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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
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• 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
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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
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• 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