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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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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

Prokaryotic cells

Protists

Spontaneous generation

Viroids

Viruses

Virusoids

Figure/ Table

Summary


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


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.


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


Members of the microbial world entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • Antony van Leeuwenhoek (1632-1723)

    • first person to observe and describe microorganisms accurately


The conflict over spontaneous generation
The Conflict over Spontaneous Generation entities studied by microbiologists.

  • 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 entities studied by microbiologists. (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 entities studied by microbiologists.

  • 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.


Earliest Molecules - RNA entities studied by microbiologists.

  • 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


Chapter 2 entities studied by microbiologists.

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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • bright-field microscope

  • dark-field microscope

  • phase-contrast microscope

  • fluorescence microscope


Microscope resolution
Microscope Resolution entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

Differential Staining

  • divides microorganisms into groups based on their staining properties

  • divides bacteria into two groups based on differences in cell wall structure


Differential Staining entities studied by microbiologists.

Figure 2.19


Electron microscopy
Electron Microscopy entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • confocal scanning laser (CLSM) microscopy and scanning probe microscopy

  • have extremely high resolution


Confocal microscopy
Confocal Microscopy entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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


Chapter 3 entities studied by microbiologists.

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


Common features of bacterial and archaeal cell structure entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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


Figure 3 2
Figure 3.2 entities studied by microbiologists.


  • largest – entities studied by microbiologists.

  • 50 μm in

  • diameter

  • smallest –

  • 0.3 μm in

  • diameter


Size – Shape Relationship entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 ( entities studied by microbiologists.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


G(-)

G(+)


Gram positive cell walls
Gram-Positive Cell Walls entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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) entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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


The Cell Wall and Osmotic Protection entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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


  • Glycocalyx ( entities studied by microbiologists.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 entities studied by microbiologists. 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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


Bacterial and entities studied by microbiologists.ArchaealCytoplasmic Structures

  • Cytoskeleton

  • Intracytoplasmic membranes

  • Inclusions

  • Ribosomes

  • Nucleoid and plasmids


Cytoplasmic matrix
Cytoplasmic Matrix entities studied by microbiologists.

  • 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)


The Procaryotic Cytoskeleton entities studied by microbiologists.

  • 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


Tubulin entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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


  • gas vacuoles entities studied by microbiologists.

    • found in cyanobacteria and some other aquatic procaryotes

    • provide buoyancy

    • aggregates of hollow cylindrical structures called gas vesicles


Inorganic inclusion bodies
Inorganic inclusion bodies entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • complex structures consisting of protein and RNA

  • sites of protein synthesis

  • smaller than eucaryoticribosomes

    • procaryoticribosomes 70S

    • eucaryoticribosomes  80S

      • S = Svedburg unit


  • bacterial and archaeal ribosomal RNA entities studied by microbiologists.

    • 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 entities studied by microbiologists.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 entities studied by microbiologists.

  • 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


Camphor entities studied by microbiologists.樟腦

Toluene甲苯


External Structures entities studied by microbiologists.

Function

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


Pili and fimbriae
Pili and Fimbriae entities studied by microbiologists.

  • 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 entities studied by microbiologists.

  • 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 entities studied by microbiologists.

The Hook and Basal Body

  • hook

    • links filament to basal body

  • basal body

    • series of rings that drive flagellar motor


Flagellar synthesis
Flagellar Synthesis entities studied by microbiologists.

  • 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


Differences of Archaeal Flagella entities studied by microbiologists.

  • 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


Motility entities studied by microbiologists.

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


Bacterial Flagellar Movement entities studied by microbiologists.

  • 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)


Mechanism of Flagellar Movement entities studied by microbiologists.

  • 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


Spirochete ( entities studied by microbiologists.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


Twitching ( entities studied by microbiologists.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 entities studied by microbiologists. (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 entities studied by microbiologists.

  • in presence of attractant (b) tumbling frequency is reduced and runs in direction of attractant are longer


The bacterial endospore
The Bacterial Endospore entities studied by microbiologists.

  • 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


What makes an endospore so resistant? entities studied by microbiologists.

  • calcium (complexed with dipicolinic acid)

  • small, acid-soluble, DNA-binding proteins (SASPs)

  • dehydrated core

  • spore coat and exosporium protect


Sporogenesis
Sporogenesis entities studied by microbiologists.

  • 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 entities studied by microbiologists.-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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