1

• Bacterial cell wall

• Plasma membrane

• Structures internal to plasma membrane

pps. 77 – 106

The BacteriaThe BacteriaThe BacteriaThe Bacteria4-b 4-b

2

http://www.microbelibrary.org/Gram%20Stain/details.asp?id=2020&Lang=

The Gram Stain: An Animated ApproachMicrobeLibrary Visual: Animation12/16/2005 by Daniel Cavanaugh, Mark Keen

This animation demonstrates at a molecular level the interaction of the Gram's stain reagents in a step-by-step process with both the gram-negative and gram-positive cell envelope.

http://www.microbelibrary.org/Search: Keen

3

Cell wall

Ribosomes

Cell membrane

Plasmid

Granules(inclusions)

DNA

Glycocalyx

FimbriaePili

Flagellum

Cytoplasm

4

• Semi-rigid structure made of peptidoglycan

• Internal to the capsule

• External to the plasma membrane

The Cell WallThe Cell Wall

Capsule

Cell wall Plasma membrane

5

• Maintain cell shape

• Protect bacteria against osmotic lysis

– Rupture of the cell resulting from movement of water into the cell

• In some species, contributes to virulence

• Chemical composition differentiates bacteria

• Site of antibiotic damage

Functions

6

Makes cell walls a good target for antibiotics

Animal cells lack cell walls

– By attacking the integrity of the cell wall

– Preventing peptidoglycan formation, cross-linking

– Example: penicillins, cephalosporins

7

Repeated units of sugars and short chains of amino acids

Peptidoglycan ‘network’

Sugars are:

– N-acetylglucosamine (NAG)

– N-acetylmuramic acid (NAM)

– Run in ‘parallel’ throughout the cell wall

– NAM links to peptide chains

– Holds the whole porous ‘mesh’ together

8

Sugars

Peptides Fig 4.13a

Small black arrows: penicillin action on peptides

9

• Thick (~ 90%) layer of peptidoglycan (20-80 nm)

• Many ‘rows’ of cross-linked peptidoglycan stacked in ‘sheets’

• Teichoic acids, lipoteichoic acid, proteins

• The cell (plasma) membrane underneath the cell wall

Gram Positive Cell Walls

10

• May regulate movement in/out of cell

• Role in growth, prevent breakdown, cell lysis

• Provide antigenic specificity– Making it possible to identify bacteria

Teichoic Acid Functions

11

• Thin layer of peptidoglycan (~ 2 nm)

– Only one to a few ‘rows’ / ‘sheets’

• Contains more protein than Gram +

• No teichoic acids

• Contains an outer membrane

– A lipid bilayer membrane ‘outside’ the cell wall but connected to it

Gram Negative Cell Walls

12

• Contains channels called porins = transport

• Contains lipopolysaccharide (LPS)

• LPS consists of polysaccharide and Lipid A

• Lipid A known as endotoxin, cause fever, shock

• O-polysaccharides – Function as antigens, cause various diseases– Useful to distinguish Gram negative bacteria– Example: E. coli O157:H7 (serovar)

Outer Membrane Functions

13

14

Gram Stain: Differential stain… used as an initial step to identify bacteria

Step Gram Positive Gram Negative

Crystal violet Dye enters cytoplasm Dye enters cytoplasm

Iodine Forms crystal with CV, too large to escape cell wall

Forms crystal with CV, too large to escape cell wall

Alcohol Dehydrates peptidoglycan, impermeable to CV-I

Dissolves outer membrane, makes holes in thin peptidoglycan, CV-I can diffuse out of cell

Color after alcohol Purple Colorless

Safranin Purple (pink is masked by the purple dye)

Pink

15

16

17

The plasma membrane is inside the cell wall and encloses the cytoplasm

Cell MembraneCell Membrane

• Phospholipid bilayer, Fluid Mosaic Model

– Similar to eukaryotes 50:50 protein to lipid

• Proteins include– Peripheral, integral proteins– Transmembrane proteins (active & passive

transport)

18Fig 4.14a

Review Membrane Proteins

19

– Production of cell wall components

– DNA replication

– Cell respiration (ATP generation via ETS)

– Act as selective or semi-permeable barrier

• Control movement of substances into & out of cells

Cell Membrane Functions

20

Antimicrobial agents damage the cell membrane

Cell Membrane Destruction

AlcoholsQuaternary ammonium compounds

Used as disinfectants

Antibiotics disrupt the membrane integrity

Example: polymyxins Causes leakage of cell contents

Use for Gram negative bacteria, e.g., Pseudomonas

21

Refers to all the contents w/in cell membrane

CytoplasmCytoplasm

– Water (~80%)– Proteins, enzymes– Inorganic and organic molecules– Nuclear area (nucleoid) containing

DNA– Ribosomes– Inclusions (granules)– Plasmids

22

Nuclear area (nucleoid)Nuclear area (nucleoid)

• Nuclear area is where the DNA is located– Single circular molecule of double stranded

DNA

• The nucleoid is NOT a membrane-enclosed region

Fig 4.6a

nucleus-like

23

24

RibosomesRibosomes

• Found in the cytoplasm not the nucleoid area

• Organelles responsible for synthesizing proteins

• Consist of protein and rRNA– Smaller than ribosomes in eukaryotic cells

• 70S (50S & 30S subunits)

25Fig 4.19

• Bind to either the 30S or 50S subunits

• Cause faulty protein synthesis

> 30S:– Streptomycin– Gentamicin – Tetracyclines

> 50S:– Erythromycin – Chloramphenicol

Antibiotic Inhibition

26

PlasmidsPlasmids

• Circular extrachromosomal DNA

• Double-stranded DNA

• Replicates autonomously

• Easily passed from bacterium to bacterium

• Plasmid genes are not necessary for the survival of the bacterium

• Carry genes coding for AB resistance

27

• Carry genes coding for AB resistance– Called resistance factors (R factor, p 246)

– Readily shared with other bacterial cells– Spread AB resistance

• Have significant medical importance

• Examples: Streptomycin, Chloramphenicol, Tetracycline

Antibiotic Resistance

28

InclusionsInclusions

Reserve deposits in the cells

Fig 4.20

29

Metachromatic granules (volutin)

Polysaccharide granules

Lipid inclusions

Sulfur granules

Carboxysomes

Gas vacuoles

Magnetosomes

Phosphate reserves

Energy reserves

Iron oxide (destroys H2O2)

Regulate buoyancy

Ribulose 1,5-diphosphate carboxylase, CO2 fixation

Energy reserves

Energy reserves

Inclusions Purpose/Function

30

EndosporesEndospores

Dormant ‘alternate’ life forms

Bacillus Clostridium

Live in soil

Obligate aerobes Obligate anaerobes

Normal flora: animal GI tract

• Under conditions of starvation (carbon, nitrogen)

– A spore forms w/in some of the bacteria

• Process called:Sporulation

31

Sporulation Sporulation

1. 2. 3.

4.

5.

6.

7.8.

9.

10.

See Fig. 4.21

32

The completed endospore consists of– Multiple layers of resistant coats

• Cortex, spore coat, sometimes an exosporium

– Nucleoid – Some ribosomes– RNA molecules– Enzymes

Structure

33

Not a reproductive structure

It is a resistant, dormant survival form

Functions

– Resistant to high temperatures– Most AB’s, disinfectants– Low energy radiation– Drying, etc.

• Can survive > thousands of years• Until environmental stimuli trigger

– Germination

34

Harmless until they germinate

But, are involved in transmission of some diseases to humans

Anthrax: Bacillus anthracis

Tetanus: Clostridium tetani

Botulism: Clostridium botulinum

Gas gangrene: Clostridium perfringens

Diseases

35

Bacillus anthracis Clostridium tetani

Clostridium botulinum

36

• Botulism caused by C. botulinum = normal flora of GI grazing animals

• Home-canned beans– Boiled, placed in jar, lids screwed on– The lids ‘popped’ indicating a vacuum formed– Upon eating beans, person contracted

botulism– Explain?

Endospore Case Study

37

• Endospores of C. botulinum survive in soil

– C. botulinum is an obligate anaerobe

• Endospores contaminating beans are – Resistant to boiling and survive– Once vacuum is formed, environment is

anaerobic– Spores are able to germinate– Vegetative cells replicate, secrete exotoxin

• Cause botulism

38

Q’sQ’s

A. Techoic acidsB. PeptidoglycanC. Porins

1. Which is NOT found in the cell walls of gram-negative bacteria?

2. Which is an incorrect pairing?

A. Metachromatic granules : polyphosphate

B. Carboxysomes : carbon dioxide fixation

C. Lipid inclusions : b-hydroxybutyrate

D. Plasmids : nucleotide reserves

E. Magnetosomes : iron oxide

D. LipopolysaccharidesE. Outer membrane

39

1.2.

3.

4.

Q’sQ’s

40

1. __________ are the primary determinants of membrane structure, while ____________ carry out membrane function.

2. Name three infections transmitted to humans by bacterial endospores.

a. __________ c. __________b. __________

Q’sQ’s

3. The following bacterial structures increase the likelihood that bacteria will be associated with disease: capsules, fimbriae, flagella, LPS.

True False

41

Appendix Appendix

42

Name the cell type:_________Name the cell type:_________ 1.

2.

3.

4.

5.

6.

7.

43

Name the cell type:_________Name the cell type:_________ 1.

2.3.

4.

5.

6.

44

‗ 1. Cell wall‗ 2. Endospore‗ 3. Fimbriae‗ 4. Flagella‗ 5. Glycocalyx‗ 6. Pili‗ 7. Plasma membrane‗ 8. Ribosomes

a. Attachment to surfacesb. Contains transmembrane

proteinsc. Motilityd. Protection from changes in

water pressuree. Protection from phagocytesf. Resting g. Protein synthesish. Susceptible to leakage of cell

contents by polymyxini. Transfer of genetic material

Structures

Functions

Q’sQ’s

Match Structures to their Functions

45

‗ 1. Inclusion ‗ 2. Volutin ‗ 3. Endospore ‗ 4. Plasmid ‗ 5. Ribosome ‗ 6. Forespore ‗ 7. Nucleoid ‗ 8. Sporulation

a. A structure consisting of chromosome, cytoplasm and endospore membrane inside a bacterial cell

b. The process of spore and endospore formation; also called sporogenesis

c. Endoflagellum d. Stored inorganic phosphate in a prokaryotic

cell e. Material held inside a cell, often consisting of

reserve deposits f. A resting structure formed inside some

bacteria g. The region in a bacterial cell containing the

chromosome h. A small circular DNA molecule that replicates

independently of the chromosome i. The site of protein synthesis in a cell,

composed of RNA and protein

Match Structures to their Functions

Structures

Functions Q’sQ’s

46

1. A vegetative cell about to enter endospore cycle2. A spore septum forms as the cytoplasmic membrane

invaginates3. Nucleoid becomes surrounded by membrane4. Cytoplasmic membrane surrounds isolated nucleoid,

cytoplasm & membrane from step 35. The forespore is completed & other DNA molecule is

degraded6. Thick protective layer, peptidoglycan, called the cortex, is

synthesized between inner & outer forespore membranes7. A second impermeable proteinaceous protective layer

called the spore coat is synthesized8. Sometimes a final layer, exosporium may be added9. Vegetative portion of cell degrades, completed endospore

released10. Bacterial endospore is completed

Sporulation Steps