Lecture #2 Prokaryotes Prokaryotes - PowerPoint Presentation

Slide1

Lecture #2

Prokaryotes

Slide2

Prokaryotes

microscopic single celled organisms

collective biomass –

10x of all eukaryotes!!!!!vast genetic diversity among membersphysical diversityshapes: spheres (coccus), rods (bacilli) and spirals

Spherical

(cocci)

Rod-shaped (bacilli)

Spiral

5 µm

2 µm

1 µm

Slide3

Prokaryotes

REMEMBER: adoption of a

three domain system of

superkingdoms1. Bacteria – prokaryotic (or Eubacteria)2. Archaea – prokaryotic3. Eukarya

- eukaryoticdivisions into protist, fungi, plants and animals

Slide4

“The Tree of Life”

Applying

molecular systematics

to the investigation of prokaryotic phylogeny has produced dramatic resultslead to a phylogenetic re-classification of prokaryotesdevelopment of Domain Bacteriaseparation into

9 major taxa of prokaryotes - based on molecular systematics

Domain Bacteria

Domain

Archaea

DomainEukarya

Universal ancestor

Proteobacteria

Alpha

Beta

Gamma

Delta

Epsilon

Chlamydias

Spirochetes

Cyanobacteria

Gram-positive

bacteria

Korarchaeotes

Euryarchaeotes

Crenarcaeotes

Nanoarchaeotes

Eukaryotes

Slide5

Role of prokaryotes

chemical recycling

ecosystems depend on a continual recycling of chemical elements between the living and nonliving components of this planet

prokaryotes function as decomposers prokaryotes also convert inorganic forms into organic forms for other living organisms

Slide6

Role of prokaryotes

symbiotic relationships

prokaryotes can possess

beneficial relationships with other prokaryotes in terms of metabolic cooperationalso hold beneficial relationships with other organisms like eukaryotesknown as symbiosisGENERAL DEFINITION: symbiosis = ecologic relationship between organisms of different speciestwo major kinds: mutualism & parasitism

Slide7

Role of prokaryotes

symbiotic relationships

mutualism

– both organisms benefithealth benefit parasitism – one organisms (parasite) benefits at the expense of the hostprokaryotes cause 50% of diseases in humanscause illness through the production of endotoxins or exotoxins

Slide8

Key Prokaryotic Adaptations

1. Cell surface structures

evolution of the cell wall

2. Motilityevolution of flagella3. Internal organization of DNAevolution of the chromosome and plasmid DNA4. Reproductionevolution of binary fission, conjugation, transformation and endospores

Slide9

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 osmolarityin a hypertonic environment – most prokaryotes will lose water and shrink = plasmolysiscell wall is NOT like the cell wall of plants and fungi – which are made of cellulose or chitinencloses the entire prokaryote

Slide10

The Bacterial Cell Wall

roles of the bacterial cell wall

structural:

forms an anchor for the attachment of many intracellular subsatncescounteracts the osmotic pressure created by the cytoplasmchanges in OP can result in the loss of water and plasmolysisinvolved in binary fission (reproduction)

protection against changes in ion and pH levels, foreign enzymes, phagocytosis by foreign pathogens

Slide11

Cell Wall & Peptidoglycans

most prokaryotic cell walls contain

peptidoglycans

(murein)presence is used to classify the two types of bacteria: gram negative and gram positivethicker in gram positive bacteria than gram negative peptidoglycan:sugar polymer modified with amino acids

cross-linked in gram-positive bacteriaforms a crystal lattice organization

Slide12

Peptidoglycan

peptidoglycan layer is a crystal lattice or mesh-like structure

formed from linear chains of two alternating sugars called

N-acetyl amino sugarsN-acetyl glucosamine (GlcNAc or NAG) N-acetyl muramic acid (MurNAc or

NAM) - 3 to 5 amino acids attachedinteractions occur between these amino acids = cross-linkingcross-linking results in a 3-dimensional structure that is strong and rigid

Slide13

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 madethis requires the assembly of more than a million new peptidoglycan subunits these subunits must be cross-linked – by enzymes called transpeptidasespenicillin & vancomycin –inhibits cell wall synthesis by preventing NAM and NAG cross-linking

Slide14

Gram positive bacteria

retain the crystal violet stain used in a Gram stain –

so they stain purple

simpler wall construction with larger amounts of peptidoglycanshigh peptidoglycan content of the cell wall take up the crystal violet dye – not washed away in subsequent stepsmost pathogens in human are gram +vedivided into cocci

and bacilli forms

Peptidoglycan

layer

Cell wall

Protein

Gram-

positive

bacteria

Gram-positive

Gram-negative

Gram-

negativebacteria

Peptidoglycan

layer

Cell wall

Plasma membrane

Lipopolysaccharide

Plasma membrane

Protein

Outer

membrane

20 µm

Slide15

Gram negative bacteria

do not retain the crystal violet dye

-

dye is washed awaycell wall of gram negative bacteria is comprised of a PG layer PLUS an outer membrane located outside the peptidoglycan layercomprised of lipopolysaccharides, lipoproteins and porins

the lipopolysaccharides are toxic to humans – endotoxin layer

Peptidoglycan

layer

PlasmaMembrane

Slide16

Gram negative bacteria

SOME WELL KNOWN GRAM NEGATIVE BACTERIA

coccobacilli

: H. influenzae, B. pertussis, L. pneumophiliacocci: N.meningitidis, N. gonorrhae

bacilli: E.coli, V. cholerae, H. pylori, S. dysenterae, Salmonella

Slide17

Gram staining

both Gram-positive and Gram-negative 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 STAINin 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% ethanol7. 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=OQ6C-gj_UHM

Slide18

Bacterial capsule

found in many prokaryotes – both +

ve

and –vefound outside the cell wallalso called the glycocalyxif it is less organized = slime layer

resists dehydrationroles in adherence to surfaces participates in colonizationmay make the bacteria resistant to the immune system

Slide19

Bacterial adhesion

via the

glycocalyx

/capsulealso through the development of specialized appendagesfimbrae – more numerous and shorter than pilipili – some can be specialized for the reproduction of the bacteria

Slide20

half of all bacteria exhibit

taxis

– the ability to move towards a specific signal

movement towards a chemical signal = chemotaxismovement toward light = phototaxismajor mobility mechanisms: flagellar and glidinggliding: movement of cells over surfaces without the aid of flagellanot completely understood

Bacterial motility

Slide21

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)

Bacterial Flagellae

Slide22

Bacterial Flagella

consist of three parts:

the basal body, the hook and the filament the filament consists of a hollow, rigid cylinder composed of a protein called flagellinattaches to a curved structure called the hookhook is attached to the basal body or basal apparatusbasal body: embedded in the cell wall down to the plasma membrane

made up of rings, a rotor and a rodthe rotor is connected to the hook via rings and a rod

stator

rotor

hook

filament

rod

basal body

http://

www.youtube.com

/

watch?v

=Ey7Emmddf7Y

Slide23

Bacterial Flagella

associated with the

basal body

is the motor made up of stationary ‘stators’ connects with the basal body’s rotating ‘rotor’ an ATP-driven proton pumps pump protons out of the bacteria (not shown)when the protons diffuse back in through the stator – turns the

rotor of the basal body and the attached rod the hook and attached filament also rotateanticlockwise rotation of flagella thrusts the cell forward with the flagellum trailing behind

stator

rotor

hook

filament

rod

basal body

Slide24

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 regionlocated in a nucleoid – a region of the cytoplasmcan also have several smaller circular pieces of DNA = plasmids

Slide25

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 recognizes2. initiators – proteins that recognize the oriC regionDnaA –binds to oriC

and unwinds a small area of the DNA helix (20 bps)DnaB –unwinds the DNA further - acts as a helicasetwo DnaB molecules move in opposite directions  replication bubble3. 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

Slide26

Prokaryotic replication

bacterial chromosome is a helix

unwinding will produce two

parent strands – sense and anti-sensethese parent strands are used a templates for the creation of new “daughter” strandsDNA daughter strands can only be made in one direction – 5’ to 3’so the enzymes run along the parent strand in the 3’  5’ directionthe anti-sense strand can be replicated continuously

= creates the leading daughter strandthe sense strand is replicated discontinuously = in fragments (Okazaki fragments) and creates the lagging daughter strand

Slide27

Prokaryotic DNA replication

at the

oriC

– a replication complex forms:1. helicase – DnaB – unwinds the DNA helix into separated parental strands2. 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 III4. DNA holoenzyme complex

– complex of several proteins including DNA polymerase III5. DNA ligase – links together Okazaki fragments into one continuous daughter strand

Slide28

Prokaryotic DNA replication

topoisomerase

DnaB

DnaG

DNA Pol III

Replication Complex

SSBs

primer

replicated

DNA

Replication Direction – daughter DNA made 5’ to 3’

for the

“

big picture

”

:

http://www.youtube.com/watch?v=-mtLXpgjHL0

Slide29

Prokaryotic Reproduction

once the DNA is replicated – the bacteria must divide

bacterial reproduction is through

binary fission = asexual reproductioneach replicated chromosome attaches to the plasma membranethe cell elongates and causes the two chromosomes to separate. the plasma membrane invaginates, or pinches inward toward the middle of the cellwhen it reaches the middle - the cell splits into two daughter cellslimited by resource availability and competition from other microorganisms (produce antibiotics)

Slide30

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.

Slide31

Genetic recombination in prokaryotes

Mutant

strain

arg

+

trp–

Mutantstrainarg+ trp–

Mixture

Mixture

Nocolonies(control)

No

colonies(control)

Coloniesgrew

Mutant

strain

arg

–

trp+

Mutantstrain

arg

–

trp

+

New strain

arg

+

trp

+

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 argininemix the two strains and grow in minimal media (lacks arginine and tryptophan)growth of the colony is observed

transfer of genetic information between the two strains

to create an arg+trp+ strain

Slide32

Genetic Recombination in Prokaryotes

Three processes bring

prokaryotic DNA

from different individuals together:1. Transformation2. Transduction3. Conjugation

Slide33

Transformation

Transformation

=

the uptake of naked, foreign DNA from the surrounding environmentExperiment: transformation of harmless Streptococcus pneumoniae bacteria into pneumonia-causing cells mix a live, nonpathogenic strain with a dead strain

non-pathogenic strain takes up a piece of DNA carrying the allele for pathogenicity foreign allele becomes incorporated into the non-pathogenic hosts chromosomecan be artificially induced in the labeither through chemical weakening of the plasma membraneOR electrical weakening

Slide34

Transduction

bacteriophages carry bacterial genes from one host cell to another

bacteriophage

– virus that infects a bacteriuminfection 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 cellif this is a random event = generalized transductionin specialized transduction – phage picks up only a few bacterial genes

A

+

Phage DNA

A+

Donor

cell

B

+

A+

B+

Crossing

over

A

+

A

–

B

–

Recipient

cell

A

+

B

–

Recombinant cell

Slide35

Conjugation

Conjugation: bacterial

“

sex”conjugation is the direct transfer of genetic material between bacterial cells that are temporarily joinedrequires the formation of a mating bridge – sex pilust

he 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)

Slide36

The F Plasmid and Conjugation

bacteria containing the F plasmid are designated F

+

cells (male)F+ cells transfer DNA to an F recipient cell (female)1. formation of the mating bridge2. a single strand of the F plasmid

breaks at a specific point and begins to move into the female bacteria

F plasmid

Bacterial chromosome

F

+ cell

Matingbridge

F

+

cell

F

+ cell

Bacterial

chromosome

F– cell

Conjunction and transfer of an F plasmid from and F

+

donor to an F

–

recipient

Slide37

The F Plasmid and Conjugation

3. the missing piece of DNA is regenerated in the male by replication – stays a double stranded plasmid despite losing it to the female

4. the female also replicates the incoming DNA – two new double stranded circular plasmids

5. two cells result that are F+ - therefore bacterial sex converts the female into a male

F plasmid

Bacterial chromosome

F

+ cell

Matingbridge

F

+

cell

F

+ cell

Bacterial

chromosome

F– cell

Conjunction and transfer of an F plasmid from and F

+

donor to an F

–

recipient

Slide38

Chromosomal Factors & Conjugation

if the F gene is part of the chromosome = cell is called the

Hfr

cell (high frequency of recombination)the Hfr cell forms a mating bridge with the F- cellsingle strand of the F factor breaks and moves into the F- cellmovement of the F factor “carries” additional genes into the F- cell – A+ and B+ allelesDNA replication begins in the Hfr and F- cell – to create double stranded DNA

F

+

cellHfr cell

F factor

Hfr cell

F

–

cell

Temporarypartialdiploid

Recombinant F–bacterium

Conjugation and transfer of part of the bacterial chromosome from an

Hfr donor to an F– recipient, resulting in recombination

the location and orientation of the F factor is important – determines what genes get transferred

Slide39

Chromosomal Factors & Conjugation

4. the

mating bridge usually

breaks before complete transfer of the chromosome-just the F factor and a few downstream genes move into the F- cell5. homologous recombination can result – B+ allele (from the Hfr cell) is switched for the B- allele (F- cell) 6. extra piece of DNA outside the chromosome is degraded over time

F

+ cell

Hfr cell

F factor

Hfr cell

F

–

cell

Temporarypartialdiploid

Recombinant F–bacterium

Conjugation and transfer of part of the bacterial chromosome from an

Hfr donor to an F– recipient, resulting in recombination

Slide40

Chromosomal Factors & Conjugation

the new

bacteria remains F- and is called a recombinant bacteria

F

+

cell

Hfr cellF factor

Hfr cell

F

–

cell

Temporarypartialdiploid

Recombinant F–

bacterium

Conjugation and transfer of part of the bacterial chromosome from anHfr donor to an F– recipient, resulting in recombination

Slide41

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

Slide42

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 fooduse the energy from either light (photo) or from electron donors in chemical reactions (chemo) to make this foodso they do NOT need organic carbon sources as a source of energy

Slide43

Adaptations in Nutritional Mode

2. Heterotrophs:

“different”, “nourishing

consumers in the food chainhave to “eat” – must obtain organic foodcannot “fix carbon” – i.e. must use organic sources of carbon as an energy source

Slide44

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 & plants2. chemoautotrophs – also need CO2 as a carbon sourceuse electron donors as their energy source – such as hydrogen sulfide, ammonia or irone.g. green sulfur bacteria

Slide45

Nutritional Mode Categories

3

.

photoheterotrophs: use light for energy but must obtain their carbon from outside organic sources4. chemoheterotrophs: must consume organic molecules for both energy and carbone.g. parasitic bacteria

Slide46

Slide47

Metabolism in Prokaryotes

prokaryotes also vary with respect to O2 utilization

1.

obligate anaerobes – cannot use O2 and are killed by the presence of O2some live exclusively by fermenting their carbon sourcessome extract energy by using something other than O2 as the ultimate electron acceptor - called anaerobic respiratione.g. nitrate ions or sulfate ions

2. obligate aerobes – require O2 for cellular respiration & growth3. facultative anaerobes

– use O2 but only if its presentcan also carry out fermentation and anaerobic respiration

Slide48

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 organismseukaryotes are limited in the nitrogenous compounds they can derive this nitrogen fromprokaryotes have more options available:some can convert atmospheric N2 to ammonia through a process called nitrogen fixatione.g. cyanobacteria - blue-green algaethis fixed nitrogen is capable of being used biochemically

Slide49

some prokaryotes

are capable of undergoing both photosynthesis and nitrogen fixation =

metabolic cooperation

e.g. cyanobacterium = Anabaenahowever a single cell must chose which pathway to use – can’t use bothAnabaena - forms a filamentous colony in which some cells use photosynthesis and other use nitrogen fixationmost cells carry out only photosynthesisthe cells that undergo nitrogen fixation are surrounded by a extra thick wall to prevent O2 diffusion = heterocytes

Metabolic Cooperation in Prokaryotes

Heterocyte

Photosynthetic

cells

20 µm

Slide50

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 presentvery fast responseenzymes respond to chemical cues in their environment and adjust their activity2. cells can adjust the amount of these enzymes that they makethrough the regulation of gene expression – transcription and translationso 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

Regulation of enzymeproduction

Enzyme 1

Regulation of gene

expression

Enzyme 2

Enzyme 3

Enzyme 4

Enzyme 5

Gene 2

Gene 1

Gene 3

Gene 4

Gene 5

Tryptophan

Precursor

Feedback

inhibition

Slide51

Bacterial groups

Bacteria or Eubacteria

include the vast majority of prokaryotes that we are aware

ofcomprised of 5 major groups:1. proteobacteria: diverse group of gram negative bacteria5 major subgroups: alpha  epsilon

2. gram-positive: very diversesolitary and colonialfree-living and parasitice.g. Bacillus, Streptococcus

Slide52

Bacterial Groups

3. cyanobacteria

:

blue-green algaephotoautotrophsO2-generating photosynthesis through chloroplasts4. chlamydias: parasitic bacteriacan only survive within animal cellscell walls lack peptidoglycan entirely5. spirochetes: helical in structureheterotrophs

most are free-livingsome can be parasitic

Slide53

Archaea

Archaea

:

share similarities with prokaryotes and eukaryotesdivided into four clades: Euryarchaeota, Crenarchaeota, Korarchaeota and Nanoarchaeota1996 - recent discovery of a new clade – Korarchaeotakoron = “young man”found in hot springs in Yellowstone

2002 – in hydrothermal vents off the coast of Iceland – found extremely small archaeaname Nanoarchaeota – smallest of the fournanos = “dwarf”

smallest genome known – only 500,000 base pairsthree other species found since then – hydrothermal vents and hot springs

Slide54

Archaea

first

Archaea

to be identified were found in extreme environments = extremophiles1. thermophiles (thermos = “hot”)clade Crenarchaeotathrive in very hot environments 2. halophiles – high saline environments (halo = “salt)clade Euryarchaeota

some tolerate the high salinity, others require itred-brown scum possess a visual pigment called bacteriorhodopsin 3. methanogens – named for the way they obtain energy

clade Euryarchaeotause CO2 to oxidize H2 and produce energy - releases methane (CH4) as a wastestrictest of anaerobes – obligate anaerobes