Ultrastructure of bacterial cell. Form and Function.

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Ultrastructure of bacterial cell. Form and
Function.
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Structure of a Prokaryotic Cell
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Bacterial Morphology and Ultrastructure

• Only two types of cells are produced by all
  living organisms on earth.
• Prokaryotes (pro. or primitive nucleus) do not
  have a membrane bound nucleus
• eubacteria (true bacteria)
• archaebacteria (ancient bacteria)
• Eukaryotes (eu, or true nucleus) have a membrane
  bound nucleus
• Algae
• fungi 
• protozoa
• plants
• animals

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Prokaryotes
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Chemical Composition of Bacteria

• Water - 70
• Dry weight - 30 composed of
• DNA - 5 MW 2,000,000,000
• RNA - 12
• protein- 70 found in
• Ribosomes(10,000) RNA
• Protein particles - MW 3,000,000
• Enzymes
• Surface structures
• polysaccharides - 5
• lipids - 6
• phospholipids - 4

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Prokaryote Structures

1. Appendages- flagella, pili, fimbrae
2. Cell envelope- glycocalyx, cell wall , cell
   membrane
3. Cytoplasm- ribosomes, granules,
   nucleoid/chromosome.

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Appendages
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Bacterial Appendages

• Pili (pl), pilus (s)
• only found in gram negative bacteria
• tubulare, hairlike structures of protein larger
  and more rare than fimbriae.
• 2 types of pili
• atacnement pilus - allow bacteria to attach to
  other cells
• sex pilus, - transfer from one bacterial cell to
  another- conjugation.

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Fimbriae

• fimbriae (pl) fimbria (s)
• Adhesion to cells and surfaces
• Responsible for biofilms.
• Pathogenesis of gonococcus and E.coli

Escherichia coli.
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Flagella

• Flagella (pl), flagellum(s)
• long appendages which rotate by means of a
  "motor" located just under the cytoplasmic
  membrane.
• bacteria may have one, a few, or many flagella in
  different positions on the cell.
• Advantages
• - chemotaxis - positive and negative.
• - motility
• All spirilla, half of bacilli, rare cocci.

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Structure of flagellaallows for 360 degree
filament rotation
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Flagella

• Three morphological regions
• Helical filament
• long outermost region composes up to 90 of its
  length
• contains the globular (roughly spherical) protein
  flagellin arranged in several chains and form a
  helix around a hollow core
• Hooked or curved area
• filament is attached consists of a different
  protein
• Basal body
• terminal portion of the flagellum 
• fix the flagellum to the cell wall and plasma
  membrane
• composed of a central rod inserted into a series
  of rings
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• Gram negative - 2 pairs of rings
• Outer pair - fixed to the outer membrane and
  peptidoglycan layer
• Inner pair - fixed to the plasma membrane (SM
  ring)
• Gram positive - only inner pair is present

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Motility

• Types of bacterial motility
• run or swim - when a bacterium moves in
  one direction for a length of time
• tumbles - periodic, abrupt random changes
  in direction
• swarming - rapid wavelike growth across a
  solid culture medium
• Mechanism of flagellar movement - relative
  rotation of the rings in the basal body of the
  flagellum
• Antigenicity
• flagellar or H antigen - useful in the
  serological identification of serotypes of
  Salmonella organisms

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Arrangements

• Flagella vary in number and arrangement.
• Polar arrangment
• Monotrichious - 1 flagellum at one end
• Fastest Pseudomonas -example
• Lophotrichious - tuft at one end
• Amphitrichious- bipolar
• Peritrichious - multiple flagella randomly
  dispersed around the bacterial cell
• E. coli - example

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Flagellar arrangements

• Monotrichous
• Lophotrichous
• Amphitrichous
• Peritrichous
• Atrichous 

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Axial filaments
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Axial filaments

• tuft of fibrils that arise at the ends of the
  cell under the outer membrane and spiral around
  the cell
• rotation an opposing of the outer membrane
  movement that propels the spirochetes by causing
  them to move like corkscrews
• Found in Spirochetes and are similar to flagella,
  but are located between the cell wall and an
  outer membrane, and are attached to one end of
  the organism.

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Evidence of motility

• Two ways by which motility can be demonstrated
• direct or microscopic
• hanging drop preparation or wet mount preparation
  by dark field mycroscope
• Distinguishes
• Brownian movement - when the bacteria show
  molecular movement
• true motility - if a bacterium describes a
  rotatory, undulatory or sinuous movement
• indirect or macroscopic
• Stab inoculation of the semisolid media
• nonmotile - growth is limited at the point of
  inoculation
• motile - growth is diffuse or moves away from the
  line of inoculation turbidity of the medium

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Detection of Motility

• Direct

• Indirect

Presence mobile bacteria
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• Bacterial motility (QuickTime movie)
• http//diverge.hunter.cuny.edu/weigang/Animations
  /SalmonellaFlagella-S.mov

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Prokaryote Structures

• Appendages- flagella, pili, fimbrae
• Cell envelope
• glycocalyx
• cell wall
• cell membrane
• Cytoplasm- ribosomes, granules,
  nucleoid/chromosome.

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2. Bacterial Surface Structure - cell envelope

• Glycocalyx - some extracellular material
  secreted by many bacterial cells in the form of
• capsule - attached tightly to the bacterium and
  has definite boundaries.
• slime layer - loosely associated with the
  bacterium and can be easily washed off
• Compositions
• layer of polysaccharide
• proteins - sometimes

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Functions of the Capsule

• Protection
• Identification
• Vaccine preparation
• Tissue attachment
• Antibiotic barrier
•  

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Medical Importance -

• rapid serological identification of
• Several groups of streptococci
• Meningococcus
• Hemophilus influenzae
• Klebsiella pneumoniae
• Some of the coliforms
• Yersinia and Bacillus specie
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Identification

• Two simple methods to distinguish the capsule
• India ink technique - most satisfactory method of
  demonstrating the capsule by Burri-Gins technique
• Bacteria is suspended in diluted India ink
• Stain with fuxin
• Bacterial cells appear to lie in a lacunae
• and red cytoplasme.
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• Quellung reaction - Homologous antibody is added
  to a preparation of capsule.
• microprecipitation at the periphery of the
  capsule altering its refractive index rendering
  the capsule to be visible

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Staining by Burri-Gins
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Neisseria meningitidis - Gram- negative coccus,
non-motile bacteria occur as two cells (orange)
in a capsule (yellow)
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Haemophilus influenza bacteria in the process of
expressing polysaccharide capsules.
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Cell wall

• Peptidoglycan (polysaccharides protein),
• Support and shape of a bacterial cell.
• The three primary shapes in bacteria are
• coccus (spherical),
• bacillus (rod-shaped)
• spirillum (spiral).
• Mycoplasma are bacteria that have no cell wall
  and therefore have no definite shape.

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Cell wall

• peptidoglycan (polysaccharides protein)
• Components of the peptidoglycan layer
• Repeating glycan chains (N acetyl glucosamine
  and N acetyl muramic acid)
• a set of identical tetrapeptide side chains
  attached to N- acetylmuramic acid
• a set of identical peptide cross bridges

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Peptidoglycan
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Differences in Cell Wall Structure

• Basis of Gram Stain Reaction
• Hans Christian Gram- 1884
• Differential Stain
• Gram Positive vs Gram Negative Cells
• Gram Positive Cells-
• Thick peptidoglycan layer with embedded
  teichoic acids
• Gram Negative Cells-
• Thin peptidoglycan layer, outer membrane of
  lipopolysaccharide.

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Cell wall
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Gram Stain Reaction

• Hans Christian Gram- 1880s
• Divides bacteria into 2 main groups-
• Gram positive
• Gram negative
• Also- gram variable
• Gram nonreactive
• Gram positive bacteria
• many layers of peptidoglycan and teichoic acids.
• form a crystal violet-iodine-teichoic acid
  complex
• Large complex, difficult to decolorize

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Gram positive bacteria
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Gram Stain Reaction

• Gram negative bacteria
• Very thin peptidoglycan
• No teichoic acids
• Alcohol readily removes the crystal violet.
• Alcohol also dissolves the lipopolysaccharide of
  the cell wall.
• Gram variable cells
• Some cells retain crystal violet some decolorize
  and take up the safranin
• 4 factors-
• Genetics- variable amount of teichoic acid.
• Age of culture- older cultures have variable
  amount of teichoic acid
• Growth medium- necessary nutrients not available
• Technique-
• smear not thin or evenly made.
• Staining procedure not done correctly-
  decolorizer left on too long.

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Gram negative bacteria
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Gram stain technique
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Gram stain
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Gram stain
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• Gram nonreactive cells
• Have peptidoglycan but have very waxy- thick
  lipids waterproof, dyes cannot enter either.
• Examples- Mycobacterium tuberculosis and leprosy.
  
• Alternative staining- acid fast stain

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Cell wall deficient forms

• L- forms ( Lister Institute where discovered)
• Bacteria loses cell wall during the life cycle
• Result of a mutation in cell wall forming genes
• Induced by treating with lysozyme or penicillin
  which disrupts the cell wall
• Protoplast-
• G bacterium with no c. wall, only a c.
  membrane
• Fragile, easily lysed
• Spheroplast-
• G bacterium loses peptidoglycan, but has outer
  membrane
• Less fragile but weakened.

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Surface structures continued

• Outer membrane
• This lipid bilayer is found in Gram negative
  bacteria and is the source of lipopolysaccharide
  (LPS) in these bacteria
• LPS is toxic and turns on the immune system.
• Not found in Gram positive bacteria.

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Lipopolysaccharide
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C. Cell membrane

• Located just under cell wall
• Very thin
• Lipid bilayer, similar to the plasma membrane of
  other cells. Transport of ions, nutrients and
  waste across the membrane
• Typical
• 30-40 phospholipids
• 60-70 proteins
• Exceptions-
• Mycoplasma- sterols
• Archaea- unique branched hydrocarbons

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Mesosome

• Extension of cell membrane
• Folding into cytoplasm internal pouch
• Increases surface area.
• Gram-positive bacteria-prominent
• Gram negative bacteria- smaller, harder to see.
• Functions-
• Cell wall synthesis
• Guides duplicated
  chromosomes into
  the daughter cells
  
  in cell division.

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Functions of Cell Membrane

• Carries out functions normally carried out by
  eukaryote organelles.
• Site for energy functions
• Nutrient processing
• Synthesis
• Transport of nutrients and waste
• Selectively permeable
• Most enzymes of respiration and ATP synthesis
• Enzyme synthesis of structural macromolecules
• Cell envelope and appendages
• Secretion of toxins and enzymes into environment.

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Prokaryote Structures

• Appendages- flagella, pili, fimbrae
• Cell envelope
• glycocalyx
• cell wall
• cell membrane
• Cytoplasm
• Nucleoid/chromosome
• Plasmid
• Ribosomes
• Granules

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3. Cell cytoplasm

• Encased by cell membrane
• Dense, gelatinous
• Prominent site for biochemical and synthetic
  activities
• 70-80 water- solvent
• Mixture of nutrients- sugar, amino acids, salts
• Building blacks for cell synthesis and energy

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A. Bacterial chromosome

• Singular circular strand of DNA
• Aggregated in a dense area- nucleiod
• Long molecule of DNA tightly coiled around
  protein molecules.

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B. Plasmids

• Nonessential pieces of DNA
• Often confer protection- resistance to drugs
• Tiny, circular
• Free or integrated
• Duplicate and are passed on to offspring
• Used in genetic engineering

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Types of plasmid

• Fertility-F-plasmids. They are capable of
  conjugation (transfer of genetic material between
  bacteria which are touching).
• Resistance-(R)plasmids, which contain genes that
  can build a resistance against antibiotics or
  poisons and help bacteria produce pili.
• Col-plasmids, which contain genes that determine
  the production of bacteriocins, proteins that can
  kill other bacteria.
• Degradative plasmids, which enable the digestion
  of unusual substances, e.g., toluene or salicylic
  acid.
• Virulence plasmids, which turn the bacterium into
  a pathogen (one that causes disease).

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Cell division in Prokaryotes

• Prokaryotes use a relatively simple form of cell
  division - binary fission.
• The diagram at 1.shows a bacterial cell.
• The cell wall and membrane are in red,
• the bacterial chromosome in blue,
• the cytoplasm in light green,
• the yellow dot represents a point of attachment
  of the chromosome to the cell membrane.

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C. Ribosomes

• Site of protein synthesis
• Thousands
• Occurs in chains polysomes
• 70S
• 2 smaller subunits
• 30S and 50S

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D. Inclusions

• If nutrients abundant- stored intracellularly
• Granules
• Crystals of inorganic compounds not enclosed by
  membranes
• Polyphosphate- corynebacterium
• Sulfur granules- photosynthetic
• Metachromatic- Mycobacterium

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Bacterial Internal Structures

• Endospores
• inert, resting, cells produced by some G genera
  Clostridium, Bacillus and Sporosarcina
• have a 2-phase life cycle
• vegetative cell metabolically active and
  growing
• endospore when exposed to adverse environmental
  conditions capable of high resistance and very
  long-term survival
• Features of spores- size, shape,
  locationidentification
• sporulation -formation of endospores
• hardiest of all life forms
• Forms inside a cell- functions in survival
• not a means of reproduction
• withstands extremes in heat, drying, freezing,
  radiation and chemicals
• germination- return to vegetative growth

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Endospores

• Resistance linked to high levels of calcium and
  dipicolinic acid
• Dehydrated, metabolically inactive thick coat
• Longevity verges on immortality - 25,250 million
  years.
• Resistant to ordinary cleaning methods and
  boiling
• Pressurized steam at 120oC for 20-30 minutes will
  destroy

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Bacterial Shapes, Arrangements, and Sizes

• Variety in shape, size, and arrangement but
  typically described by one of three basic shapes
• coccus - spherical
• bacillus rod
• coccobacillus very short and plump
• vibrio gently curved
• spirillum - helical, comma, twisted rod,
• spirochete spring-like

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