CC5 - Bacterial Warfare

Question 1

How does the Sec translocon differ from the BAM machinery in terms of structure, substrates, energy sources and origin?

Answer 1

1. Structure: Sec is 3 proteins forming a channel. BAM is 5 proteins that aids folding and insertion.
2. Substrate: Sec translocates both membrane proteins and soluble proteins. BAM is only for beta-barrel membrane proteins.
3. Energy sources: Sec uses ATP hydrolysis. Not fully understood how BAM gets its energy but it’s not ATP.
4. Origin: Sec is conserved across all 3 domains; BAM is only in bacteria and is thought to have evolved from Sec.

Question 2

What are bacteroides and how can they be both commensal and pathogenic bacteria?

Answer 2

They’re a diverse group of Gram-negative, strictly anaerobic bacteria found in the gut of many animals, including humans.

Some species are considered commensal because they have a mutualistic relationship with their host and provide important functions such as aiding in digestion and synthesis of essential vitamins. However, other species can be opportunistic pathogens by causing abscesses if found elsewhere in the body e.g., B. fragilis.

Question 3

Describe the structure of the gram-negative cell envelope. What is the function of each layer?

Answer 3

1. OM: asymmetrical lipid bilayer, containing LPS in the outer leaflet and phospholipids in the inner leaflet.
   - Barrier from toxins in the environment
   - Responsible for interactions with environment
2. PERIPLASM: thin layer containing enzymes and proteins for nutrient uptake, cell wall synthesis, etc.
   (Thin layer of peptidoglycan for structural support)
3. IM: symmetric lipid bilayer of phospholipids and proteins.
   - Maintains electrochemical gradients

Question 4

Describe the structure of the gram-positive cell envelope.

Answer 4

1. CELL WALL: thick layer of peptidoglycan and teichoic acids.
   - Structural support
   - Teichoic acids are negatively charged that help regulate cation movement.
2. CELL MEMBRANE: phospholipid bilayer.
   - Permeability barrier
   - Regulates the movement of molecules

Question 5

What is the role of Lipid A in bacterial infections?

Answer 5

Lipid A is a component of LPS found in the OM of gram-negative bacteria. It’s a potent endotoxin i.e., it can stimulate the host immune system to trigger inflammatory signals.

The resulting immune response can cause fever, septic shock, tissue damage and in severe cases organ failure and death. The severity of this depends on the structure of lipid A and how immunogenic they are.

Question 6

Describe the structure and function of peptidoglycan.

Answer 6

Structure:
- repeating units of alternating GlcNAc and MurNAc residues, connected by a B-1,4 glycosidic bond
- 20-40 disaccharide units per glycan strand in E. coli
- The peptide chain is cross-linked by short peptides of both L- and D- amino acids

Function:
- structural support
- protection from osmotic stress

Question 7

How is peptidoglycan synthesised in gram-negative bacteria?

Answer 7

1. Synthesis of the precursor molecules
2. Assembly of the PG subunits
3. Cross-linking
4. Remodeling

Stage 1: (CYTOPLASM) A pento-peptide unit is formed in the cytoplasm through synthesis reactions involving glutamine, sugar molecules and phosphate transfers.This results in UDP-GlcNAc, some of which is then converted to UDP-MurNAc. Each of these reactions requires ATP for energy. Bactoprenol transports these PG subunits across the cell membrane.

Stage 2: (CYTOPLASMIC MEMBRANE) Further reactions cause the pento-peptide unit to form Lipid II, a disaccharide precursor to PG. Lipid II is transported across the membrane by flippase (MurJ), where it can be added to the growing glycan chain by PG glycosyltransferase.

In the final step, a penicillin-binding proteins (transpeptidases) crosslink the individual glycan chains.

Question 8

How is peptidoglycan remodelled during growth and division in E. coli?

Answer 8

The rate of PG synthesis and remodeling is tightly regulated to ensure proper cell growth and division. PG growth during cell elongation and division involves two distinct machineries: the elongasome and the divisome.

The elongasome, organized by the actin homologue MreB, facilitates the insertion of new material during growth along the lateral region of the cell cylinder.

The divisome, under the ultimate control of the tubulin homologue FtsZ, facilitates peptidoglycan synthesis during cell division. FtsZ forms a constricting ring that separates dividing cells.

PG hydrolases also play important roles in active turnover of PG by breaking and re-using the fragments (~50% of PG is turned over in E. coli per cell cycle).

Question 9

What is the function of the Lol pathway? Give an overview of the steps involved.

Answer 9

The Lol pathway is responsible for the localization of lipoproteins to the OM in gram-negative bacteria.

1. Lipoproteins are synthesized in the cytoplasm as protein precursors with an N-terminal signal sequence for transport. This contains a lipobox consensus sequence.
2. Translocation across the IM by Sec (in E. coli - other organisms use Tat).
3. Addition of a diacylglycerol to a conserved cys residue (Lgt).
4. This signals for SS cleavage by a signal peptidase (LspA).
5. Cys residue is acylated again by Lnt, generating a triacylated lipoprotein.
6. Recognition by LolCDE complex which powers extraction of the lipoprotein using ATP.
7. Lipoprotein chaperoned by LolA to the OM acceptor LolB
8. LolB inserts the lipoprotein into the inner leaflet of the OM, returning LolA to LolCDE.

Question 10

What is the structure and function of Lpp?

Answer 10

A highly conserved, major OM protein found in many Gram-negative bacteria.

Structure:
- signal peptide, short N-terminal domain, central domain enriched in Ala and Lys, and hydrophobic C-terminal anchor domain.
- forms a trimeric, helical coiled-coil

Function:
- stabilizes OM and cell wall
- sets distance between PG and OM
- Lpp mutants have defects in cell division
- Lpp mutants are more susceptible to certain antibiotics

Question 11

What is the structure and function of the Bam complex?

Answer 11

Structure:
- 5 proteins with a central BamA channel of 16-strands
- BamA has 5 polypeptide transport-associated (POTRA) domains that BamB and BamC interact with
- Water-filled lumen
- Lipoproteins form a ring beneath BamA cavity
- Lateral gate between B1 and B16
- Loops forms a dome over the barrel

Function:
OMP folding and insertion.

Question 12

What structural features of BamA underpin its role as a catalyst for OMP folding?

Answer 12

The B-strands are much shorter at the lateral gate (20A vs 12A), likely destabilizing the membrane bilayer.

Loop6 near the gate acts as a clamp during OMP folding, and may undergo conformational changes that help stabilize the OMPs for proper folding and insertion.

Question 13

What are the two models for beta-barrel assembly within the Bam complex and what is the evidence for each?

Answer 13

The budding model: the lateral gate pairs by hydrogen bonding with substrate B-strands to nucleate B-sheet formation (similar to Sec model).
- Structures show both an open and closed state of BamA
- Recent research into the MOA for darobactin also suggests a lateral gate

The assisted model: BamA has an indirect role in distorting the membrane bilayer to reduce the kinetic barrier for pre-folded OMP insertion.
- In vitro folding studies.

Question 14

What is meant by the terms ‘murein’ and ‘murein sacculus’

Answer 14

Murein: peptidoglycan

Murein sacculus: bacterial cell wall

Question 15

Describe the structure and function of MurJ, involved in PG biosynthesis.

Answer 15

MurJ is a flippase that moves PG precursors from the cytoplasm into the periplasm.

Crystal structures show is has 14 TM helices, and 2A structures identified a hydrophobic groove that leads into a large central cavity that’s mostly cationic. This is suggestive of an alternating access mechanism.

Question 16

What are the associated functions of bacterial lipoproteins?

Answer 16

• Maintaining attachment to PG
• Regulation of PG synthesis via PBPs
• Assembly of OMPs
• Biogenesis of LPS
• Cell surface adhesion

Question 17

What is the Lol avoidance signal?

Answer 17

The Lol avoidance signal is a short amino acid sequence at the N-terminus of the lipoprotein that prevents recognition by the Lol pathway. This sequence is thought to interfere with the binding of LolA to the lipoprotein, which prevents its transport to the outer membrane. Lipoproteins that contain the Lol avoidance signal are typically anchored to the inner membrane by their lipid moiety, and they are not exposed to the external environment.

Question 18

How can antibiotics inhibit protein synthesis? Give an example and how resistance can develop against this.

Answer 18

Binding to the 30S subunit can inhibit protein synthesis.

E.g., tetracycline binds to 16S rRNA above the A-site which blocks rotation of the aatRNA after codon-anticodon pairing. This site is different to that found in eukaryotes, but mutations in the bacterial binding site can impair drug binding.

Question 19

What is the mechanism of action of B-lactam antibiotics? Give an example of one. How has resistance occurred against them?

Answer 19

These target the transpeptidase centers of PBPs that cross-link the glycans within the periplasm. e.g., penicillin.

Beta-lactams have a similar structure to the dipeptide found at the end of the PG precursor molecule. PBPs bind this dipeptide and bind irreversibly at the active site. This induces oxidative stress, as well as weakening of the cell wall.

B-lactamases hydrolyze the amide bond in the beta-lactam ring, rendering the antibiotic inactive.

Question 20

What are the different classes of B-lactamases? How do they function? Why are they so severe?

Answer 20

1. Serine-B-lactamases (most common) - uses a Ser residue to attack the B-lactam ring.
2. Metallo-beta-lactamases - require Zn in their active site.

Both of these confer resistance to many B-lactam antibiotics, and metallo-beta-lactamases are even resistant to carbapenems, the last-resort antibiotics.

Question 21

How are B-lactamases being targeted to prevent antibiotic resistance? Give an example.

Answer 21

Combination therapies use both B-lactams and B-lactamase inhibitors.

B-lactamase inhibitors are suicide substrates, irreversibly modifying active site groups.

E.g., clavulanic acid is used in combination with amoxicillin.

Question 22

What are carbapenems? How do they work? What is the problem with even these antibiotics?

Answer 22

A class of broad-spectrum antibiotics that are often considered to be last resort antibiotics.

Like other beta-lactam antibiotics, they inhibit PBPs.

Carbapenemases have already begun to arise and are resistant to B-lactamase inhibitors such as clavulanic acid.

Question 23

How are porins involved in antibiotic resistance? What are the 3 main methods of porins conferring antibiotic resistance?

Answer 23

Many antibiotics rely on porins to reach their targets in the bacterial cell. Therefore, changes in the structure or expression of porins can lead to decreased antibiotic uptake and increased resistance.

1. Porin loss
2. Narrowing of channels
3. Reduced expression

Question 24

What is TolC and how is it involved in antibiotic resistance? What is the role of AcrAB in this?

Answer 24

TolC is an OM protein involved in the efflux pump system found in Gram-negative bacteria.

Many bacteria can increase TolC expression in response to antibiotic exposure, allowing them to rapidly pump out antibiotics and reduce their intracellular concentration.

AcrAB acts as the inner membrane transporter and TolC acts as the outer membrane channel, forming a continuous conduit across the two membranes.

Question 25

What is vancomycin? How is resistance developing against it?

Answer 25

A glycopeptide antibiotic that targets the dipeptide of lipid II in PG biosynthesis. This disrupts cell wall crosslinking.

It’s only effective against gram-positive bacteria due to gram-negative bacteria having an impermeable OM.

Resistance is occurring through enzyme-catalyzed substitutions at the dipeptide alanine, preventing vancomycin from binding.

Question 26

What are newer antibiotics targeting, now that efflux systems are becoming far more sophisticated? Give an example of one of these antibiotics.

Answer 26

Larger antibiotics are being developed to kill Gram-negative bacteria using surface-accessible targets, such as BamA.

Darobactin forms a rigid B-strand that binds at the BamA lateral gate and prevents OMP insertion.

Question 27

What is the structure of porins? What is their role in the outer membrane of gram-negative bacteria? Give an example of one and the structural property that confers its specificity.

Answer 27

Trimers of B-barrels tend to form porins that allow passive diffusion of small molecules across the outer membrane. These have charged, narrow eyelets.

e.g., OmpF.
A loop goes into the barrel and the amino acid composition of this loop dictates the size/charge of the molecules that can diffuse through.

LPS is essential in the oligomerization of OmpF in the outer membrane, although we don’t know why.

Question 28

How do we know that OMPs are mostly immobile? How are OMPs turned over in bacteria?

Answer 28

FRAP experiments and the use of non-covalent labels on OMPs showed their lack of movement. However, bacteria are able to change their OMPs to enable adaptation to a particular habitat…without an energy source…

This is thought to occur through binary partitioning, in which old OMPs are displaced to the poles of growing cells as new OMPs take their place.

Question 29

What are the 6 structural characteristics of OMPs?

Answer 29

1. Beta-barrel structure
2. Amphipathic nature (hydrophobic outside, hydrophilic inside)
3. Porin-like channels
4. Periplasmic domains
5. Variable loop regions
6. Aromatic girdles

Question 30

What are aromatic girdles? What is the role in OMPs?

Answer 30

A conserved structural feature of OMPs, composed of two rings of aromatic amino acids that stack on top of one another. The first ring is located near the extracellular surface and the second is deeper within the barrel, near the periplasmic space.

• stabilize the barrel structure by creating a hydrophobic environment and hydrogen bonds with adjacent beta-strands to maintain integrity of the barrel.
• prevent diffusion of hydrophilic molecules

Question 31

What interactions aid in OM stabilization with the underlying peptidoglycan? What do they all interact with for this stabilization?

Answer 31

Covalent: Lpp
Non-covalent: Pal and OmpA

mDAP is an amino acid commonly found in bacterial cell walls. It’s a component of the peptide cross-linking that occurs in the PG layer, and all of the stabilizing interactions occurs to it.

Question 32

How do OMPs interact with the outer membrane? What does this aid with?

Answer 32

Extracellular loops contain positively charged residues to interact with negatively charged phosphate groups of LPS. These help to stabilize the OMP’s position in the membrane.

Some OMPs also have conserved polar LPS binding sites that directly contact the phosphate groups.

Question 33

What enzymes can be found in the outer membrane of gram-negative bacteria? Give a specific example, and state its function.

Answer 33

B-lactamases, proteases, transporters…

OmpT protease cleaves basic antimicrobial peptides from the host immune system, with preferential cleavage of dibasic amino acids.

Question 34

How are OMPs organised spatially and temporally in the bacterial outer membrane? How do we know this?

Answer 34

Spatially: OMPs cluster to form islands where their diffusion is restricted by interactions with other OMPs. These are distributed throughout the cell and contain Bam complexes to catalyze the insertion of more OMPs.
- Shown using fluorescent microscopy of live bacteria.

Temporally: OMP biogenesis occurs as a gradient, with old OMP islands being pushed towards the poles of growing cells due to OMP biogenesis occuring at the mid-body, leading to binary distribution when cells divide.

These are separated from LPS-rich regions.
- Shown via atomic force microscopy.

Question 35

Why do bacteria switch their OMPs, such as switching between OmpF and OmpC?

Answer 35

1. Glucose availability: OmpC is upregulated when glucose is abundant, likely because it’s more selective for small molecules which are important for maintaining osmotic balance, while OmpF is more permeable to larger molecules such as glucose.
2. Osmotic stress: high osmotic stress causes upregulation of OmpF as it’s more permeable to larger molecules, helping the bacteria to take up nutrients and restore osmotic balance.

Question 36

What is the role of FHA domains in CDIs?

Answer 36

In CDI systems, the FHA domain is found in the CdiA protein, serving as the receptor-binding domain.

Question 37

How do type VI/T6SS secretion nanomachines assemble and puncture cells?

Answer 37

The machinery is assembled in an orderly manner, starting with membrane complex formation which allows baseplate positioning. The baseplate serves as a platform for contractile tail elongation.

A tube, made up of a repeating subunit called the HCP, forms and the sheath component wraps around the inner tube - made up of a protein called VipA/VipB. The sheath contracts in response to a signal, which drives the tube to puncture the target cell membrane.

Inside the tube, or loaded onto the tip, are various effector proteins that can be delivered into the target cell upon puncture.

The ATPase ClpV recycles the sheath.

Question 38

What is T6SS? Is it species selective?

Answer 38

A large contractile machine that injects effectors into the pathogen cell with no species selectivity.

Question 39

What are CDI systems in gram-negative bacteria? What do they consist of? How do bacteria protect themselves from their own CDI?

Answer 39

(Contact-dependent inhibition) Bacterial warfare systems used to inhibit the growth of other bacteria through physical contact.

The CDI system typically consists of CdiA, a large extracellular filamentous protein. CdiA has a receptor-binding domain at its C-terminus that recognizes specific receptors on target bacteria. The N-terminus contains a toxic effector domain that can be delivered into the target bacteria upon contact, leading to growth inhibition or cell death.

The CDI system is often accompanied by an immunity protein, which is co-expressed with CdiA and protects the bacteria from self-intoxication.

Question 40

What are bacteriocins? How are they released? How do they then enter target cells and function?

Answer 40

• Antimicrobial peptides with the ability to selectively target and kill other bacteria.
• Diffusion, secretion (e.g., colicins), export, cell lysis.
• Receptor-mediated endocytosis, pore formation, enzymatic activity… and must use the PMF to get across the inner membrane.

Some bacteriocins are PTM’d with unusual amino acids to make them resistant to proteases. Others, such as colicins, are large proteins that have enzymatic activity and can disrupt essential cellular processes in target bacteria.

Question 41

What is the difference between a class A and a class B colicin?

Answer 41

A: uses the Tol system
B: uses the TonB system

Question 42

What is the function and structure nuclease colicins? Give examples.

Answer 42

Function:
A type of colicins that cleave DNA, RNA or both in target cells.

Structure:
Elongated hairpins with unstructured N-termini and a tightly-bound immunity protein.

Examples:
- ColE3 cleaves rRNA within the A site of the ribosome.
- ColE9 binds and cuts dsDNA in the minor groove in a non-specific manner.

Question 43

How does ColE9 exploit the PMF to translocate into cells? What experimental evidence is there for this?

Answer 43

It assembles an outer membrane translocon to activate cellular transport. It threads through two pores of OmpF to deliver a TolB-binding epitope to the periplasm, connecting the surface-bound ColE9 to the PMF.

Evidence:
- Cryo-EM structures of OmpF occupied by ColE9
- Labelling of ColE9 with bulky fluorophores slowed passage

Question 44

How are CDIs able to confer specificity against target species?

Answer 44

CDIs use a variety of receptors, often BamA. The extracellular loops of BamA are highly variable, explaining species specificity.

Question 45

What is tit-for-tat killing?

Answer 45

Assembly of the T6SS at the exact point where the cell is initially attacked.

Question 46

What is meant by the term ‘molecular forcefield’ in MD?

Answer 46

Refers to a mathematical description of the intermolecular forces that govern the behaviour of the atoms or molecules in the simulation.

It defines the potential energy of the system as a function of the positions and velocities of the atoms/molecules.

Question 47

Why was an accurate simulation of LPS required for studying the outer membrane? What did it show and how was this discovery studied further?

Answer 47

The use of oversimplified models with only phospholipids is highly inaccurate as it would omit any interactions LPS is having with OMPs.

Once LPS was accurately simulated, it showed divalent cations that provide stabilizing electrostatic interactions with the phosphate groups of LPS.

Further studies involved replacing the cations with twice the number of monovalent cations which lead to the loss of membrane structure and mixing of inner and outer leaflets.

Question 48

What is umbrella sampling?

Answer 48

A computational method used in MD to calculate the free energy profile of a system along a reaction coordinate.

Question 49

How has molecular dynamics shown that bacteria can use their OM to protect against hydrophobic AND polar molecules?

Answer 49

MD simulations and umbrella sampling were used to calculate the free energy of transferring a molecule from one side of the membrane to another.

Hydrophobic molecules can’t interact with lipid A headgroups. Polar molecules face equal barriers in the core of the membrane.

Question 50

What is polymixin B1? How does it interact with the outer membrane of gram-negative bacteria?

Answer 50

A last-resort antibiotic that binds lipid A, causing the membrane to become destabilized and leading to cell death.

Simulations show that its fatty acid tail inserts spontaneously into the core of the membrane and some water molecules are able to enter with the polar regions of PB1, creating a defect. The NH3+ groups form very tight electrostatic interactions with the phosphate groups of lipid A, suggesting there’s a large kinetic barrier to overcome before PB1 can form a pore. Hence entry into a membrane containing LPS is much slower than entry into phospholipid-only membranes.

Question 51

How do proteins impact the width of the membrane?

Answer 51

Proteins thin the membrane, likely because the membrane it trying to match its width to the hydrophobic surface of the protein.

Question 52

What is clumping in yeast and how does it happen? What triggers this?

Answer 52

The aggregation of yeast cells into clusters, often due to flocculins. These proteins can bind to other yeast cells or to specific substrates, like sugars, causing the cells to aggregate and form clumps.

High levels of sugars can promote clumping by increasing the expression of floccation genes. This means the clump can work together to produce invertase, the enzyme needed to break down the sugar.

Question 53

Give 3 reasons why the division of labour in bacteria is important, and a key example of where this is vital.

Answer 53

1. Allows for task specialization
2. Reduces time moving between tasks
3. Allows machines to be built to do tasks

Colicin toxin release requires cell suicide. As you don’t want all cells to do this, division of labour is crucial.

Question 54

What determines if bacteria cooperate or compete?

Answer 54

When mixed, if the secretor cell is being cooperative, it is paying the cost for secretion unlike the non-secretor cells and so we would expect that eventually is would become competitive. When patchy, the secretor cells are benefiting from one another whilst the non-secreting cells are being somewhat deprived, and so we might expect the cooperative trait remains.

Question 55

How can bacteria exhibiting group behaviour act as a target for antibacterial therapy? Why can’t resistance occur against these?

Answer 55

Some bacteria make protective biofilms that envelope the entire bacterial community. Substances that block biofilm production weakens the community, making it easier to remove.

Resistant bacteria producing biofilms costs energy, while non-resistant bacteria benefit from it ‘free-of-charge’. As a result, the non-resistant bacteria grow faster than the resistant ones, reducing the occurrence of the latter.

Question 56

Why is genotype arrangement important in bacterial clonal patches?

Answer 56

If some have ‘attacking’ genotypes, they are better on the outskirts of a colony, whilst bacteria that produce necessary nutrients are protected within.

Question 57

How do antibiotics that prevent bacteria from cooperating differ from conventional antibiotics?

Answer 57

Preventing cooperation is intrinsically a selection against resistance.

Question 58

Which would you expect to have greater competition: within species or between species? Why?

Answer 58

Within species as they inhabit the same niches and require the same nutrients.

Question 59

When should bacteria use weapons?

Answer 59

When in close contact with competition as most weapons (other than bacteriocins) are short-range.

Question 60

Does strain mixing always favor T6SS? Why?

Answer 60

No

• Increasing T6SS takes a lot of energy without significant benefits
• Killed cells form a protective barrier around other competitor cells

Hence, excessive T6SS cells actually do worse than cells in large colonies that have no T6SS.

Question 61

Why are effector toxins essential for T6SS success?

Answer 61

These burst cells, removing them to prevent protective barrier formation.

Question 62

How can a retaliator overcome the bacteria firing first?

Answer 62

If the retaliator fires back multiple times, rather than once. The retaliator also has the advantage of knowing where the first attack took place and hence can accurately target the bacteria.

Question 63

What is the role of BtuB in protecting bacterial colonies from a colicin attack?

Answer 63

Colicins bind BtuB to enter cells and it’s highly abundant within the cell membrane. When BtuB is deleted, colicin is able to get past the previously formed ‘dead-cell-barrier’ and kill the next line of susceptible cells.

Hence, the abundance of BtuB acts as a mop and takes up all of the colicins.

Question 64

How is the SOS response involved in colicin release? Why is this important? How does this affect neighbouring cells?

Answer 64

DNA damage upregulates the SOS response and toxin production.

If a cell is being attacked and results in DNA damage, it can undergo suicide and release colicins as it was going to die anyway. This forms a line of defence.

Colicins can autoregulate one another such that neighbouring cells are more likely to produce colicins and launch a large-scale counter attack.

Question 65

What happens when you perturb competitive and cooperative bacterial communities? What is then predicted to form the most stable community?

Answer 65

Competitive: the system remains stable by negative feedback from regulatory networks.

Cooperative: system collapses due to co-dependence on one another

Having non-cooperative and weak interactions results in a more stable and ideal community.

Question 66

What evidence suggests cooperation between some Bacteroides species?

Answer 66

Bacteroides ovatus breaks down inulin but only in the presence of glucose and fructose. When the inulin-cleaving enzyme is deleted, ovatus actually grows better.

That’s because they import inulin and cleaving them actually works against this, suggesting there must be cooperation at hand but that this is costly.

In the presence of other species, there’s a benefit from breaking down the inulin that initially doesn’t help the bacterium to grow. This suggests there’s evolution of costly cooperation between some Bacteroides species.

Question 67

What 4 mechanisms are proposed to define species within the microbiome?

Answer 67

Ecosystem on a leash
Host control
Symbiont control
Open ecosystem

Question 68

How can we watch the evolution of antibiotic resistance in a lab? What different parameters can be put in place to study this?

Answer 68

A growth plate is set up that contains E. coli at either end of the plate. There’s an increasing concentration of antibiotic from the ends to the center. Images are then taken over the course of bacterial growth showing that mutations occur rapidly to allow for growth at the center of the plate where antibiotic resistance is highest.

Sequencing of the bacteria at each concentration can then provide more information.

Parameters:
- Width of the plate (changes effective population exposed to antibiotic)
- Width of each concentration zone (time for new mutants to acculumate)
- Concentration differences (intermediate concentrations seem to allow for accumulation of mutations for higher resistance)

Question 69

How does trimethoprim act as an antibiotic?

Answer 69

It inhibits bacterial metabolism at the dihydrofolate to tetrahydrofolate step, which is key to forming essential amino acids.

Question 70

How do stress responses allow for bacteria to mutate?

Answer 70

They provide an initial tolerance barrier to stress (e.g., using drug efflux) which provides time for accumulation of mutations before the stress becomes to great and kills the bacteria.

Question 71

What is the mechanism of action of quinolones? What happens when sub-lethal levels of this are given?

Answer 71

Inhibit DNA topoisomerases, interfering with changes in DNA supercoiling.

Sub-lethal levels induces the SOS response.

Question 72

What are the 3 main secondary effects associated with sub-lethal levels of antibiotics?

Answer 72

1. RpoS stress response
   (RpoS is a sigma factor)
2. (p)ppGpp stringent response
   (Changes gene transcription levels)
3. Reactive oxygen species

Question 73

Describe the SOS response.

Answer 73

The SOS response is a DNA damage response mechanism used by bacteria to repair damaged DNA and prevent the transmission of mutations to the next generation.

The SOS response is mediated by the LexA repressor protein, which normally binds to specific DNA sequences and represses the expression of genes involved in DNA repair and recombination. When DNA damage occurs, the RecA protein is activated and binds to single-stranded DNA produced at the site of damage, forming a nucleoprotein filament that promotes the cleavage of LexA. This causes a derepression of genes involved in DNA repair and recombination, allowing for the activation of various DNA repair mechanisms.

Question 74

How does LexA undergo self-cleavage in the SOS response?

Answer 74

In the non-cleavable LexA form, there’s a serine protease active site that is located far away from the site of cleavage. When LexA interacts with RecA, a conformational change arises that pushes the cleavage site into the serine protease active site, causing self-cleavage.

Question 75

What is the importance of horizontal gene transfer and the SOS response in antibiotic resistance?

Answer 75

HGT: allows for resistant genes to be moved from bacteria to bacteria.

SOS: required for resistance evolution during infection (when SOS is knocked out, bacteria cannot become resistant to antibiotics)
- SOS triggers HGT

Question 76

How do lysogenic phage use the SOS response?

Answer 76

As an escape signal

The expression of SOS response genes results in synthesis of an antirepressor the cleaves phage repressors. This allows for expression of lytic phage genes, essentially notifying a prophage that the cell is in trouble and may have suffered irreparable DNA damage, so by synthesizing prophages it acts as an escape signal.

Question 77

How is the SOS response induced in persistence of bacteria?

Answer 77

Persistence involves lying dormant until the antibiotic goes away, at which point growth can continue.

SOS response can trigger toxin-antitoxin systems that are able to deplete membrane potentials. This could account for the decrease in ATP concentration, causing slowing of cellular processes and inhibited cell growth.