1
Q
Prokaryotes are defined as “before nucleus”, what does that mean?
A
DNA isn’t enveloped in any internal membrane but is free in the cytoplasm.
2
Q
Eukaryotes are defined as “true nucleus”, what does that mean?
A
Cell’s DNA is surrounded by a membrane.
3
Q
What is the difference between bacteria and archaea?
A
Archaea:
- Lack peptidoglycan
- Non-pathogenic
4
Q
How did eukaryotes come to exist? How was that proven?
A
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.
5
Q
What is the prokaryotic cell size in comparison to eukaryote’s?
A
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.
6
Q
What are the different types of prokaryotic shapes?
A
- Coccus (Strepto- plural)
- Rod/Bacillus
- Spirillum/Spirochete (:)
~ Vibrios (curved rods/comma)
7
Q
What are the structures/features of a prokaryotic cells?
A
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.
8
Q
What is a cell envelope?
A
It is what’s around a bacteria/archaea.
* Can be a single layer or complex multilayer structure.
9
Q
Describe the tree of life. What are the three major domains of life?
A
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
10
Q
What organism takes up the majority of the tree of life? State why.
A
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).
11
Q
Describe the three major domains of life in a phylogeny tree.
A
- 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.
12
Q
What is a cell envelope?
A
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.
13
Q
What is a plasmid?
A
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)).
14
Q
What is a nucleoid?
A
Where the main chromosome is.
15
Q
How do scientists classify the bacterial cell envelope?
A
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.
16
Q
What is the peptidoglycan cell layer?
A
A cell wall that consists of alternating sugars (polysaccharides / glycan) that are cross-linked together for strength by chains of amino acids (peptides).
17
Q
What is the structural difference between Gram +ve and -ve bacteria?
A
- 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.
18
Q
What is the outer membrane?
A
Made out of lipopolysaccharides and proteins.
19
Q
What is lipopolysaccharide? What is its purpose?
A
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.
20
Q
What is the function of the cell wall?
A
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.
21
Q
What is the function of the prokaryotic membrane?
A
- 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.
22
Q
What is the proton motive force?
A
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.
23
Q
Describe prokaryotic chromosomes.
A
- 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.
24
Q
What is DNA domain?
A
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).
25
Describe ribosomes and transcription & translation in prokaryotes.
* 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*).
26
What does it mean by transcription and translation are coupled?
They are happening at the same time.
Translation is initiated before transcription concluded.
27
What is a polysome?
A cluster of ribosomes held together by a strand of messenger RNA which each is translating.
28
What does “s” unit mean in microbiology?
**Svedberg unit.**
It’s a measure at which proteins sediment at when you centrifuge.
A sedimentation coefficient —> determines density & size.
Lighter —> sediment slower.
29
Why is translation & transcription coupling important?
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.
30
What is EPS/Glycocalyx?
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.
31
What is a capsule?
* 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.*
32
What is a slime layer? Describe it.
* 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.
33
What is the biofilm?
An assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance matrix.
34
What are common characteristics/purposes of capsules & slime layers?
* 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.
35
What is fimbriae?
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.
36
What is pili? How do they differ to fimbriae?
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.
37
Define and explain horizontal gene transfer.
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).
38
What is the purpose of horizontal gene transfer.
* 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.
39
What is flagella?
* 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).
40
How does flagella work?
Feed protons to it and it will spin around.
At the bottom of the flagella, there are energy producing reactions that makes it spin.
41
Relate flagella to movement.
* 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.
42
Why are genes not transcribed all the time?
Waste of energy, a cell doesn’t need its genes all the time.
43
What does it mean by RNAP complex being a holoenzyme?
A group of bunch of different proteins.
Enzyme + coenzyme (nonprotein) = Holoenzyme, active form of an enzyme.
RNAP + sigma factor = RNAP holoenzyme.
44
What is the sigma (σ) factor?
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.
45
Describe transcription and mention sigma factor.
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.
46
How can a cell produce only some genes at once?
* **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).
47
What is the function of σ70 / σD / RpoD?
It transcribes most genes in growing cells. It’s a major σ factor for normal growth (metabolic enzymes).
48
What is the function of σ32 / σH / RpoH?
Heat shock response.
Activated when the bacteria is experiencing a heat shock.
49
How does σH, heat shock factor, activate heat shock genes?
1. At 30°C, *rpoH* (gene of the σ factor) is transcribed (all the time), but is **not translated** effective,g into σH protein.
2. Special proteins that fold other proteins shunt σH to **degradation**.
3. At 42°C, σH 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, σH 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).
50
What’s the difference between σ factors and regulator proteins?
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.
51
There are many different σ factors for different contexts/situations, how does the bacteria know which σ to activate?
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.
52
Why is Gram positive or negative classification important?
*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.
53
What is a regulator protein?
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.
54
What is an operon?
* 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).
55
What is the operator region?
* 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.
56
What’s a repressor?
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.
57
How can operator sequences allow repression or induction of expression?
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.
58
What is positive and negative regulation?
* **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).
59
Provide an example of enzyme repression via negative control.
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).
60
Provide an example of enzyme induction via negative control.
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.
61
Provide an example of enzyme induction via positive control.
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 ).
Cells To Organisms
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