Lecture 1: Prokaryotes

Question 1

Prokaryotes are defined as “before nucleus”, what does that mean?

Answer 1

DNA isn’t enveloped in any internal membrane but is free in the cytoplasm.

Question 2

Eukaryotes are defined as “true nucleus”, what does that mean?

Answer 2

Cell’s DNA is surrounded by a membrane.

Question 3

What is the difference between bacteria and archaea?

Answer 3

Archaea:

• Lack peptidoglycan
• Non-pathogenic

Question 4

How did eukaryotes come to exist? How was that proven?

Answer 4

They arose from an archaean ancestor.

Certain kind of molecular mechanisms (e.g., division of cell into parts) that indicated they were the stepping stone to eukaryotes.

Question 5

What is the prokaryotic cell size in comparison to eukaryote’s?

Answer 5

Most prokaryotes are 1-3 micrometers.
Typical Eukaryotic cell is 10x the size (100x the volume).

Exceptions exist, Oscillatoria (a cyanobacterium) is 8x50 micrometres (thin but long), it’s almost as big as a eukaryotic cell.

Question 6

What are the different types of prokaryotic shapes?

Answer 6

• Coccus (Strepto- plural)
• Rod/Bacillus
• Spirillum/Spirochete (:)
  ~ Vibrios (curved rods/comma)

Question 7

What are the structures/features of a prokaryotic cells?

Answer 7

Outside
* Fimbrae
* Pili
* Flagellum

Inside
* Capsule – Cell Envelope
* Cell wall – Cell Envelope
* Plasma membrane – Cell Envelope
* Nucleoid
* Ribosome (Float around unpartitioned)
* Plasmid

~ Sometimes you’ll get:
* Little granules that would be found within the generalised sac that contains the inside of the internal structure.

• Invaginations in the cell membrane for a bigger surface area, but it’s not a separate internal structure (an invagination of an existing membrane).

Features
* Prokaryotes don’t have separate internal structures like eukaryotes.
* Don’t have a nucleus.
* No endoplasmic reticulum.
* No membranous organelles.

Question 8

What is a cell envelope?

Answer 8

It is what’s around a bacteria/archaea.
* Can be a single layer or complex multilayer structure.

Question 9

Describe the tree of life. What are the three major domains of life?

Answer 9

Made from genomes of prokaryotes and eukaryotes.
The more similar the genome is then the closer together they are on the little trees (subdivisions).

• Bacteria & Archaea (Prokaryotes)
• Eukaryotes

Question 10

What organism takes up the majority of the tree of life? State why.

Answer 10

Bacteria are very diverse genetically & phenotypically and have a lot of ecological niches.
Some infect but most don’t do anything (they convert chemicals into metabolites).

Archaea come 2nd and eukaryotes are the minority (single celled eukaryotes exist, such as amoeba).

Question 11

Describe the three major domains of life in a phylogeny tree.

Answer 11

• Bacteria is the last (universal) common ancestor. They originated from sacs of replicating chemicals that got more complicated ever since.
• Archaea are a little more specialised. While they have a lot in common with bacteria, some elements are more molecularly similar to eukaryotes.
• Eukaryotes share the same phylogenic clade with archaea.

~ Eukaryotic cell organisation is far more recent than the prokaryotic cell organisation.

Question 12

What is a cell envelope?

Answer 12

Defines what’s around a bacteria/archaea.
Can be a single layer or complex multilayer structure.

Consists of:
* Capsule (external slimy coat)
* Cell wall
* Plasma membrane

! ! !
* Not always in that order, or that all 3 of the structures are present.

• Some have cell walls made of a different material (peptidoglycan, peptide).
• Some bacteria & archaea lack cell walls, some have particular modifications.

Question 13

What is a plasmid?

Answer 13

Accessory genetic information.

(Some important for metabolism, but often they are accessory proteins —> improved proteins that already exist.
(e.g., antibiotic resistance plasmids have different forms of RNAP & important proteins that aren’t vulnerable to antibiotics)).

Question 14

What is a nucleoid?

Answer 14

Where the main chromosome is.

Question 15

How do scientists classify the bacterial cell envelope?

Answer 15

By classifying them into gram positive & gram negative bacteria, which are named after the ability to take up purple/Gram stain (there are exceptions though, some bacteria are neither + nor - since they only have a cell membrane).

• Gram +ve — take up the purple stain into the cell wall. Stain due to presence of thick peptidoglycan cell layer.
• Gram -ve — take up the counter stain into the cell membrane (appear pink). Do not take up primary stain. They are resistant due to a hard outer shell.
  ~ Purple stain won’t be able to stain the peptidoglycan structure. Counter stain will stain the outer membrane pink.

Question 16

What is the peptidoglycan cell layer?

Answer 16

A cell wall that consists of alternating sugars (polysaccharides / glycan) that are cross-linked together for strength by chains of amino acids (peptides).

Question 17

What is the structural difference between Gram +ve and -ve bacteria?

Answer 17

• Gram-negative — surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide.
• Gram-positive – lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram-negatives.

Question 18

What is the outer membrane?

Answer 18

Made out of lipopolysaccharides and proteins.

Question 19

What is lipopolysaccharide? What is its purpose?

Answer 19

Amphipathic glycoconjugate made of a lipid domain attached to a core ogliosaccharide (polysaccharide made out of small amount of monosaccharides) and a distal polysaccharide.

• Important in terms of take up of the cell stain, but also of different classes of bacteria (-/+).
  It will repel the stain. Purple stain will never get through the external membrane.
• Important in terms of interacting with the environment or the immune system.
  + What antibiotics can cross this cell membrane (might repel certain classes of antibiotics, some antibiotics may get into the cell really easily).
  Some antibiotic target LPS, others target peptidoglycan.

Question 20

What is the function of the cell wall?

Answer 20

It maintains rigidity (resisting osmotic pressure, changes in salt concentrations).

Bacteria with no peptidoglycan have an alternative:

• Cell membrane is packed with proteins.
• An over layer of a protein.
• Outer cell membrane without LPS.

Question 21

What is the function of the prokaryotic membrane?

Answer 21

• Permeability Barrier — Prevents leakage and functions as a gateway for transport of nutrients into and out if the cell.
  It has specialise proteins to control permeability (pumps, channels, etc).
• Protein Anchor — Site of many proteins involved in transport, bioenergetics (energy production), and chemotaxis (sensing chemicals that bacteria wants to go towards, like food, or away from, like antibiotics).
  Vital for bacterial interaction with host.
  Site of ATPase —> converts proton/electric motor force into ATP, which it can use as an energy source for all of its bioenergetic interactions.
• Energy conservation — Site of generation and use of the proton motive force.
  Makes sure that + ions stay outside & - ions stay inside, and generate ATP.
  Because there’s no organelles like eukaryotes, the membrane is an important site for energy production, etc.

Question 22

What is the proton motive force?

Answer 22

The force that promotes movement of protons across membranes downhill the electrochemical potential.

The transfer of H⁺ through a proton pump that generates an electrochemical gradient of protons.

Question 23

Describe prokaryotic chromosomes.

Answer 23

• Mostly circular (some exceptions do exist, some are linear).
• ‘Supercoiled’ — condensed around histones (imagine twisting an elastic band, end up with a bundle).
• Contain DNA binding proteins that help/relieve supercooling and can even end up with organising the genetic material into DNA domains.
  ( They don’t necessarily need all of that machinery to compact them down. There’s a natural tension in them and they tend to twist up ).
• Only a single copy.
• Plasmids carry accessory information — used in horizontal gene transfer.
• Some structure from proteins.

Question 24

What is DNA domain?

Answer 24

Contains structural motif that recognises double-/single- stranded DNA. It regulates transcription or plays a role in DNA replication, repair, storage, and modification (e.g., methylation).

Question 25

Describe ribosomes and transcription & translation in prokaryotes.

Answer 25

• Ribosomes are smaller than in Eukaryotes:
  ~ Prokatyotic 70S (30S+50S)
  ~ Eukaryotic 80S (40S+50S)
• No separate compartments in Prokaryotes (thus, no separate place for translation to happen).
• So, transcription & translation are coupled.
  ~ mRNA:RNAP:Ribosome complex (all these sorts of proteins interact with each other, and can be present together).
• Multiple ribosomes get loaded onto a single mRNA (polysome).

Question 26

What does it mean by transcription and translation are coupled?

Answer 26

They are happening at the same time.
Translation is initiated before transcription concluded.

Question 27

What is a polysome?

Answer 27

A cluster of ribosomes held together by a strand of messenger RNA which each is translating.

Question 28

What does “s” unit mean in microbiology?

Answer 28

Svedberg unit.

It’s a measure at which proteins sediment at when you centrifuge.
A sedimentation coefficient —> determines density & size.

Lighter —> sediment slower.

Question 29

Why is translation & transcription coupling important?

Answer 29

The control of transcription & translation is coupled a lot more in prokaryotes.
* Because if RNAP is slow, ribosomes will run out of mRNA to translate.
The speed control at which proteins shuttle along the nucleic acids is rigidly controlled.

Question 30

What is EPS/Glycocalyx?

Answer 30

A sticky or slimy material that many bacteria and archaea secrete on their surface.
Important in terms of their adherence to surfaces + structural integrity.

• Consists of polysaccharides, protein, glycoproteins, or glycolipids.
• Usually sub-divided into capsules or slime layers.

Question 31

What is a capsule?

Answer 31

• Layer organised into tight matrix that excludes small particles.
• Tightly associated with the prokaryote.
• Virulence factor (something that can contribute to pathogenicity).
• Stop antibiotics from reaching the bacteria.

Softer capsule can be used for adhering to surfaces.

Question 32

What is a slime layer? Describe it.

Answer 32

• Easily deformed and loosely attached.
• Tend to encompass more than one bacteria.
• Help stick to surfaces.
• Prevent bacteria from being phagocytosed.
• Help form communities and form biofilm.
• It is made up of glycoproteins, glycolipids, and exopolysaccharides.
• It may serve to trap nutrients.
• Aid in cell motility.
• Bind cells together or to adhere to smooth surfaces.

Question 33

What is the biofilm?

Answer 33

An assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance matrix.

Question 34

What are common characteristics/purposes of capsules & slime layers?

Answer 34

• Play a major role in the adherence of bacteria to surfaces in their environment.
• Involved in the formation of biofilms.
• Prevent dehydration.
• Capsules act as virulence factors for some diseases —> Prevent the bacteria from being phagocytosed.
• Help some bacteria avoid destruction by host immune system —> Big capsule, can’t be engulfed easily or antibiotics can’t get into them.

Question 35

What is fimbriae?

Answer 35

Bristle-like, short fibre.

• Plays a role in the adherence of bacteria to host cells in either symbiotic or pathogenic situations.
• Help bacteria to clump together, adhere to surfaces.

Question 36

What is pili? How do they differ to fimbriae?

Answer 36

Long hair-like tubular microfibres.

• Pili are longer than Fimbriae and there will only be a few per cell (exceptions exist).
• Important for mating (the ability of bacteria to exchange genetic information).
• Sex pili are responsible for the attachment of donor and recipient cells in bacterial conjugation.
• Type IV pili support adhesion and twitching motility.
• Pili are also used in adherence to surfaces.
• Once the Pili find another bacteria, they’ll pierce the bacteria.

Question 37

Define and explain horizontal gene transfer.

Answer 37

Donor bacteria posts/injects a plasmid own the Pili into the recipient cell.

Plasmid —> accessory genes/proteins
(not necessarily required for the functioning of the cell, but might be a different form of protein, e.g. that’s antibiotic resistant).

Question 38

What is the purpose of horizontal gene transfer.

Answer 38

• Increases diversity of the bacterial population.

If the recipient wasn’t antibiotic resistant, but the donor was:
After the genetic transfer, the recipient is resistant.

Question 39

What is flagella?

Answer 39

• Used for locomotion —> molecular motor.
• Hollow tube like appendages composed entirely of a protein called flagellin that are 12-30 nm in diameter.
• Connected to the bacterial cell body by a complex structure consisting of a hook and basal body.
• There are different forms of flagella arrangement:
• Monotrichous (single one).
• Lophotrichous (bunch on one side).
• Amptrichous (1 at either end).
• Peritrichous (All around the outside; look like fimbri but they’re motorised).

Question 40

How does flagella work?

Answer 40

Feed protons to it and it will spin around.
At the bottom of the flagella, there are energy producing reactions that makes it spin.

Question 41

Relate flagella to movement.

Answer 41

• Can be in response to stimuli.
  ~ Towards food source.
  ~ Away from toxins (UV light (sterilises & damages bacteria), excess heat).
• Can be a virulence factor (any kind of factor that tends to make the prokaryotes more pathogenic or more virulent) — helps bacteria to spread.

Flagella can be peritrichous (located in a random assortment across the cell) or polar:
* Peritrichous flagella allows the prokaryotes to move any direction and bundle/push apart.

• Polar flagella (on one side) can be reversible or unidirectional.

Question 42

Why are genes not transcribed all the time?

Answer 42

Waste of energy, a cell doesn’t need its genes all the time.

Question 43

What does it mean by RNAP complex being a holoenzyme?

Answer 43

A group of bunch of different proteins.

Enzyme + coenzyme (nonprotein) = Holoenzyme, active form of an enzyme.

RNAP + sigma factor = RNAP holoenzyme.

Question 44

What is the sigma (σ) factor?

Answer 44

A subunit in prokaryotes that controls binding. It is a bit of RNAPc that bonds to DNA.

RNAPc binds when σ factor is present, because it does the scanning & binding to promoter.

• It can be used to control the transcription of genes.
• Needed for initiation; enables specific binding of RNAP to gene promoters.

Question 45

Describe transcription and mention sigma factor.

Answer 45

1. RNAP holoenzyme scans DNA for promoter sequences.
2. Binding to the specific promoter sequence forms the closed complex.

(If present, the antibiotic rifamycin will bind to this form of polymerase and prevent formation of the subsequent open complex.)

3. RNA polymerase unwinds DNA and begins _transcribing _ RNA from ribonucleoside troiphosphates (rNTPs).
4. Sigma factor leaves the complex.
   It can leave the complex, once polymerisation has happened, so it’s free to go back to the beginning and recruit some more RNAP holoenzyme to promoters.

Question 46

How can a cell produce only some genes at once?

Answer 46

• Method 1 (Not so common in prokaryotes):
  Only activate some genes at once.

~ Requires a way to tell the difference between the genes.

~ Requires different proteins for the different genes.

Multiple σ factors in prokaryotes enables selectivity of gene activation.
Not common in prokaryotes because not a lot of genes have different σ factors.

• Method 2: Inhibit genes until you need them.

~ Can involve other DNA binding proteins that prevent σ binding to the promoters.

~ Can involve inhibition or degradation of σ factors, or preventing their production.
(E.g. anti-σ factor, bind to σ factor & inhibit transcription. There are anti-anti-σ factors that knock the anti-σ off the σ factor to activate it).

Question 47

What is the function of σ70 / σD / RpoD?

Answer 47

It transcribes most genes in growing cells. It’s a major σ factor for normal growth (metabolic enzymes).

Question 48

What is the function of σ32 / σH / RpoH?

Answer 48

Heat shock response.
Activated when the bacteria is experiencing a heat shock.

Question 49

How does σ^H, heat shock factor, activate heat shock genes?

Answer 49

1. At 30°C, rpoH (gene of the σ factor) is transcribed (all the time), but is not translated effective,g into σ^H protein.
2. Special proteins that fold other proteins shunt σ^H to degradation.
3. At 42°C, σ^H protein is translated at high levels.
4. At 42°C, proteins denature from their native folded forms to unfolded forms. The unfolded forms are bound by chaperon proteins.
   (σ factor no longer turned over — Chaperons are distracted because, rather than degrading σ factor, instead they refold denatured proteins and are thus not available to degrade σ anymore).
5. Freed from the chaperone proteins, σ^H is not degraded and can drive expression of heat-shock genes.

*(Heat-shock genes might be more chaperones, or specialised (heat stable version) form of proteins that are more stable at higher temperatures, or flagella proteins to let the cell swim away from the heat, or proteins that make the cell wall thicker (so it undergoes less osmotic pressure), or more stable form of polymerase).

Question 50

What’s the difference between σ factors and regulator proteins?

Answer 50

A regulator protein is any other kind of protein that’s capable of binding DNA that’s not a σ factor.

σ factors are a part of the RNA polymerase complex. They are there to guide RNAP to the promoter region to initiate transcription.

Question 51

There are many different σ factors for different contexts/situations, how does the bacteria know which σ to activate?

Answer 51

One of the ways in which cells can do this is the reception of signals and the transduction of those signals into genetic change.
So, the activation or the inhibition of certain genes.

Question 52

Why is Gram positive or negative classification important?

Answer 52

The stain is not important, it’s what the stain tells us about the bacteria.

Knowledge of making a cell envelope in 2 different ways.

Important because it can be a virulence factor:
* It’s important in terms of antibiotic choice.
or
* How LPS can get cleaved off and become endotoxins (protective and structural function), and they can stop clotting and stop the recruitment of immune cells to a particular site.

Question 53

What is a regulator protein?

Answer 53

Regulate different processes and activities.

Transcription factors are a type of regulatory proteins that activate/inhibit transcription of DNA by binding to specific DNA sequences.

It’s more frequent that other proteins will help to regulate gene expression than sigma factors.

Question 54

What is an operon?

Answer 54

• A cluster of genes (of different regulatory elements/bunch of different genes that are involved in the same bio synthetic pathway) on the same functioning unit.
• The gene, or genes, which get transcribed when the operator is bound.
• Named after the operator region.
• Multiple proteins can be produced from a single mRNA (polycistronic — 2 or more proteins are encoded on a single mRNA).

Question 55

What is the operator region?

Answer 55

• A binding site for regulator proteins.
• Useful for co-regulating several genes in the same pathway.
  (Useful because the cell can switch sugar substrates and respond to changes in the environment really quickly).
• Can be downstream (3’ of the promoter region) or upstream (5’ of the promoter region).

~ If you have 4 genes on the same pathway:
If you regulate one gene, you regulate all of them.

Question 56

What’s a repressor?

Answer 56

A regulatory protein that can completely switch off a pathway.
If it’s downstream of the promoter region, the suppressor might act like a steric block —> So, no proteins can be produced.

Question 57

How can operator sequences allow repression or induction of expression?

Answer 57

Through negative or positive control.

Depending on where the operon is in relation to the promoter (which orientation the operator sequences are to the promoter), there can be different mechanisms of control.
Depending on whether our sensing of change in the environment involves the activation of that repressor/repression of activator.

• Negative control — operator sequences are downstream of the RNAP binding site.
• Positive control — operator sequences are upstream of RNAP binding site.

Question 58

What is positive and negative regulation?

Answer 58

• Positive regulation —> regulation in which the presence of specific regulatory element increases the expression of genetic information quantitatively.
  (More common in eukaryotes).
  ~ Involves activators; activating the activators or inhibiting the activators.
• Negative regulation —> regulation in which the presence of specific regulatory elements diminishes the expression of genetic information.
  ~ Involves inhibitors; either inhibiting the inhibitor or activating the inhibitor.
  (Using an operator/operon to negatively regulate the expression of enzymes).

Question 59

Provide an example of enzyme repression via negative control.

Answer 59

Bacteria synthesise arginine because there is none in the media.
After arginine is added, the bacteria no longer need to synthesise their own. So to save energy, they “switch off” the arginine biosynthesis enzymes.

• Add arginine to media of a growing culture.
• Production of arginine synthesis enzymes repressed.
• Total protein/growth unaffected.
  (Bacteria don’t need to make their own arginine.
  Arginine is not getting turned over. The level of arginine biosynthesis enzymes doesn’t fall, it just stops getting produced.).
• Arginine acts as a co-repressor.
• Binds to repressor protein that binds operator.
• Inhibits RNAP transcription (the RNAP is still binding but it’s no longer transcribing DNA).

Question 60

Provide an example of enzyme induction via negative control.

Answer 60

Culture of bacteria has the right amount of sugars. But β-galatosidase isn’t being synthesised until we add lactose (probably because it is easier to digest, or energy dense, or glucose ran out (preferential)).

• Add lactose to media of a growing culture.
• Production of β-galatosidase (lactose digesting) enzymes activated.
• Total protein/ growth unaffected.
• Allolactose acts as an inducer (Repressor gets kicked off the operator, inducer is repressing a repressor).
• Binds to repressor protein that binds operator.
• Allows RNAP transcription.

Question 61

Provide an example of enzyme induction via positive control.

Answer 61

In positive control there is no inhibitor, because we recruit RNAP.
Operon is 5’ —> Doesn’t work to stop the progression of the polymerase. It works to recruit the polymerase to the promoter.

• Add maltose to media of a growing culture.
• Production of maltose digesting enzymes activated.
• Total protein / growth unaffected,
• Maltose acts as an inducer (1).
• Binds to activator protein that binds (2) operator.
• Changes DNA structure to allow Polymerase to bond (or recruit RNAP somehow ).