Transcript Document

Lecture #2
Prokaryotes
Prokaryotes
• microscopic single celled organisms
• collective biomass – 10x of all
eukaryotes!!!!!
• vast genetic diversity among
members
• physical diversity
– shapes: spheres (coccus), rods
(bacilli) and spirals
1 µm
Spherical
(cocci)
2 µm
Rod-shaped
(bacilli)
5 µm
Spiral
Prokaryotes
• REMEMBER: adoption of
a three domain system of
superkingdoms
– 1. Bacteria – prokaryotic
(or Eubacteria)
– 2. Archaea – prokaryotic
– 3. Eukarya - eukaryotic
• divisions into protist, fungi,
plants and animals
“The Tree of Life”
•
separation into 9 major taxa of prokaryotes - based on molecular systematics
Universal ancestor
Crenarcaeotes
Korarchaeotes
Gram-positive
bacteria
Cyanobacteria
Spirochetes
Chlamydias
Epsilon
Delta
Gamma
Beta
Alpha
Proteobacteria
Euryarchaeotes
Domain
Archaea
Domain Bacteria
Domain
Eukarya
Eukaryotes
Applying molecular systematics to the investigation of prokaryotic phylogeny has
produced dramatic results
– lead to a phylogenetic re-classification of prokaryotes
– development of Domain Bacteria
Nanoarchaeotes
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Bacterial groups
• Bacteria or Eubacteria
– include the vast majority of prokaryotes that we are aware of
• comprised of 5 major groups:
• 1. proteobacteria: diverse group of gram negative bacteria
– 5 major subgroups: alpha  epsilon
• 2. gram-positive: very diverse
– solitary and colonial
– free-living and parasitic
– e.g. Bacillus, Streptococcus
Bacterial Groups
• 3. cyanobacteria: blue-green algae
– photoautotrophs
– O2-generating photosynthesis through chloroplasts
• 4. chlamydias: parasitic bacteria
– can only survive within animal cells
– cell walls lack peptidoglycan entirely
• 5. spirochetes: helical in structure
– heterotrophs
– most are free-living
– some can be parasitic
Archaea
– Archaea: share similarities with prokaryotes and
eukaryotes
– divided into four clades: Euryarchaeota, Crenarchaeota,
Korarchaeota and Nanoarchaeota
– 1996 - recent discovery of a new clade – Korarchaeota
• koron = “young man”
• found in hot springs in Yellowstone
– 2002 – in hydrothermal vents off the coast of Iceland –
found extremely small archaea
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name Nanoarchaeota – smallest of the four
nanos = “dwarf”
smallest genome known – only 500,000 base pairs
three other species found since then – hydrothermal vents and hot
springs
Archaea
– first Archaea to be identified were found in extreme
environments = extremophiles
– 1. thermophiles (thermos = “hot”)
• clade Crenarchaeota
• thrive in very hot environments
– 2. halophiles – high saline environments (halo = “salt)
• clade Euryarchaeota
• some tolerate the high salinity, others require it
• red-brown scum possess a visual pigment called bacteriorhodopsin
– 3. methanogens – named for the way they obtain energy
• clade Euryarchaeota
• use CO2 to oxidize H2 and produce energy - releases methane (CH4) as a
waste
• strictest of anaerobes – obligate anaerobes
Role of prokaryotes
• chemical recycling
– ecosystems depend on a continual recycling of chemical
elements between the living and nonliving components of
this planet
– chemotrophic prokaryotes function as decomposers
– prokaryotes also convert inorganic forms into organic
forms for other living organisms
• autotrophs use CO2 for energy but make organic compounds
which are stored and passed up through the food chain
Role of prokaryotes
• symbiotic relationships
– prokaryotes can possess beneficial relationships
with other prokaryotes in terms of metabolic
cooperation
– also hold beneficial relationships with other
organisms like eukaryotes
– known as symbiosis
– GENERAL DEFINITION: symbiosis = ecologic
relationship between organisms of different
species
• two major kinds: mutualism & parasitism
Role of prokaryotes
• symbiotic relationships
– mutalism – both organisms benefit
• health benefit
– parasitism – one organisms (parasite) benefits at the
expense of the host
• prokaryotes cause 50% of diseases in humans
• cause illness through the production of endotoxins or exotoxins
Key Prokaryotic Adaptations
• 1. Cell surface structures
– evolution of the cell wall
• 2. Motility
– evolution of flagella
• 3. Internal organization of DNA
– evolution of the chromosome and plasmid DNA
• 4. Reproduction
– evolution of binary fission, conjugation,
transformation and endospores
The Bacterial Cell Wall
• key feature – prokaryotes are surrounded by a cell wall
• maintains cell shape, provides physical protection and allows the cell to
control its osmolarity
– in a hypertonic environment – most prokaryotes will lose water and shrink
= plasmolysis
• cell wall is NOT like the cell wall of plants and fungi – which are
made of cellulose or chitin
• encloses the entire prokaryote
The Bacterial Cell Wall
• roles of the bacterial cell wall
• structural: forms an anchor for the attachment of many
intracellular subsatnces
• counteracts the osmotic pressure created by the
cytoplasm
– changes in OP can result in the loss of water and plasmolysis
• involved in binary fission (reproduction)
• protection against changes in ion and pH levels, foreign
enzymes, phagocytosis by foreign pathogens
Cell Wall & Peptidoglycans
• most prokaryotic cell walls
contain peptidoglycans
(murein)
– presence is used to classify the two
types of bacteria: gram negative and
gram positive
– thicker in gram positive bacteria than
gram negative
• peptidoglycan:
– sugar polymer modified with amino
acids
– cross-linked in gram-positive bacteria
– forms a crystal lattice organization
Peptidoglycan
• peptidoglycan layer is a crystal lattice or mesh-like structure
• formed from linear chains of two alternating sugars called N-acetyl amino sugars
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N-acetyl glucosamine (GlcNAc or NAG)
N-acetyl muramic acid (MurNAc or NAM) - 3 to 5 amino acids attached
interactions occur between these amino acids = cross-linking
cross-linking results in a 3-dimensional structure that is strong and rigid
high degree of cross-linking in gram-positive bacteria
Antibiotic actions
• Antibacterial drugs such as penicillin interfere with
the production of peptidoglycan by binding to the
enzymes that perform the cross-linking
– for a bacterial cell to reproduce – new cell walls must be
made
– this requires the assembly of more than a million new
peptidoglycan subunits
– these subunits must be cross-linked – by enzymes called
transpeptidases
– penicillin & vancomycin –inhibits cell wall synthesis by
preventing NAM and NAG cross-linking
Gram positive bacteria
• retain the crystal violet stain used in a Gram stain – so they stain purple
• simpler wall construction with larger amounts of peptidoglycans
• high peptidoglycan content of the cell wall take up the crystal violet dye – not
washed away in subsequent steps
• most pathogens in human are gram +ve
• divided into cocci and bacilli forms
Lipopolysaccharide
Cell
wall
Outer
Cell membrane
wall Peptidoglycan
layer
Plasma membrane
Peptidoglycan
layer
Plasma membrane
Protein
Protein
Grampositive
bacteria
Gramnegative
bacteria
20 µm
Gram-positive
Gram-negative
Gram negative bacteria
• do not retain the crystal violet dye - dye is washed away
• cell wall of gram negative bacteria is comprised of a PG layer
PLUS an outer membrane
– located outside the peptidoglycan layer
– comprised of lipopolysaccharides, lipoproteins and porins
– the lipopolysaccharides are toxic to humans – endotoxin layer
Peptidoglycan
layer
Plasma
Membrane
Gram negative bacteria
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SOME WELL KNOWN GRAM NEGATIVE BACTERIA
coccobacilli: H. influenzae, B. pertussis, L. pneumophilia
cocci: N.meningitidis, N. gonorrhae
bacilli: E.coli, V. cholerae, H. pylori, S. dysenterae,
Salmonella
Gram
staining
– both Gram-positive and Gramnegative bacteria take up the
same amounts of crystal violet
(CV) and iodine (I).
– in Gram-positive bacteria - the
ethanol used in washing the
bacteria dehydrates the
bacteria and traps the CV-I in
the cell wall– PURPLE STAIN
– in gram negative bacteria – the
thinner cell wall does not
prevent extraction of the CV-I
complex
– plus the outer membrane limits
the amount of CV-I complex
that can reach the PG layer –
CLEAR STAIN
1. Place a slide with a bacterial smear on a staining rack.
2. STAIN the slide with crystal violet for 1-2 min.
3. Pour off the stain and rinse with water thoroughly.
4. Flood slide with Gram's iodine for 1-2 min.
5. Pour off the iodine and rinse with water thoroughly..
6. Decolourize by washing the slide briefly with acetone (2-3
seconds) – alternatively use 95% ethanol
7. Wash slide thoroughly with water to remove the acetone
8. Flood slide with safranin counterstain for 2 min.
9. Wash with water.
10. Blot excess water and dry by hand over bunsen flame.
http://www.youtube.com/watch?v=OQ6Cgj_UHM
Bacterial capsule
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found in many prokaryotes – both +ve and –ve
found outside the cell wall
also called the glycocalyx
if it is less organized = slime layer
resists dehydration
roles in adherence to surfaces
participates in colonization
may make the bacteria resistant to the immune system
Bacterial adhesion
• via the glycocalyx/capsule
• also through the
development of specialized
appendages
– fimbrae – more numerous and
shorter than pili
– pili – some can be specialized
for the reproduction of the
bacteria
Bacterial motility
• half of all bacteria exhibit taxis – the ability to move towards a
specific signal
– movement towards a chemical signal = chemotaxis
– movement toward light = phototaxis
• major mobility mechanisms: flagellar and gliding
• gliding: movement of cells over surfaces without the aid of flagella
– not completely understood
Bacterial Flagellae
• most motile bacteria propel themselves by flagella that are
structurally and functionally different from eukaryotic flagella
– major types of flagellar bacteria:
• monotrichous (one flagella)
• lophotrichous (tuft at one end)
• peritrichous (found evenly over the surface)
– consist of three parts: the motor, the hook and the filament
– the filament consists of a hollow, rigid cylinder composed of a protein called
flagellin
• attaches to a curved structure called the hook
– hook is attached to the basal body or basal apparatus
– basal body: embedded in the cell wall down to the plasma membrane
hook
filament
Bacterial
Flagella
basal body
stator
http://www.youtube.com/watch?v=Ey7Emmddf7Y
rotor
rod
– a portion of the basal body is called the motor
• made up of stationary ‘stators’ and a rotating ‘rotor’ connected to the hook via a
rod
• an ATP-driven proton pumps pump protons out of the bacteria
• when the protons diffuse back in through the stator – turns the rotor and the
attached rod
• the hook and attached filament also rotate
– anticlockwise rotation of flagella thrusts the cell forward with the flagellum
trailing behind
hook
hook
filament
filament
Bacterial
Flagella
basal body
basal body
stator
stator
rotor
rotor
rod
rod
Prokaryotic genome organization
• lack the compartmentalization of eukaryotic cells
• do have specialized membranes that perform specific functions
• genome is a single circular chromosome contained in a nucleoid region
– located in a nucleoid – a region of the cytoplasm
– can also have several smaller circular pieces of DNA =
plasmids
DNA replication – the prokaryotic players
• prokaryotic replication requires 3 things:
• 1. initiation sequence– DNA sequence that initiates DNA synthesis – called
oriC
– region of DNA that the replication machinery recognizes
• 2. initiators – proteins that recognize the oriC region
– DnaA –binds to oriC and unwinds a small area of the DNA helix (20 bps)
– DnaB –unwinds the DNA further - acts as a helicase
– two DnaB molecules move in opposite directions  replication bubble
• 3. termination sites – DNA synthesis stops when the regions of DNA being
replicated meet each other
– alternatively – can stop at specific sequences of DNA = termination
sequences
Prokaryotic replication
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bacterial chromosome is a helix
unwinding will produce two parent strands – sense and anti-sense
these parent strands are used a templates for the creation of new “daughter” strands
DNA daughter strands can only be made in one direction – 5’ to 3’
– so the enzymes run along the parent strand in the 3’  5’ direction
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the anti-sense strand can be replicated continuously = creates the leading daughter strand
the sense strand is replicated discontinuously = in fragments (Okazaki fragments) and creates the
lagging daughter strand
Prokaryotic DNA replication
• at the oriC – a replication complex forms:
– 1. helicase – DnaB – unwinds the DNA helix into separated parental strands
– 2. single, strand binding proteins (SSBs) – bind to the unwinding DNA to prevent
rehybridization back into a helix
– 3. primase – DnaG (or RNA polymerase II) - makes a small RNA primer for the binding of DNA
polymerase III
– 4. DNA holoenzyme complex – complex of several proteins including DNA polymerase III
– 5. DNA ligase – links together Okazaki fragments into one continuous daughter strand
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for the “big picture”: http://www.youtube.com/watch?v=-mtLXpgjHL0
topoisomerase
DNA Pol III
replicated
DNA
DnaB
primer
DnaG
SSBs
Replication Complex
Replication Direction – daughter DNA made 5’ to 3’
• once the DNA is replicated – the bacteria
must divide
• bacterial reproduction is through binary
fission = asexual reproduction
• each replicated chromosome attaches to
the plasma membrane
• the cell elongates and causes the two
chromosomes to separate.
• the plasma membrane invaginates, or
pinches inward toward the middle of the
cell
• when it reaches the middle - the cell splits
into two daughter cells
• limited by resource availability and
competition from other microorganisms
(produce antibiotics)
Prokaryotic
Reproduction
• Budding helps some
prokaryotes to replicate.
– The bud is an outgrowth of the
parent cell.
– The bud has an exact duplicate
copy of the parent cell’s
genome.
– The bud falls off and a mature
parent cell arises.
Prokaryotic
Reproduction
Genetic recombination in prokaryotes
• prokaryotes can transfer information to each other
– Experiment: two mutant strains of E.coli with different nutritional
requirements grown on minimal media (sugars, salts, no amino acids)
– one strain trp- will NOT grow in the absence of tryptophan
– second strain arg- will NOT grow in the absence of arginine
– mix the two strains and grow in minimal media (lacks arginine and
tryptophan)
– growth of the colony is observed
Mixture
Mutant
strain
arg+ trp–
transfer of genetic information
between the two strains to
create an arg+trp+ strain
Mutant
strain
arg– trp+
Mixture
Mutant
strain
arg+ trp–
No
colonies
(control)
Colonies
grew
New strain
arg+ trp+
No
colonies
(control)
Mutant
strain
arg– trp+
Genetic Recombination in Prokaryotes
• Three processes bring prokaryotic DNA
from different individuals together:
– 1. Transformation
– 2. Transduction
– 3. Conjugation
Transformation
• Transformation = the uptake of naked, foreign DNA from the
surrounding environment
• Experiment: transformation of harmless Streptococcus pneumoniae
bacteria into pneumonia-causing cells
– mix a live, nonpathogenic strain with a dead strain (lysis in dead strain results and the
release of genetic material into the surrounding environment)
– non-pathogenic strain takes up a piece of DNA carrying the allele for pathogenicity
– foreign allele becomes incorporated into the non-pathogenic hosts chromosome
• can be artificially induced in the lab
– either through chemical weakening of the plasma membrane
– OR electrical weakening
Transduction
Phage DNA
A+ B+
• bacteriophages carry bacterial genes from one
host cell to another
• bacteriophage – virus that infects a bacterium
• infection of another bacterium results in the
introduction of the new piece of DNA – if it
contains a new gene – alters the genetic makeup of
the recipient cell
• if this is a random event = generalized
transduction
• in specialized transduction – phage picks up only a
few bacterial genes
A+ B+
Donor
cell
A+
Crossing
over
A+
A– B–
Recipient
cell
A+ B–
Recombinant cell
Conjugation
– Conjugation: bacterial “sex”
• conjugation is the direct transfer of
genetic material between bacterial
cells that are temporarily joined
• requires the formation of a mating
bridge – sex pilus
• the transfer is one-way: One cell
(“male”) donates DNA, and its “mate”
(“female”) receives the genes
• “Maleness,” the ability to form a sex
pilus and donate DNA, results from a
gene called = F (for fertility) factor
• F factor can be part of the
chromosome or found on a plasmid (F
plasmid)
The F Plasmid and Conjugation
•
bacteria containing the F plasmid are designated F+ cells (male)
– function as DNA donors during conjugation
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F+ cells transfer DNA to an F recipient cell (female) through conjugation:
1. formation of the mating bridge
2. a single strand of the F plasmid breaks at a specific point and begins to move into the
female/F- bacteria
3. the missing piece of DNA is regenerated in the male/F+ by replication
4. the female replicates the incoming DNA – two new double stranded circular plasmids
result
5. two cells result that are F+ - therefore bacterial sex converts the F- into an F+ bacteria
F plasmid
F+ cell
Mating
bridge
F– cell
Bacterial chromosome
F+ cell
F+ cell
Bacterial
chromosome
Conjunction and transfer of an F plasmid from and F+ donor to an F– recipient
Chromosomal F factors and Conjugation
-if the F gene is part of the chromosome = cell is called the Hfr cell (high
frequency of recombination)
1. the Hfr cell forms a mating bridge with the F- cell
2. single strand of the chromosome breaks and moves into the F- cell
-movement of the F factor “carries” additional genes into the F- cell
e.g. A+ and B+ alleles
3. DNA replication begins in the Hfr and F- cell – to create double stranded DNA
Hfr cell
F+ cell
F factor
Hfr cell
F– cell
Temporary
partial
diploid
Conjugation and transfer of part of the bacterial chromosome from an
Hfr donor to an F– recipient, resulting in recombination
Recombinant F–
bacterium
Chromosomal F factors and Conjugation
4. the mating bridge usually breaks before complete transfer of the chromosome
-so just the F factor and a few downstream genes move into the F- cell
5. homologous recombination can result – B+ allele (from the Hfr cell) is
switched for the B- allele in the F- cell
6. extra piece of DNA outside the chromosome is degraded over time
7. new bacteria remains F- and is called a recombinant bacteria
Hfr cell
F+ cell
F factor
Hfr cell
F– cell
Temporary
partial
diploid
Conjugation and transfer of part of the bacterial chromosome from an
Hfr donor to an F– recipient, resulting in recombination
Recombinant F–
bacterium
Bacterial adaptation
• prokaryotes are very successful because they
are able to adapt to many environments
• because of rapid reproduction rates – natural
selection in overdrive
• numerous metabolic adaptations have
evolved in prokaryotes
Adaptations in Nutritional Mode
• one adaptation is in “food metabolism”
• broken down into two major categories:
• 1. Autotrophs: “self”, “nourishing”
– producers in the food chain
– able to make their own food
– use the energy from either light (photo) or from electron
donors in chemical reactions (chemo) to make this food
– so they do NOT need organic carbon sources as a source
of energy
• 2. Heterotrophs: “different”, “nourishing
– consumers in the food chain
– have to “eat” – must obtain organic food
– cannot “fix carbon” – i.e. must use organic sources of
carbon as an energy source
Nutritional Mode Categories
– 1. photoautotrophs: photosynthetic organisms that capture light
energy and use it to drive synthesis of organic compounds from
inorganic carbon sources (e.g.CO2)
• e.g. blue-green algae & plants
– 2. chemoautotrophs – also need CO2 as a carbon source
• use electron donors as their energy source – such as hydrogen sulfide,
ammonia or iron
• e.g. green sulfur bacteria
– 3. photoheterotrophs: use light for energy but must obtain their
carbon from outside organic sources
– 4. chemoheterotrophs: must consume organic molecules for both
energy and carbon
• e.g. parasitic bacteria
Metabolism in Prokaryotes
• prokaryotes also vary with respect to O2 utilization
– 1. obligate anaerobes – cannot use O2 and are killed by
the presence of O2
• some live exclusively by fermenting their carbon sources
• some extract energy by using something other than O2 as the
ultimate electron acceptor - called anaerobic respiration
– e.g. nitrate ions or sulfate ions
– 2. obligate aerobes – require O2 for cellular respiration &
growth
– 3. facultative anaerobes – use O2 but only if its present
• can also carry out fermentation and anaerobic respiration
Metabolism in Bacteria
• prokaryotes can also utilize nitrogen for
metabolic pathways = nitrogen metabolism
• nitrogen is essential for the production of
amino acids and nucleic acids in all
organisms
• eukaryotes are limited in the nitrogenous
compounds they can derive this nitrogen
from
• prokaryotes have more options available:
• some can convert atmospheric N2 to
ammonia through a process called nitrogen
fixation
– e.g. cyanobacteria - blue-green algae
– this fixed nitrogen is capable of being used
biochemically
Metabolic Cooperation in Prokaryotes
• some prokaryotes are capable of undergoing both photosynthesis
and nitrogen fixation = metabolic cooperation
– e.g. cyanobacterium = Anabaena
– however a single cell must chose which pathway to use – can’t use both
– Anabaena - forms a filamentous colony in which some cells use photosynthesis
and other use nitrogen fixation
– most cells carry out only photosynthesis
– the cells that undergo nitrogen fixation are surrounded by a extra thick wall to
prevent O2 diffusion = heterocytes
Photosynthetic
cells
Heterocyte
20 µm
Bacterial adaptation and gene expression
• bacteria can respond to changes in their
environment by exerting metabolic control
at two levels
– 1. cells can adjust the activity of the
enzymes already present
• very fast response
• enzymes respond to chemical cues in
their environment and adjust their
activity
– 2. cells can adjust the amount of these
enzymes that they make
• through the regulation of gene
expression – transcription and
translation
• so genes in bacteria can be switched on
and off based on changes in the metabolic
status of the cell
• basic mechanism for this control = operon
model
Regulation of enzyme
activity
Precursor
Regulation of enzyme
production
Feedback
inhibition
Enzyme 1 Gene 1
Enzyme 2 Gene 2
Regulation
of gene
expression
Enzyme 3 Gene 3
Enzyme 4 Gene 4
Enzyme 5Gene 5
Tryptophan