Microbiology 微生物学 Spring-Summer

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2 Microbiology 微生物学 2017 Spring-Summer

3 Relevant Information and Resources Course slides can be found at 教学工作 Course-related questions will be answered through s. Textbook: Brock Biology of Microorganisms (13 th )

4 Metabolic Diversity Energy: PHOTOTROPHS (use light as energy source) Carbon: CO 2 Organic C Photoautotrophs Photoheterotrophs Heterotroph: Carbon source is NOT CO 2 ; Autotroph: Carbon source is CO 2 ;

5 Metabolic Diversity Energy Sources Chemicals Light Aerobe: with oxygen; Organic chemicals (glucose, acetate, etc.) Chemotrophy Chemoorganotrophs Inorganic chemicals (H 2, H 2 S, Fe 2+, NH 4+, etc.) Phototrophy Chemolithotrophs Phototrophs (glucose O 2 CO 2 H 2 O) (H 2 O 2 H 2 O) (light) Anaerobe: without oxygen; Facultative: with or without oxygen; Only In Prokaryotes

6 Cell Envelope

7 Cell Envelope Cell Envelope O-polysaccharide Core polysaccharide Lipid A Protein Out Cell wall Outer membrane Porin Lipopolysaccharide (LPS) 8 nm Periplasm Cytoplasmic membrane Peptidoglycan Phospholipid Lipoprotein In

8 Cytoplasmic Membrane

9 Cytoplasmic Membrane 6 8 nm Phospholipids Out Hydrophilic groups Hydrophobic groups In Integral membrane proteins Phospholipid molecule

10 Cytoplasmic Membrane - Function 1. Permeability Barrier Prevents leakage and functions as a gateway for transport of nutrients into and out of the cell 2. Protein Anchor Site of many proteins involved in transport, bioenergetics, and chemotaxis 3. Energy Conservation Site of generation and use of the proton motive force

11 Transport and transport systems Simple transport: Driven by the energy in the proton motive force Out In Phosphoanhydride Phosphate ester Ribose Transported substance Group translocation: Chemical modification of the transported substance driven by phosphoenolpyruvate 1 ABC transporter: Periplasmic binding proteins are involved and energy comes from ATP 2 3 Phosphates Adenosine TriPhosphate (ATP) Adenine The three classes of membrane transport systems

12 Cell Wall

13 Cell Wall O-polysaccharide Core polysaccharide Lipid A Protein Out Cell wall Outer membrane Porin Lipopolysaccharide (LPS) 8 nm Periplasm Cytoplasmic membrane Peptidoglycan Phospholipid Lipoprotein In

14 Cell Wall Gram-positive Peptidoglycan Gram-negative Outer membrane Cytoplasmic membrane Protein Protein Peptidoglycan Cytoplasmic membrane Cytoplasmic membrane Peptidoglycan Outer membrane

15 Cell Wall Gram Negative

16 Cell Wall Gram Positive

17 Cell Wall Escherichia coli Bacillus subtilis

18 Cell Wall Peptidoglycan (PG) -- Only in bacteria, over 100 different PG; -- Missing in some bacteria, such as Mycoplasmas

19 Cell Wall - PG N-Acetylglucosamine (G) N-Acetylmuramic acid (M) Invariable (1,4) (1,4) (1,4) N-Acetyl group Variable Peptide cross-links Lysozymesensitive bond L-Alanine D-Glutamic acid Meso-diaminopimelic acid D-Alanine Gram negative Structure of the repeating unit in peptidoglycan

20 Cell Wall - PG Peptidoglycan in Escherichia coli and Staphylococcus

21 Cell Wall - PG

22 Cell Wall - PG N-Acetylglucosamine (G) N-Acetylmuramic acid (M) (1,4) (1,4) (1,4) N-Acetyl group Gram Positive Peptide cross-links Lysozymesensitive bond L-Alanine D-Glutamic acid Meso-diaminopimelic acid D-Alanine Structure of the repeating unit in peptidoglycan Diaminopimelic acid Lysine Cross-linking amino acids in peptidoglycan

23 Cell Wall - PG Only in Gram Positive Common to have teichoic acids embedded in the cell wall (lipoteichoic acids, with covalently bound membrane lipids)

24 Cell Wall - PG Target of some antibiotics, such as penicillin - biosynthesis, and lysozyme glycosidic bond

25 Cell Wall - PG Ribitol (replaceable by glycerol) Teichoic Acid

26 Cell Wall - PG Teichoic Acid biosynthesis and transport

27 Cell Wall - PG LipoTeichoic Acid

28 Lysozyme and Protoplasts Wall Membrane Lysozyme digests wall H 2 O enters Lysis H 2 O enters H 2 O enters Low solute solution Lysozyme digests wall Protoplasts and their formation: Lysozyme breaks the -1, 4, glycosidic bonds in peptidoglycan Protoplast Isotonic solute solution

29 PG Synthesis

30 Cell Wall the Outer Membrane -LPS replaces most of phospholipids in outer half of outer membrane - Endotoxin: the toxic component of LPS (Lipid A)

31 Cell Wall the Outer Membrane O-specific polysaccharide Core polysaccharide Lipid A X n

32 Cell Wall the Outer Membrane Lipid A: 1. Variable in different bacteria 2. Glucosamine unit 3. β(1 6)

33 The Outer Membrane O polysaccharide Core polysaccharide Lipid A Protein Out Lipopolysaccharide (LPS) Cell wall Outer membrane 8 nm Porin Periplasm Peptidoglycan Phospholipid Cytoplasmic membrane In Lipoprotein -Porins : channels for movement of hydrophilic lowmolecular weight substance - Abundant

34 The Periplasm - 15 nm wide - Contents have gel-like consistency - Houses many proteins

35 Cell Walls of Archaea Lysozyme-insensitive (1,3) N-Acetyl group N-Acetylglucosamine (G) N-Acetylmuramic acid (M) (1,4) (1,4) (1,4) N-Acetylglucosamine N-Acetyltalosaminuronic acid N-Acetyl group Peptide cross-links Peptide cross-links Lysozymesensitive bond L-Alanine D-Glutamic acid Pseudomurein - Typically no outer membrane - Pseudomurein (T vs. M; L-amino acids only) - Cell walls of some Archaea lack pseudomurein Meso-diaminopimelic acid D-Alanine

36 Part 3 Part 2 Part 1 Cell Walls of Archaea UA: uronic acid Glu: Glucose Gal: Galactose Gly: Glycine GluNac: N- acetylglucosamine GlaNac: N- acetylgalactosamine GulNUA: N- acetylgulosaminuron ic acid Polysaccharide cell walls of Archaea Man: Mantose

37 Cell Walls of Archaea The outermost layer Possible function: Protection layer from osmotic lysis; Selective sieve Protein anchor S-layer (proteins or glycoproteins) found in most Archaea and some Bacteria

38 Cell Walls of Archaea Methanomicrobia bacterium Aquaspirillum serpens

39 Other Cell Surface Structure and Inclusions

40 Other Cell Surface Structures Bacterial capsules and Slime layers (a) Acinetobactor by negative staining cells with India ink. (phase contrast) Cell Capsule (b) Rhodobacter capsulatus (TEM) (c) Rhizobium trifolii stained with ruthenium red. (TEM)

41 Other Cell Surface Structure 1. Capsule 2. Slime layer

42 Other Cell Surface Structures Flagella Fimbriae Fimbriae (Salmonella) -Filamentous protein structures -Enable organisms to stick to surfaces or form pellicles

43 Other Cell Surface Structures Pili (Escherichia coli) Viruscovered pilus

44 Motility on agar plates Flagellum & Swimming Agar < 0.3% Flagellum & Swarming 0.3% < Agar < 0.5% Type IV Pili & Twitching Agar 1%

45 Other Cell Surface Structures Type IV Pili & Twitching

46 Cell Inclusions PHB Poly- -hydroxyalkanoate (PHA) 聚羟基脂肪酸酯

47 Cell Inclusions -carbon Poly- hydroxylbutyrate (PHB), a common PHA, Rhodovibrio sodomensis Stained by Nile red Polyhydroxyalkanoate Also Glycogen, a polymer of glucose

48 Cell Inclusions Polyphosphate (a) Polyphosphate granules in Heliobacterium modesticaldum (b) Sulfur globules. Isochromatium buderi (in the periplasm) Sulfur

49 Cell Inclusions Magnetotactic bacteria and magnetosomes a. Coccoid magnetotactic bacteria b. Magnetosomes from Magnetaspirillum magnetotacticum c. Magnetosomes from a magnetic coccus Magnetotactic is not genuine tactic!

50 Cell Inclusions Cyanobacteria and Gas vesicles

51 Cell Inclusions Ribs Gas vesicle proteins GvpA GvpC

52 Endospores Bacillus & Clostridium The bacterial endospores

53 Endospores

54 Germination Sporulation and Endospores Vegetative cell Sporulating cell Developing spore Mature spore The life cycle of an endospore-forming bacterium

55 Endospores a. Endospore b. Refractility is being lost c. & d. The new vegetative cell is emerging. Endospore germination in Bacillus

56 Endospores Exosporium Spore coat Core wall Cortex DNA Structure of the bacterial endospore a. TEM b. Fluorescent by malachite green

57 Endospores (a) Dipicolinic acid (DPA) (b) Ca 2+ crosslinks DPA Carboxylic acid groups Small acid-soluble proteins (SASPs) a. Transforming DNA into more compact A form, enhancing resistance; b. Carbon and energy source during germination

58 Endospores Sporulation in Bacillus subtilis: A, B, C, D, Wild-type; E, F, G, H, pyruvate dehydrogenase E1 deletion strains; I, J, K, L, E2 deletion strains Stained by FM4-64 (Red) and/or MTG (Green). DAPI was used to stain chromosomes

59 Sporulation and Endospores Coat Free endospore Maturation, cell lysis Spore coat, Ca 2 uptake, SASPs, dipicolinic acid Growth Germination Stage VI, VII Stage V Cortex Vegetative cycle Cell division Asymmetric cell division; commitment to sporulation, Stage I Sporulation stages Cell wall Cytoplasmic membrane Cortex formation Stage IV Prespore Septum Engulfment Mother cell Stage II Stage III

60 Spore Companioned Crystal Parasporal Endotoxins of B. thuringiensis

61 Spore Companioned Crystal

62 Endospores B. anthracis and Anthrax

63 Microbial Locomotion

64 Microbial Locomotion

65 Flagella and Motility Bacterial flagella. (a) Peritrichous, (b) Polar, (c) Lophotrichous

66 Flagella and Motility Rhodospririllum centenum (a)polar, (b)peritrichous

67 Flagella and Motility Flagella tuft Amphitrichous Rhodospririllum photometricum Lophotrichous

68 Flagella and Motility Shewanella oneidensis Bacillus subtilis

69 Flagella and Motility Flagellum in bacteria.

70 Flagella and Motility 14 nm Filament Flagellin L P MS Structure and function Outer membrane (LPS) Hook Proton gradient Sodium gradient L Ring 1000 H + /turn. Periplasm P Ring Rod Peptidoglycan MS Ring Rod MS Ring Mot protein C Ring Fli proteins (motor switch) Mot protein C Ring Mot protein 45 nm

71 Archaeal Flagella Thinner and ATP-dependent (From limited studies),

72 Flagella and Motility Hookfilament junction Filament synthesis Outer membrane MS/C ring Motor (Mot) proteins P ring L ring Early hook Cap Late hook Filament Peptidoglycan Cytoplasmic membrane Flagella biosynthesis

73 Assembly of Flagella

74 Flagella and Motility Reversible flagella Bundled flagella (CCW rotation) Peritrichous Tumble flagella pushed apart (CW rotation) Flagella bundled (CCW rotation) CCW rotation Unidirectional flagella CW rotation Polar CW rotation Cell stops, reorients CW rotation Movement in peritrichous and polarly flagellated prokaryotes

75 Gliding Motility Gliding bacteria. a. Oscillatoria princeps b. O. princeps on an agar surface c. Flavobacterium johnsoniae d. Non-gliding mutant

76 Gliding Motility In Cytoplasmic membrane Peptidoglycan Outer membrane Out Movement of cell Movement of outer membrane proteins Surface Gliding motility in Flavobacterium johnsoniae

77 Gliding Motility Gliding motility in Flavobacterium johnsoniae

78 Gliding Motility

79 Gliding Motility

80 Gliding Motility

81 Microbial Taxes

82 Microbial Taxes - Cell Motion as a Behavioral Response Tumble Run Tumble Attractant Run No attractant present: Random movement Attractant present: Directed movement Chemotaxis in a peritrichously flagellated bacterium such as Escherichia coli

83 Chemotaxis Repellants Transducer (MCP) Cell wall Flagellar motor Cytoplasm Cytoplasmic membrane Interactions of MCPs, Che proteins, and the flagellar motor in Bacterial chemotaxis.

84 Cells per tube Chemotaxis Measuring chemotaxis using a capillary tube assay. Attractant Control Repellent Time Tracks of motile bacteria in seawater swarming around an algal cell

85 Chemotaxis

86 Phototaxis Wavelength nm Light Phototaxis of phototrophic bacteria a. Thiospirillum jenense b. Rhodospirillum centenum Time (h)

87 This Concludes Chapter 3!