Main drivers of microbial communities

Question 1

The ‘theatre of activity’ within a microbiome includes what in addition to the microbes?

Answer 1

• Microbial structural element: Proteins/peptides, lipids, polysacharides, nucleic acids
• Mobile genetic elements (including viruses/phage relic DNA)
• Environmental conditions
• MIcrobial metabolites: signalling molecules, toxins, (an)organic molecules

Question 2

why is extracellular DNA an important part of the microbiome?

Answer 2

Extracellular DNA is one of the most key elements of building up this extracellular matrix that allows the build-up of this complexity.

eDNA are extracellular nucleic acid biopolymers critical to the integrity of the biofilm matrix by stabilizing charges, providing structural rigidity, and protecting the matrix from host defense responses
- interactions with these sugars is really what allows this interaction with other chemical compounds and environmental conditions
- It allows you to have differences between these bacteria that compose this complex community as well

Question 3

what are the main drivers?

Answer 3

• Abiotic factors and biodiversity: balance between nutrients and structure, host diversity and phage diversity as well. Mobile genetic elements are also drivers and the reason there is a metabolism associated that helps development
• Phenotypic resistance: Phenotypic resistance and sensitivity host making it harder to stop infection or successful survival –
• Spatial refuges

Question 4

whats the importance of predateor and host interaction in a mature community?

Answer 4

When you have a mature community, the interaction between the predator and the element on the host will always be the tipping point between having a cost/ benefit, to now having a resistant element to the virus that drives the diversity
If a virus is able to predate the entire population then you have population collapse and therefore you have a reduction of phage diversity. (phage diversity will usually be as diverse as the host)

Question 5

how does balance drive evolution within a microbiome?

Answer 5

• Balance is required, depends on the mutations on both sides of these two components (predator and host)
  o on bacteria that enable to overcome predator and mutations on the predator that enables overcome bacteria
   this balance is what drives evolution

Composition and evolution of phage communities, controls bacterial population and evolutionary dynamics of microbial communities.

Question 6

brefily describe the lytic bacteriophage lifecycle

Answer 6

Bacteriophages adhere to cell surface and the bacterial cell is being recognised by the tail fibres, a change in the conformation of that elements that allows the nucleic acids to be inserted. The nucleic acids are injected into the cell, the cell is hijacked by that phage and there is degradation of the bacterial chromosome that can initiate the production of virions. The virions are being produced, throughout their production they release enzymes that leads to the release of the progeny into the environment – this is called the lytic lifecycle

Question 7

what phages have lytic lifecycles

Answer 7

Virulent phages

(& temperate phages when under environmental stress)

Question 8

what phages have the lysogenic lifecycle

Answer 8

temperate phages

Question 9

what is the major difference between the lytic and lysogenic lifecycles

Answer 9

temperate phages integrate into the host (bacterial) chromosome and replicate with the bacterium. (without killing the cell)
Only through stress on the environment are they release and begin their lytic lifecycle.

Question 10

why are the phages long tail fibres retracted?

Answer 10

Capsids have association with long tail fibres that are retracted for conservation of energy for interaction with bacteria in a random way

Question 11

how do viruses come into contact with cells

Answer 11

Viruses DO NOT SEEK CELLS. They are non-living entities and do not infect host by simply being present with them. They will need to bump into them for them to interact. Interaction takes place by recognition of different peptides

Question 12

what two ways can a phage attach to a cell?

Answer 12

The tail fibres will be able to recognise the proteins or sugars on the bacteria.
A primary receptor – only one will be recognised
Secondary receptor – both will be recognised
So you will have phages that will be depending on the lipopolysaccharides for an infection or you have the one that can recognise lipopolysaccharides and outer membrane proteins

Question 13

how is the flagellum exploited by phages?

Answer 13

The flagellum is exploited by phages as their entryway, typically using curled tail fibres that wrap around a rotating flagellum (used to come into proximity)
via a tail fibre they approximate themselves with the flagellum to get to the bacteria cell and initiate the attachment procedure and infection state

Question 14

Phages that use flagellum to bind to a target bacteria are known as…

Answer 14

flagellotropic phages

Question 15

describe the way one group of flagellotropic phages infect using their head

Answer 15

1. A flexible filament extending from their heads is used to wrap around the bacteriums rotating flagellum
2. then spin clockwise along the flagellum towards the cell pole
3. where the tail fibre contacts its receptor on the cell surface and starts the infection

Question 16

what is the significance of the way flagellotropic phages infect?

in terms of evolution and success

Answer 16

The flagellum is not indispensable for infection, it simply increases the chance of the phage tail finding the receptors on the moving bacteria
There are mutations that then will lead to the ability of these viruses to have this tipping point of the host – in this case the additional tail fibre increases the ability to infect
However this increase in changes does NOT mean it is essential for a new infection to take place ( the additional fibre is not required for this virus to complete its infection cycle

Question 17

what mechanism do bacteria use to evade being injected

Answer 17

super infection exclusion

Question 18

how do microviruses pentrate?

Answer 18

Penetration capacity comes from the presence of the inner tube – in the case of these micro-viruses, they have a needle type event that penetrates through, vis degradation of the peptidoglycan layer they release the DNA

Question 19

what is super infection exclusion?

Answer 19

the ability to carry mobile element into the bacterial cell allowing the infection to be stopped by the presence of another virus

Question 20

describe the entry of Pseudomonas phage phi6

Answer 20

1. The phage uses the protein spikes that protrude from its capsid to adsorb to the side of a pilus in the bacteria. Bacteria pili alternately extend and retract to allow the bacteria to move, and the phage uses pili retraction to get closer to the outer membrane of the cell.
2. Then protein P6 from the phage fuses the phage lipid envelope with the bacterial outer membrane
3. The resulting nuclecapsid (no lipid) has an exposed endopeptidase that digests a path through the peptidoglycan layer (fusion release the enzyme)
4. At this point the phage has an inner and outer capsid surrounding its genome. The outer layer causes a region of the cell membrane to invaginate and contrct to bud off from the cell membrane
5. it then loses the outer layer by an unknown process
6. the inner capsid remains to protect the dsRNA from cleavage by ribonucleases. On each vertice of the inner capsid there is an RNA-dependent RNA polymerase (RdRP)
7. using the dsRNA as template the RdRPs transcribe positive-sense RNAs that exit from the capsid into the cytoplasm where they are directly used for translation of phage proteins or packaged into progeny phages (hijacking the bacteria to produce new virions)

Question 21

how does the entry of Pseudomona phage phi6 differ from most phages

Answer 21

During infection by most phages, only the phage DNA enters the bacterial cell; the capsid remains outside. (normal)
Phage phi6 has a dsRNA genome that woul7d be rapidly cleaved by host ribonucleases inside the bacteria.
To avoid this, the phage uses a strategy in which the capsid containing the dsRNA enters the cell

Question 22

How does a temperate phage genome decide what to do when it arrives in a host cell?

Answer 22

• Individual decisions and voting – e.g. phage lambda
• Group decision – e.g. phage phi3T, (arbitrium)

Question 23

what is the first thing lamda phage does when it enters a cell?

Answer 23

Once inside the cell, phage lambda quickly initiates synthesis of early proteins including regulatory protein CII

Question 24

how does CII lead to lysogeny?

Answer 24

Increase of the CII protein leads to increase of CI which leads to lysogeny – CI represses cro stopping entry into the lytic pathway

If you have release or loss of CII then you don’t have much CI and so Cro can initiate the lysis event
CII’s primary role in the lambda phage regulatory network is to initiate the repressor establishment cascade
The CI protein of bacteriophage lambda is both a repressor and activator of transcription

Question 25

what factor is the lytic/lysogenic decision based upon?

Answer 25

• Concentration of phages in the infected cell
  Higher phage concentration leads to more CII synthesized – more probability of lysogeny
  More phages inside the cell indicate more phages in the environment, so host strains may become scarce
• Metabolic state of the host cell
  Starving cells are smaller, so CII concentration will be higher, favouring lysogeny when the host is starved

Question 26

Higher phage concentration lead to more… sythesised meaning more probability of…

Answer 26

Higher phage concentration leads to more CII synthesized – more probability of lysogeny

Question 27

how is the decision made when multiple phages infect a single cell?

Answer 27

when multiple phages infect a single cell, the decision of all phages is integrated within the cell, such that **only a unanimous vote for lysogeny by all phages eventually leads to a lysogenized bacterium. **
If one phage follows the lytic cycle, the cell will be lysed (dies) even if the other phages follow the lysogenic cycle.
Only one needs to ‘decide’ to take the lytic cycle - individual decision drive the consequence to these cells
You need a synchronised response for the lysogeny cycle to take place

Question 28

how do phages make a group decision?

Answer 28

For some phages (e.g. phi3T), the choice is influenced by communication between phages infecting different cells
During infection phages produce the phage protein AimP
AimP is cleaved to form a small peptide – Arbitrium
Arbitrium is exported from the bacterial cell
This is how they communicate the concentration
Arbitrium is taken up by neighbouring cells via a bacterial transport protein (OPP)

Question 29

what happens if there are low levels of arbitrium present?

Answer 29

Low levels of arbitrium present in a cell: phage infection likely follows the lytic cycle
Not that many phages in the environment, so it is still advantageous to the phage to replicate. Enough host compared to virus.

Question 30

what happens if there are high levels of arbitrium in a cell

Answer 30

High levels of arbitrium present in a cell: phage infection likely follows the lysogenic cycle
Too many phages in the environment can lead to scarcity of host cells, so it is more advantageous to remain in a dormant state

Question 31

can all phages communicate via arbitrium?

Answer 31

Arbitrium is a language that only one type of virus can decode
Different phages use different arbitrium’s, thus communicating only with phages of their own kind

Question 32

what is the molecular mechanism behind arbitrium?

Answer 32

Early genes aimR and aimP are expressed immediately upon infection
AimR activates AimX expression. AimX is an inhibitor of lysogeny, directing the phage to a lytic cycle
AimP is also expressed, secreted and processed to produce Arbitrium (proteases present in bacterial cells activate arbitrium, by cleaving AimP)
Arbitrium is internalized into other bacteria by the OPP transporter
Now, when the phage infects the bacterium, AimR binds the Arbitrium molecules and cannot activate the expression of AimX, leading to lysogeny preference
(Arbitrium releases the repression over the aimR )

Question 33

what happens when arbitrium enters the cell

Answer 33

AimR binds the Arbitrium molecules and cannot activate the expression of AimX, leading to lysogeny preference
(Arbitrium releases the repression over the aimR )

Question 34

Early genes aimR and aimP are expressed immediately upon infection.
what does aimR do?

Answer 34

AimR activates AimX expression. AimX is an inhibitor of lysogeny, directing the phage to a lytic cycle

Question 35

what is the third type of phage lifecycle?

Answer 35

Chronic lifecycle

Question 36

Chronic lifecycles are normally driven by viruses known as…

Answer 36

inoviridae

Actually considered to be abundant in the environment. Commonly present in lung infections, especially for those with infections as a result of cystic fibrosis susceptibility

Question 37

describe a mechanism in which bacterial viruses are used to protect the host generalyl giving the bacterial cells an advantage

Answer 37

bacteria will releases these viruses (inoviridae) from their chromosome to protect themselves against antibiotics

Question 38

what genome do filamentous phages have?

Answer 38

f1 phages have a ssDNA genome inside a long cylindrical protein coat.

Question 39

describe the filamentous phage lifecycle

Answer 39

When the f1 genome enters the cell, the capsid disassembles, depositing the proteins in the cell membrane.(to be used upon exit)
f1 then relies on host proteins for replication - they convert the phage ssDNA into dsDNA to use in transcription and synthesis of new ssDNA genomes.
f1 also produces a ssDNA-binding protein that coats the ssDNA genomes, protecting them from degradation. Protection from bacterial defense
The new f1 genomes find the capsid components at the cell membrane (some deposited during infection by a parent phage, but most freshly made and anchored in the membrane in anticipation).
Most filamentous phages exit through a borrowed host secretion channel, but f1 encodes a secretion channel of its own (gp4 secretin).
Using this mechanism of extrusion, filamentous phages leave the host cell intact – this means they can continuously reproduce inside the cell without causing significant harm.
So they can continuously infect the cell without causing cell death

Question 40

how does pseudomonas initiate biofilm cycle?

Answer 40

This process of infecting and continuously is what Pseudomonas uses to initiate the biofilm cycle
It will sacrifice itself and inject the extracellular DNA into the cell in order to create enough extracellular DNA to create the building blocks to create the extracellular matrix to initiate the biofilm formation
These filamentous phages are important for this and also to create crystalline-like structures that prevent the infection states as well

Question 41

what are the tasks that a phage must accomplish to keep their lytic cycle turning?

Answer 41

• Capsid must protect the genome from environmental dangers (e.g. UV, nucleases)
• Quickly recognize a host when it collides with one
• Irreversibly bind to its receptor and deliver the phage genome into the cell
• Redirect the cell metabolism for the production of many progeny phages
• Replicate the phage genome and synthesise a correct number of structural proteins (e.g. capsid)
• Pack the new genomes into the capsids
• Time the lysis of the host for release of the new phages

Question 42

once the phage adsorbs to the cell, the phage and cell together are known as what?

Answer 42

virocell

Question 43

Leviviridae Qβ has a ssRNA genome that encodes how many genes

Answer 43

4

Question 44

why is an RNA genome efficient?

Answer 44

efficient because copies of the positive-sense RNA genome function also as mRNA for translation of phage proteins – DNA synthesis is expendable!

Question 45

Leviviridae Qβ has a ssRNA genome that encodes only 4 genes
what are each of them encoding?

Answer 45

• 1 gene for RNA replication – the replicase
• 2 genes for structural proteins that form the capsid
• 1 gene for a multifunctional protein termed maturation protein

Question 46

how does Phage Qβ further economize by overlapping the genes

Answer 46

When translating the major capsid protein, ~5% of the time the ribosome reads through the leaky stop codon and continues translating, thus yielding the longer minor capsid protein. (creates another protein by making mistakes and passing the stop codon- mechanism that continues evolving by translating information in the shortest space possible to be able to efficiently continue replicating)

Question 47

the smallest DNA phage genomes are found among what type of phage

Answer 47

(tailess) microphage

Question 48

Microviridae φX174 has a circular ssDNA genome of how many nucleotide and genes

Answer 48

5,386 nt that encodes 11 genes

Question 49

what phage is used by illumina to increase diversity wihtin sequencing projects?

Answer 49

Microviridae φX174

Question 50

describe how we know that overlapping genes help with fitness

Answer 50

Decompression the genome - separating overlapping genes to see how the virus acts, reduces the fitness advantage of the virus

Question 51

what is the bp size of the largest phage

Answer 51

The largest known phage has a genome of 735 kbp

Question 53

most phages have larger genome 50-100kbp - what are the potential functions of their genes?

Answer 53

• Structural proteins (capsid, tail, tail fibres, etc)
• Allow phage to carry out the basic life cycle steps with greater fitness
• Counter host defences (lecture 2)
• Compete with other phages wanting the same host or battle other mobile genetic elements
• Obtain extracellular resources needed for phage replication (e.g. phosphate)
• Precisely manipulate the host’s metabolism to provide for the needs of the phage
• Expand the host range
• Thrive under other environmental conditions
• Temperate phages carry metabolic genes that benefit the host, thereby serving the phage’s interests as well

Question 54

what explains the mosaic structure that arises when the genomes of two phages are compared?

Answer 54

Phages, evolve by accumulating mutations, but they also evolve through recombination events in which they exchange genetic material with other phages.

These events have been suggested to explain the mosaic structure that arises when the genomes of two phages are compared: nearly identical sequences alternate with sequences that are merely similar or even completely divergent

Question 55

what does the mosaic structure (when you compare phage genomes) tell you?

Answer 55

Mosaic structure tells you that the viruses have overlapping elements and this similarity that they have among each other allows comparison but more importantly these nearly identical structures are what allows them to keep evolving and providing that bacteria the ability to continue responding to their fitness environment

Question 56

what ways can phage helps the bacterial host

Answer 56

• Protecting bacteria against lytic infection of other phages, by superinfection exclusion
• Providing virulence factors (e.g. extracellular toxins)
• Providing genes encoding for antimicrobial (mobile genetic elements are carrying more of these antimicrobial resistance genes)
• Aiding with biofilm formation
• Suppressing mammalian immunity against the bacterium

Question 57

What type of phages are important vectors for horizontal gene transfer among bcteria

Answer 57

Temperate phages are important vectors of horizontal gene transfer among bacteria.
By doing so, they can alter and benefit the bacteria by

Question 58

how do PF phages give stability to biofilms

Answer 58

• Causing lysis of a few of the cells, which leads to the release of DNA into the extracellular space (eDNA), which adds structural integrity to the biofilm
• Accumulating in the biofilm matrix where they spontaneously align and give the biofilm matrix liquid crystalline properties that
• Enhances biofilm adhesion
• Survival to desiccation
• Provides high antibiotic tolerance (by sequestering antibiotics)

Question 59

After bacterial ligands stimulate the immune response in mammals, what happens if a Pf phage is present?

Answer 59

If Pf phage is present, it is taken up by the mammalian cells by endocytosis
This leads to the production of phage RNA that triggers specific signalling pathways that inhibit the immune response to bacteria (TNF production and phagocytosis)
Provides the cell capacity to avoid being detected by the LPS
1. recognition of phage RNA by TLR3-mediated TRIF signalling
2. TRIF activation leads to type 1 interferon production
3. Type 1 interferon inhibits TNK secretion and bacterial phagocytosis