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微生物學. 許勝傑 博士 長庚大學 生物醫學系 助理教授 E-mail: [email protected] 電話 (03)211-8800#3690. Chapter 1 The Evolution of Microorganisms and Microbiology. CHAPTER GLOSSARY Archaea Bacteria Eukarya Fungi Genome Genomic analysis Koch’s postulates Microbiology Microorganism Prions

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slide1

微生物學

許勝傑 博士

長庚大學

生物醫學系 助理教授

E-mail: [email protected]

電話 (03)211-8800#3690

slide5

Chapter 1

The Evolution of Microorganisms and Microbiology

CHAPTER GLOSSARY

Archaea

Bacteria

Eukarya

Fungi

Genome

Genomic analysis

Koch’s postulates

Microbiology

Microorganism

Prions

Prokaryotic cells

Protists

Spontaneous generation

Viroids

Viruses

Virusoids

Figure/ Table

Summary

slide6

What is microbiology?

  • study of organisms too small to be clearly seen by the unaided eye (i.e., microorganisms)
  • these organisms are relatively simple in their construction and lack highly differentiated cells and distinct tissues
slide7

The Importance of Microorganisms

  • most populous and diverse group of organisms
  • found everywhere on the planet
  • play a major role in recycling essential elements
  • source of nutrients and some carry out photosynthesis
  • benefit society by their production of food, beverages, antibiotics, and vitamins
  • some cause disease in plants and animals

Microbes are estimated to contains 50% of biological carbon and 90% of biological nitrogen on Earth.

slide8

Fig. 1.1 Concept map showing the types of biological entities studied by microbiologists.

slide9
Members of the microbial world
  • prokaryoticcells lack a true membrane-delimited nucleus
  • eukaryoticcells have a membrane-enclosed nucleus, are more complex morphologically and are usually larger than prokaryotic cells

Classification schemes

  • five kingdom scheme includes Monera原核界, Protista, Fungi, Animalia and Plantae with microbes placed in the first three kingdoms
  • three domain alternative, based on a comparison of ribosomal RNA, divides microorganisms into Bacteria(true bacteria),Archaeaand Eucarya (eucaryotes)
discovery of microorganisms
Discovery of Microorganisms
  • Antony van Leeuwenhoek (1632-1723)
    • first person to observe and describe microorganisms accurately
the conflict over spontaneous generation
The Conflict over Spontaneous Generation
  • spontaneous generation
    • living organisms can develop from nonliving or decomposing matter
  • Francesco Redi (1626-1697)
    • disproved spontaneous generation for large animals
    • showed that maggots on decaying meat came from fly eggs
louis pasteur 1822 1895
Louis Pasteur (1822-1895)
  • his experiments
    • placed nutrient solution in flasks
    • created flasks with long, curved necks
    • boiled the solutions
    • left flasks exposed to air
  • results: no growth of microorganisms
koch s postulates
Koch’s postulates
  • The microorganism must be present in every case of the disease but absent from healthy individuals.
  • The suspected microorganism must be isolated and grown in a pure culture.
  • The same disease must result when the isolated microorganism is inoculated into a healthy host.
  • The same microorganism must be isolated again from the diseased host.
slide14

Earliest Molecules - RNA

  • original molecule must have fulfilled protein and hereditary function
  • ribozymes
    • RNA molecules that form peptide bonds
    • perform cellular work and replication
  • earliest cells may have been RNA surrounded by liposomes
slide15

Chapter 2

The Study of Microbial Structure: Microscopy and Specimen Preparation

CHAPTER GLOSSARY

Atomic force microscope

Bright-field microscope

Confocal scanning laser microscope (CSLM)

Dark-field microscope

Differential interference contrast (DIC) microscopy

Differential staining

Fixation

Fluorescence microscopy

Gram stain

Negative staining

Parfocal

Phase-contrast microscope

Refractive index

Resolution

Scanning electron microscope (SEM)

Simple staining

Transmission electron microscope (TEM)

lenses
Lenses
  • focus light rays at a specific place called the focal point
  • distance between center of lens and focal point is the focal length
  • strength of lens related to focal length
    • short focal length more magnification
the light microscope
The Light Microscope
  • bright-field microscope
  • dark-field microscope
  • phase-contrast microscope
  • fluorescence microscope
microscope resolution
Microscope Resolution
  • ability of a lens to separate or distinguish small objects that are close together
  • wavelength of light used is major factor in resolution

shorter wavelength  greater resolution

preparation and staining of specimens

Fixation

  • preserves internal and external structures and fixes them in position
  • organisms usually killed and firmly attached to microscope slide
    • heat fixation – routine use with procaryotes
      • preserves overall morphology but not internal structures
    • chemical fixation – used with larger, more delicate organisms
      • protects fine cellular substructure and morphology
Preparation and Staining of Specimens
  • increases visibility of specimen
  • accentuates specific morphological features
  • preserves specimens
dyes and simple staining
Dyes and Simple Staining
  • dyes
    • Ionizable dyes have charged groups
      • basic dyes have positive charges
      • acid dyes have negative charges
      • chromophore groups
        • chemical groups with conjugated double bonds
  • simple stains
    • a single stain is used
    • use can determine size, shape and arrangement of bacteria
gram staining
Gram staining

Differential Staining

  • divides microorganisms into groups based on their staining properties
  • divides bacteria into two groups based on differences in cell wall structure
electron microscopy
Electron Microscopy
  • beams of electrons are used to produce images
  • wavelength of electron beam is much shorter than light, resulting in much higher resolution
newer techniques in microscopy
Newer Techniques in Microscopy
  • confocal scanning laser (CLSM) microscopy and scanning probe microscopy
  • have extremely high resolution
confocal microscopy
Confocal Microscopy
  • laser beam used to illuminate a variety of planes in the specimen
  • computer compiles images created from each point to generate a 3-dimensional image
  • used extensively to observe biofilms
scanning probe microscopy
Scanning Probe Microscopy
  • atomic force microscope
    • sharp probe moves over surface of specimen at constant distance
    • up and down movement of probe as it maintains constant distance is detected and used to create image
slide28

Chapter 3

Bacteria and Archaea

CHAPTER GLOSSARY

Lipopolysaccharides (LPSs)

Nucleoid

Peptidoglycan

Periplasmic space

Plasmid

Porin proteins

Sex pili

S-layer

Spirillum螺旋菌屬

Spirochete螺旋體

Bacillus

Capsule

Cell envelope

Chemotaxis

Coccus

Endospore

Fimbriae

Fluid mosaic model

Gas vacuole

Glycocalyx

Inclusions

slide29

Common features of bacterial and archaeal cell structure

  • prokaryotes differ from eukaryotes in size and simplicity
    • most lack internal membrane systems
  • prokaryotes are divided into Bacteria and Archaea
  • Bacteria are divided into 2 groups based on their Gram stain reaction
size shape and arrangement
Size, Shape, and Arrangement
  • cocci (s., coccus) – spheres
    • diplococci (s., diplococcus) – pairs
    • streptococci – chains
    • staphylococci – grape-like clusters
    • tetrads – 4 cocci in a square
    • sarcinae – cubic configuration of 8 cocci
  • bacilli (s., bacillus) – rods
    • coccobacilli – very short rods
    • vibrios – resemble rods, comma shaped
  • spirilla (s., spirillum) – rigid helices
  • Spirochetes 螺旋體– flexible helices
  • Mycelium 菌絲– network of long, multinucleate filaments
  • Pleomorphic多形性– organisms that are variable in shape
  • Archaea
    • pleomorphic, branched, flat, square, other unique shapes
slide32

largest –

  • 50 μm in
  • diameter
  • smallest –
  • 0.3 μm in
  • diameter
slide33

Size – Shape Relationship

  • important for nutrient uptake
  • surface to volume ratio (S/V)
  • small size may be protective mechanism from predation掠食
bacterial cell envelope
Bacterial Cell Envelope
  • Plasma membrane
  • Cell wall
  • Layers outside the cell wall

Functions of the plasma membrane

  • encompasses the cytoplasm
  • selectively permeable barrier
  • interacts with external environment
    • receptors for detection of and response to chemicals in surroundings
    • transport systems
    • metabolic processes
fluid mosaic model of membrane structure
Fluid Mosaic Model of Membrane Structure
  • Lipid bilayerin which proteins float

Membrane proteins

  • peripheral proteins
    • loosely associated with the membrane and easily removed
  • integral proteins
    • embedded within the membrane and not easily removed
the asymmetry of most membrane lipids
The asymmetry of most membrane lipids
  • polar ends
    • interact with water
    • hydrophilic
  • nonpolar ends
    • insoluble in water
    • hydrophobic

Bacterial Membranes

  • differ from eukaryotes in lacking sterols
    • do contain hopanoids, sterol-like molecules
  • a highly organized, asymmetric system which is also flexible and dynamic
the bacteria cell wall
The Bacteria Cell Wall
  • rigid structure that lies just outside the plasma membrane

Functions of cell wall

  • provides characteristic shape to cell
  • protects the cell from osmotic lysis
  • may also contribute to pathogenicity
  • very few procaryotes lack cell walls
bacterial cell walls
Bacterial Cell walls
  • bacteria are divided into two major groups based on the response to gram-stain procedure.
    • gram-positive bacteria stain purple
    • gram-negative bacteria stain pink
  • staining reaction due to cell wall structure
peptidoglycan murein structure
Peptidoglycan (肽聚糖)(Murein, 胞壁質) Structure
  • meshlike polymer of identical subunits forming long strands
    • two alternating sugars
      • N-acetylglucosamine (NAG) (N-乙酰葡萄糖胺)
      • N- acetylmuramic acid (NAM) (N-乙酰胞壁酸)
    • alternating D- and L- amino acids

Diaminoacids present in peptidoglycan

slide41

peptidoglycan strands have a helical shape

  • peptidoglycan chains are crosslinked by peptides for strength

G(-)

G(+)

gram positive cell walls
Gram-Positive Cell Walls
  • composed primarily of peptidoglycan
  • may also contain large amounts of teichoic acids (negatively charged)
    • help maintain cell envelop
    • protect from environmental substances
    • may bind to host cells
  • some gram-positive bacteria have layer of proteins on surface of peptidoglycan
  • teichoic acids
  • polymers of glycerol or ribitol (核糖醇) joined by phosphate groups

Isolated gram+ cell wall

periplasmic space of gram bacteria
Periplasmic Space of Gram + bacteria
  • lies between plasma membrane and cell wall and is smaller than that of Gram - bacteria
  • periplasm has relatively few proteins
  • enzymes secreted by Gram + bacteria are called exoenzymes
    • aid in degradation of large nutrients
gram negative cell walls
Gram-Negative Cell Walls
  • consist of a thin layer of peptidoglycan surrounded by an outer membrane
  • outer membrane composed of lipids, lipoproteins, and lipopolysaccharide (LPS)
  • no teichoic acids
  • peptidoglycan is ~5-10% of wall weight
  • periplasmic space differs from that in Gram + cells
    • may constitute 20-40% of cell volume
lipopolysaccharides lpss
Lipopolysaccharides (LPSs)
  • consists of three parts
    • lipid A
    • core polysaccharide
    • O side chain (O antigen)

Importance of LPS

  • may contribute to attachmentto surfaces and biofilm formation
  • creates apermeability barrier
  • contributes to negative charge on cell surface (core polysaccharide)
  • protection from host defenses (O antigen)
  • helps stabilizeouter membrane structure (lipid A)
  • can act as an endotoxin(lipid A)
other characteristics of the outer membrane
Other Characteristics of the Outer Membrane
  • more permeable than plasma membrane due to presence of porin proteins and transporter proteins
    • porin proteins form channels through which small molecules (600-700 daltons) can pass
the mechanism of gram staining
The Mechanism of Gram Staining
  • thought to involve shrinkage of the pores of the peptidoglycan layer of gram-positive cells
    • constriction prevents loss of crystal violet during decolorization step
  • thinner peptidoglycan layer and larger pores of gram-negative bacteria does not prevent loss of crystal violet
slide48

The Cell Wall and Osmotic Protection

  • osmotic lysis
    • can occur when cells are in hypotonicsolutions
    • movement of water into cell causes swelling and lysis due to osmotic pressure
  • cell wall protects against osmotic lysis
  • lysozymebreaks the bond between N-acetyl glucosamine and N-acetylmuramic acid
  • penicillin inhibits peptidoglycan synthesis
  • If cells are treated with either of the above they will lyse if they are in a hypotonic solution
  • protoplast – cell completely lacking cell wall
  • spheroplast – cell with some cell wall remaining
capsules slime layers and s layers
Capsules, Slime Layers, and S-Layers
  • capsules
    • usually composed of polysaccharides
    • well organized and not easily removed from cell
  • slime layers (黏液層)
    • similar to capsules except diffuse, unorganized and easily removed
    • slime may aid in motility
  • S-layers
    • regularly structured layers of protein or glycoprotein
    • In bacteria the S layer is external to the cell wall
    • common among Archaea, where they may be the only structure outside the plasma membrane

S-layers

functions of capsules slime layers and s layers
Functions of capsules, slime layers, and S-layers
  • protection from host defenses (e.g., phagocytosis)
  • protection from harsh environmental conditions (e.g., desiccation)
  • attachment to surfaces

More functions…

  • protection from viral infection or predation by bacteria
  • protection from chemicals in environment (e.g., detergents)
  • facilitate motility of gliding bacteria
  • protection against osmotic stress
slide51

Glycocalyx (醣外被)

    • network of polysaccharides extending from the surface of the cell
    • a capsule or slime layer composed of polysaccharides can also be referred to as a glycocalyx
archaeal membranes
Archaeal membranes
  • composed of unique lipids
    • isoprene units (five carbon, branched)
    • ether linkages rather than ester linkages to glycerol
  • some have a monolayer structure instead of a bilayer structure
archaeal cell walls differ from bacterial cell walls
Archaeal Cell Walls Differ from Bacterial Cell Walls
  • lack peptidoglycan
  • most common cell wall is S layer
  • may have protein sheath external to S layer
  • S layer may be outside membrane and separated by pseudomurein
  • pseudomurein may be outermost layer – similar to gram-positive microorganisms
archaeal cell walls
Archaeal cell walls
  • lack peptidoglycan
  • cell wall varies from species to species but usually consists of complex heteropolysaccharides
  • Methanogens have walls containing pseudomurein

pseudomurein

Cell envelopes of Archaea

slide55

Bacterial and ArchaealCytoplasmic Structures

  • Cytoskeleton
  • Intracytoplasmic membranes
  • Inclusions
  • Ribosomes
  • Nucleoid and plasmids
cytoplasmic matrix
Cytoplasmic Matrix
  • substance in which nucleoid, ribosomesand inclusion bodies are suspended
  • lacks organelles bound by unit membranes
  • composed largely of water
  • is a major part of the protoplasm (the plasma membrane and everything within)
slide57

The Procaryotic Cytoskeleton

  • homologs of all 3 eucaryotic cytoskeletal elements have recently been identified in Bacteria and one has been found in Archaea
  • functions include roles in cell division, protein localization and determination of cell shape
slide58

Tubulin

  • FtsZ – many bacteria and archaea
    • forms ring during septum formation in cell division
  • MreB – many rods, some archaea
    • maintains shape by positioning peptidoglycan synthesis machinery
  • CreS – rare, maintains curve shape

Actin

Intermediate filament

intracytoplasmic membranes
Intracytoplasmic Membranes
  • plasma membrane infoldings
    • observed in many photosynthetic bacteria
      • analogous to thylakoids of chloroplasts
      • reactions centers for ATP formation
    • observed in many bacteria with high respiratory activity
  • anammoxosome in Planctomycetes
    • organelle – site of anaerobic ammonia oxidation
inclusions
Inclusions
  • granules of organic or inorganic material that are stockpiled by the cell for future use
  • some are enclosed by a single-layered membrane
    • membranes vary in composition
    • some made of proteins; others contain lipids

Organic inclusion bodies

  • glycogen
    • polymer of glucose units
  • poly-β-hydroxybutyrate (PHB)
    • polymers of β-hydroxybutyrate
  • cyanophycin granules
    • large polypeptides containing about equal quantities of arginine and aspartic acid
  • carboxysomes
    • contain the enzyme ribulose-1,5,-bisphosphate carboxylase (Rubisco), enzyme used for CO2 fixation
slide61
gas vacuoles
    • found in cyanobacteria and some other aquatic procaryotes
    • provide buoyancy
    • aggregates of hollow cylindrical structures called gas vesicles
inorganic inclusion bodies
Inorganic inclusion bodies
  • polyphosphate granules
    • also called volutin granules and metachromatic granules
    • linear polymers of phosphates
  • sulfur granules
  • Magnetosomes Fe3O4
    • found in aquatic bacteria
    • magnetite particles for orientation in Earth’s magnetic field
    • cytoskeletal protein MamK
    • helps form magnetosome chain
ribosomes
Ribosomes
  • complex structures consisting of protein and RNA
  • sites of protein synthesis
  • smaller than eucaryoticribosomes
    • procaryoticribosomes 70S
    • eucaryoticribosomes  80S
      • S = Svedburg unit
slide64

bacterial and archaeal ribosomal RNA

    • 16S small subunit
    • 23S and 5S in large subunit
    • archaea has additional 5.8S (also seen in eukaryotic large subunit)
  • proteins vary
    • archaea more similar to eukarya than to bacteria
the nucleoid
The Nucleoid (類核體)

Procaryotic chromosomes are located in the nucleoid, an area in the cytoplasm

  • irregularly shaped region
  • location of chromosome
    • usually 1/cell
  • not membrane-bound
the procaryotic chromosome
The procaryotic chromosome
  • a closed circular, double-stranded DNA molecule
  • looped and coiled extensively
  • nucleoid proteins probably aid in folding
    • nucleoid proteins differ from histones

Plasmids

  • usually small, closed circular DNA molecules
  • exist and replicate independently of chromosome
  • have relatively few genes present
  • genes on plasmids are not essential to host but may confer selective advantage (e.g., drug resistance)
  • curing is the loss of a plasmid
  • classification of plasmids based on mode of existence, spread, and function
slide67

Camphor樟腦

Toluene甲苯

slide68

External Structures

Function

protection, attachment to surfaces, horizontal gene transfer, cell movement

pili and fimbriae
Pili and Fimbriae
  • fimbriae (s., fimbria)
    • short, thin, hairlike, proteinaceous appendages
      • up to 1,000/cell
    • mediate attachment to surfaces
    • some (type IV fimbriae) required for twitching motility or gliding motility that occurs in some bacteria
  • sex pili (s., pilus)
    • similar to fimbriae except longer, thicker, and less numerous (1-10/cell)
    • required for mating
patterns of flagella distribution
Patterns of Flagella Distribution
  • monotrichous – one flagellum
  • polar flagellum – flagellum at end of cell
  • amphitrichous – one flagellum at each end of cell
  • lophotrichous – cluster of flagella at one or both ends
  • peritrichous – spread over entire surface of cell
  • extends from cell surface to the tip
  • hollow, rigid cylinder
  • composed of the protein flagellin
  • some procaryotes have a sheath around filament
  • filament
flagellar ultrastructure
Flagellar Ultrastructure

The Hook and Basal Body

  • hook
    • links filament to basal body
  • basal body
    • series of rings that drive flagellar motor
flagellar synthesis
Flagellar Synthesis
  • an example of self-assembly
  • complex process involving many genes and gene products
  • new molecules offlagellinare transported through the hollowfilament
  • growth is from tip, not base
slide73

Differences of Archaeal Flagella

  • flagella thinner
  • more than one type of flagellin protein
  • flagellum are not hollow
  • hook and basal body difficult to distinguish
  • more related to Type IV secretions systems
  • growth occurs at the base, not the end
slide74

Motility

Flagellar movement

Spirochete motility

Twitching motility

Gliding motility

  • Bacteria and Archaea have directed movement
  • chemotaxis
    • move toward chemical attractants such as nutrients, away from harmful substances
  • move in response to temperature, light, oxygen, osmotic pressure, and gravity
slide75

Bacterial Flagellar Movement

  • flagellum rotates like a propeller
    • very rapid rotation up to 1100 revolutions/sec
    • in general, counterclockwise (CCW) rotation causes forward motion (run)
    • in general, clockwise rotation (CW) disrupts run causing cell to stop and tumble (打滾)
slide76

Mechanism of Flagellar Movement

  • flagellum is 2 part motor producing torque (項鍊,轉矩)
  • rotor (轉子)
    • C (FliG protein) ring and MS ring turn and interact with stator
  • stator (發電機的定子;定片)- Mot A and Mot B proteins
    • form channel through plasma membrane
    • protons move through Mot A and Mot B channels and produce energy through proton motive force
    • torque powers rotation of the basal body and filament
slide77

Spirochete (螺旋體) Motility

  • multiple flagella form axial fibril which winds around the cell
  • flagella remain in periplasmic space inside outer sheath
  • corkscrew shape exhibits flexing and spinning movements
slide78

Twitching (抽動) and Gliding (滑行) Motility

  • may involve Type IV pili and slime
  • twitching
    • pili at ends of cell
    • short, intermittent, jerky motions
    • cells are in contact with each other and surface
  • gliding
    • smooth movements
chemotaxis
Chemotaxis (化學趨化作用)
  • movement towards a chemical attractant or away from a chemical repellent
  • concentrations of chemical attractants and chemical repellents detected by chemoreceptors on surfaces of cells
chemotaxis towards attractant
Chemotaxis Towards Attractant
  • in presence of attractant (b) tumbling frequency is reduced and runs in direction of attractant are longer
the bacterial endospore
The Bacterial Endospore
  • formed by some bacteria
  • dormant
  • resistant to numerous environmental conditions
    • heat
    • radiation
    • chemicals
    • desiccation 乾燥

Endospore Structure

  • spore surrounded by thin covering called exosporium
  • thick layers of protein form the spore coat
  • cortex皮層, beneath the coat, thick peptidoglycan
  • core has nucleoid and ribosomes
slide82

What makes an endospore so resistant?

  • calcium (complexed with dipicolinic acid)
  • small, acid-soluble, DNA-binding proteins (SASPs)
  • dehydrated core
  • spore coat and exosporium protect
sporogenesis
Sporogenesis
  • Also called endospore formation or sporulation
  • normally commences when growth ceases because of lack of nutrients
  • complex multistage process
germination transformation of dormant spores into active vegetative cells
Germination-Transformation of dormant spores into active vegetative cells
  • activation
    • prepares spores for germination
    • often results from treatments like heating
  • germination
    • spore swelling
    • rupture of absorption of spore coat
    • loss of resistance
    • increased metabolic activity
  • outgrowth
    • emergence of vegetative cell
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