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

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)

3

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

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

Spiral:

Spirilla, Spirillum

Rod-shaped:

bacilli, bacillus

Round:

Cocci, coccus

Bacteria are classified by shape into 3 groups:

Shapes of Bacteria

Shapes of Bacteria

• Coccus

– Chain = Streptoccus

– Cluster = Staphylococcus

– Diplopcoccus

• Bacillus

– Chain = Streptobacillus

• Coccobacillus

• Vibrio = curved

• Spirillum

• Spirochete

Bacillus

Spiral bacterium

Vibrio Spirillum Helicobaterium

Spirochete

Bacterial Structures

• Cell Wall

-Lipopolysaccharides

-Teichoic Acids

• Cell Membrane & Cytoplasm

-Inclusions

• Ribosomes

• Nucleoid

-Chromosome & Plasmids

• Capsule

• Flagella

• Pili

• Spores

Chapter 2

Every bacterium has

The Cell Wall

Gram Positive Gram Negative

1884

Hans Christian Gram

- outside of cell

membrane

- rigid, protecting cell

from osmotic lysis

Gram staining

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

11

Chapter 2 p. 24Gram stain related with cell wall

12

Gram -

Gram +

Cell wall

-peptidoglycan

NucleoidCell membrane

Flagellum

Cell (inner) membrane Outer membraneRibosomes

Granule

Cell wall

-peptidoglycan

Capsule

Pili

The Cell Wall

G+ Bacteria (~90%) G- Bacteria(~10%)

PeptidoglycanThe Cell Wall

Peptidoglycan

More than 40 sheets

in Gram positive bacteria

Only 1-2 sheets

In Gram negative bacteria

The Cell Wall

• 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

16

Fig. 2-16

Lysozyme target

Where

lysozyme

exits?

17

G- Peptidoglycan

G+ Peptidoglycan

Peptidoglycan recognition protein, PGRP

Pentapeptide (vary from species)

meso-diaminopimelic acid

m-Dpm

DD-transpeptidase

DD-transpeptidase

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

Synthesis of cell wall is inhibited

-bacteria undergoing cytolysis

Sensitive to osmotic pressure

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

21

Fig. 2-17

(WTA wall associated)LTA, membrane associated

22

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

23Fig. 2-18

A lipid component

of endotoxin

(LPS=

endotoxin)

G-

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

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

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+

27

Wall-less forms (bacteria)

Polymorphic

G+ G-

Grow slowly

Coney as tried egg

StaphylococcusStaphylococcus

L form

28

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)

Cell Membrane

• Bilayer Phospholipid

• Water can penetrate

• Exchange material

• Flexible

• Not strong, ruptures easily

– Osmotic pressure created by cytoplasm

31

Cytoplasm

• 80% Water, 20% Salts-Proteins)

– Osmotic Shock important

• Inclusion body

-granules for identification of bacteria

• Chromosome

• Plasmids

• No organelles (Mitochondria, Golgi, etc.)

33

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

34

Nucleoid

Ribosome

• Protein synthesis；• Targets of antibiotics

70S :

30S (16S rRNA)

50S (5S & 23S rRNA)

Erythromycin

Streptomycin

36

Procaryotic ribosome

37

Antibiotics target to

bacteria ribosome

Bacterial Structures

• Cell Wall

-Lipopolysaccharides

-Teichoic Acids

• Cell Membrane & Cytoplasm

-Inclusions

• Ribosomes

• Nucleoid

-Chromosome & Plasmids

• Capsule

• Flagella

• Pili

• Spores

Chapter 2some bacteria

All bacteria

39

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

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

Monotrichous

Amphitrichous

Lophotrichous

Peritrichous

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

43

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

44

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

46

F-Pilus for Conjugation

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)

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

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”)

51

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

Bacillus anthracis G+

Aerobic spore-forming bacteria

(form spores in O2 )

Starting1week after the

9/11/2001 attack,

letters containing anthrax

were sent to media offices

and to Senators Tom Daschle

and Patrick Leahy

54

Cell wall, G+/G-

Chromosome, Plasmid, Ribosome（70S）

Flagella (Chemotaxis)

Pili (fimbriae)

Capsule (slime layer, glycocalyx)

Spore (resistant)

Summary :

Growth & Culture of Bacteria

56

Figure 2-20