1 Bacterial cell wall Plasma membrane Structures internal to plasmamembrane pps. 77 – 106 The Bacteria 4- b

2 The Gram Stain: An Animated Approach The Gram Stain: An Animated Approach MicrobeLibrary Visual: Animation 12/16/2005 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. Search: Keen

3 Cell wall Ribosomes Cell membrane Plasmid Granules (inclusions) DNA Glycocalyx Fimbriae Pili Flagellum Cytoplasm

4 Semi-rigid structure made of peptidoglycan Internal to the capsule External to the plasma membrane The Cell Wall Capsule Cell wallPlasma 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

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14 Gram Stain: Differential stain… used as an initial step to identify bacteria StepGram PositiveGram Negative Crystal violetDye enters cytoplasm IodineForms crystal with CV, too large to escape cell wall AlcoholDehydrates peptidoglycan, impermeable to CV-I Dissolves outer membrane, makes holes in thin peptidoglycan, CV-I can diffuse out of cell Color after alcoholPurpleColorless SafraninPurple (pink is masked by the purple dye) Pink

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17 The plasma membrane is inside the cell wall and encloses the cytoplasm Cell Membrane Phospholipid bilayer, Fluid Mosaic Model –Similar to eukaryotes 50:50 protein to lipid Proteins include –Peripheral, integral proteins –Transmembrane proteins (active & passive transport)

18 Fig 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 Alcohols Quaternary 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 Cytoplasm –Water (~80%) –Proteins, enzymes –Inorganic and organic molecules –Nuclear area (nucleoid) containing DNA –Ribosomes –Inclusions (granules) –Plasmids

22 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

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24 Ribosomes 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)

25 Fig 4.19 Bind to either the 30S or 50S subunits Cause faulty protein synthesis >30S: –Streptomycin –Gentamicin –Tetracyclines >50S: –Erythromycin –Chloramphenicol Antibiotic Inhibition

26 Plasmids 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 Inclusions 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 Inclusions Purpose/Function

30 Endospores Dormant ‘alternate’ life forms BacillusClostridium 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 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’s A.Techoic acids B.Peptidoglycan C.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.Lipopolysaccharides E.Outer membrane

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

41 Appendix

42 Name the cell type: _________

43 Name the cell type: _________

44 ‗1. Cell wall ‗2. Endospore ‗3. Fimbriae ‗4. Flagella ‗5. Glycocalyx ‗6. Pili ‗7. Plasma membrane ‗8. Ribosomes a.Attachment to surfaces b.Contains transmembrane proteins c.Motility d.Protection from changes in water pressure e.Protection from phagocytes f.Resting g.Protein synthesis h.Susceptible to leakage of cell contents by polymyxin i.Transfer of genetic material Structures Functions Q’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’s

46 1.A vegetative cell about to enter endospore cycle 2.A spore septum forms as the cytoplasmic membrane invaginates 3.Nucleoid becomes surrounded by membrane 4.Cytoplasmic membrane surrounds isolated nucleoid, cytoplasm & membrane from step 3 5.The forespore is completed & other DNA molecule is degraded 6.Thick protective layer, peptidoglycan, called the cortex, is synthesized between inner & outer forespore membranes 7.A second impermeable proteinaceous protective layer called the spore coat is synthesized 8.Sometimes a final layer, exosporium may be added 9.Vegetative portion of cell degrades, completed endospore released 10.Bacterial endospore is completed Sporulation Steps