; NAG & NAM (backbone)
- N-acetylglucosamine
- N-acetylmuramic acid
(The same in all bacterial species)
• Tetrapeptide (vary from species)
-D form of Amino acids (not L form)
D form aa. is hard to be break down
• Pentapeptide (vary from species)
- cross link with tetrapeptide over NAG & NAM as 3D
PeptidoglycanThe Cell Wall
Glycosidic bond
b-1,4 linkage
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Fig. 2-16
Lysozyme target
Where
lysozyme
exits?
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G- Peptidoglycan
G+ Peptidoglycan
Peptidoglycan recognition protein, PGRP
Pentapeptide (vary from species)
meso-diaminopimelic acid
m-Dpm
DD-transpeptidase
DD-transpeptidase
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Structure of penicillin DD-transpeptidase cannot
catalyze formation of the cross-
links, and an imbalance
between cell wall production
and degradation develops,
resulting in the cell die rapidly.
Penicillin irreversibly binds to
DD-transpeptidase
DD-transpeptidase catalyzes
cross-links of tetrapeptides
and Pentapeptide
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Synthesis of cell wall is inhibited
-bacteria undergoing cytolysis
Sensitive to osmotic pressure
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20Cytoplasm
Cytoplasmic membrane
GRAM POSITIVE CELL WALL
Special components: Teichoic acid TA
Teichoic acid (WTA wall associated)
Lipoteichoic acid (LTA, membrane associated)
-Negatively charged
-Surface antigen, attachment of bacteria to animal cells
Lipoteichoic acid Peptidoglycan-teichoic acid
Peptidoglycan
Figure 2-16 p.24
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Fig. 2-17
(WTA wall associated)LTA, membrane associated
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GRAM NEGATIVE CELL WALL
Cytoplasm
Inner (cytoplasmic) membrane
Outer Membrane
(Major permeability barrier) LipopolysaccharidePorin
Braun lipoprotein
Periplasmic binding proteinPermease
Special components: outer membrane,LPS,lipoprotein
Peptidoglycan
Figure 2-17 p.25
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23Fig. 2-18
A lipid component
of endotoxin
(LPS=
endotoxin)
G-
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Chemical structure of lipid A in E. Coli
Lipid A (LPS) has been
demonstrated to activate cells
via Toll-like receptor 4 (TLR4),
MD-2 and CD14 on the cell
surface
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Outer Membrane
Gram negative bacteria
• major permeability barrier
• space between inner and outer membrane
– periplasmic space
store degradative enzymes
b-lactamas
• Gram positive bacteria : no periplasmic space
β-Lactam antibiotics
b-lactamas
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Wall-less forms (bacteria)
• Result from action of:
- Lysozyme lytic for cell wall
- antibiotics block peptidoglycan biosynthesis
• In osmotically protective media (isotonic)
- spheroplasts (with outer membrane)
- protoplasts (no outer membrane)
• If wall-less bacteria can grow and divide
– L forms bacteria chronic infection
Induced by antibiotic (penicillin…)
-resistant to antibiotic treatment
-reversion (to normal with wall)
-relapses of the overt infection drug resistant
G-
G+
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Wall-less forms (bacteria)
Polymorphic
G+ G-
Grow slowly
Coney as tried egg
StaphylococcusStaphylococcus
L form
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Cell Wall Summary
• Unique to bacteria
• Determine shape of bacteria
(L form bacteria’s shape ?)
• Strength prevents osmotic rupture
• G+ -peptidoglycan +TA
• G- -out membrane (LPS) + peptidoglycan
• Antibiotics targets, some antibiotics effect
directly:
– Lysozyme (disrupt peptidoglycan)
– Penicillin (Inhibit peptidoglycan synthesis)
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Cell Membrane
• Bilayer Phospholipid
• Water can penetrate
• Exchange material
• Flexible
• Not strong, ruptures easily
– Osmotic pressure created by cytoplasm
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Cytoplasm
• 80% Water, 20% Salts-Proteins)
– Osmotic Shock important
• Inclusion body
-granules for identification of bacteria
• Chromosome
• Plasmids
• No organelles (Mitochondria, Golgi, etc.)
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Nuclear material (nucleoid)
Chromosome
circular, Haploid
Advantages of 1N DNA over 2N DNA
-more efficient, grows quicker
-mutations allow adaptation to environment quicker
Plasmids
Extra-chromosomal DNA
Independent replication
multiple copy number,horizontal transfer
coding
- pathogenesis factors
- antibiotic resistance factors superbug
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Nucleoid
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Ribosome
• Protein synthesis;• Targets of antibiotics
70S :
30S (16S rRNA)
50S (5S & 23S rRNA)
Erythromycin
Streptomycin
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Procaryotic ribosome
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Antibiotics target to
bacteria ribosome
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Bacterial Structures
• Cell Wall
-Lipopolysaccharides
-Teichoic Acids
• Cell Membrane & Cytoplasm
-Inclusions
• Ribosomes
• Nucleoid
-Chromosome & Plasmids
• Capsule
• Flagella
• Pili
• Spores
Chapter 2some bacteria
All bacteria
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Capsules and slime layers
• Envelope outside cell wall
-Well defined: capsule
-Not defined: slime layer
glycocalyx- polysaccharide on external surface
• Polymer: usually polysaccharide (Table 2-1), but
often lost during in vitro culture
• Protective in vivo
-adhere bacteria to surface
S. mutans (teeth)
-prevent phagocytosis
complement can’t penetrate capsules
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Chapter 2
Flagella
• Some bacteria have Flagella, motile
Arrangement basis for classification
– Monotrichous; 1 flagella
– Lophotrichous; tuft at one end
– Amphitrichous; both ends
– Peritrichous; all around bacteria
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Monotrichous
Amphitrichous
Lophotrichous
Peritrichous
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Flagella
• Locomotory organelles- flagella
• Swarming occurs with some bacteria
-Spread across Petri Dish
Proteus species most evident
• Sense environment
• Chemotaxis
-respond to food/poison
or unfriendly environments
Proteus
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Flagella – embedded in cell membrane
– project as strand
– Flagellin (protein) subunits
– move cell by propeller like action
E. Coli with flagella
Shigela no flagella
Flagella antigen
Hauch
H antigen
O antigen
Ohne Hauchpropeller
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Pili (fimbriae)
• Short protein appendages (hair like)
– smaller than flagella
– pilins (protein)- vary/species
• Common pilus (pili)
• Adhere bacteria to host epithelium
– E. coli has numerous types
• K88, K99, F41, etc.
– Anti-pili antibodies to block adherence
• Flotation, increase boyancy
– Pellicle (scum on water)
– More oxygen on surface
• Sex Pilus, (F-pilus Fertility factor)
-Used in conjugation (“sex” conjugation)
for exchange of genetic information
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F-Pilus for Conjugation
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Endospores (spores)
An endospore is a dormant, tough, and non-
reproductive structure produced by certain bacteria.
Endospore formation is usually triggered by a lack
of nutrients, and usually occurs in Gram-positive
bacteria.
•Dormant cell (non-reproductive structure )
-a thick celled structure formed inside the cell,
-encloses all the nuclear materials and some
cytoplasm
Location important in classification
Central, Subterminal, Terminal
Sporulation (Fig. 2-28)
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The endospore consists of the bacterium's DNA,
ribosomes and large amounts of dipicolinic acid.
Dipicolinic acid is a spore-specific chemical that help in
the ability for maintaining dormancy and resistance.
-comprises up to 10% of the spore's dry weight
Resistant to adverse conditions
desiccation, high temperature, extreme freezing,
irradiation, and chemical disinfectants
Boiling >1 hr still viable
Sterilization, autoclave
Allows the bacteria to survive for many years or
centuries. Viable bacterial spores have been found
that are 40 million years old on the earth.
Endospores
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Endospores
• Produced when starved…under unfavorable condition
Bacillus anthracis form spores in O2
anthrax-corpse - no necropsy
• In suitable condition, endospores activate
Dormant cell germination (vegetative form)
(reproductive form)
• Activation conditions ?
(home work, -related with medical practice, Clostridium
tetani -tetenus )
- Bacillus stearothermophilus -spores
Used for quality control of heat sterilization equipment
- Bacillus anthracis - spores (Bacillus and Clostridium)
Used in biological warfare (“911”)
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Sporulation cells of bacillus species.
Unidentified
bacillus from soil
Bacillus cereus
Related with
foodborne disease
Bacillus megaterium
Used to be a model
organism for Gram-
positive bacteria
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Bacillus anthracis G+
Aerobic spore-forming bacteria
(form spores in O2 )
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Starting1week after the
9/11/2001 attack,
letters containing anthrax
were sent to media offices
and to Senators Tom Daschle
and Patrick Leahy
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Cell wall, G+/G-
Chromosome, Plasmid, Ribosome(70S)
Flagella (Chemotaxis)
Pili (fimbriae)
Capsule (slime layer, glycocalyx)
Spore (resistant)
Summary :
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Growth & Culture of Bacteria
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Figure 2-20