Drivers of microbiol communities

Question 1

Which factors determine the phage lytic vs lysogenic decision?

Answer 1

The concentration of phages and the metabolic status of the cell

Question 2

Which of the following mechanisms do phages use to determine if they follow the lytic or lysogenic life cycle? Choose all the correct ones.

A) Communication between phages infecting different cells via tail fibers
B) Making individual decisions independently of the number of phages in the cell and environment
C) Using small peptides to communicate between infected cells.
D) Making individual decisions based, among others, on the phage concentration inside a cell

Answer 2

C, D

Question 3

The smallest phages have a genome that is:

A) Single-stranded RNA (ssRNA)
B) Double-stranded RNA (dsRNA)
C) Double-stranded DNA (dsDNA)

Answer 3

A?

Question 4

What is the microbiome?

Answer 4

Microbiota + “theatre of activity”

Question 5

Name the different microbiota

Answer 5

Bacteria
Archaea
Fungi
Protists
Algae

Question 6

Parts of the “theatre of activity”

Answer 6

Microbiol structural elements: Proteins/peptides, lipids, polysacharides, nucleic acids (DNA/RNA)

Internal/external elements: Environmental conditions, mobile genetic elements, microbial metabolites (signalling moleciles, toxins, (An)organic molecules

Question 7

Define biome

Answer 7

A reasonably well-defined habitat which has distinct bio-physico-chemical properties

Question 8

What controls bacterial population and evolutionary dynamics of microbial communities?

Answer 8

Composition and evolution of phage communities

Question 9

What represent the most abundant biological entities on our planet?

Answer 9

Viruses

Question 10

in terms of geneic diversity, viruses are

Answer 10

a large reservoir of novel genetic diversity

Question 11

What drives evolution?

Answer 11

Balance of mutations between bacteria and viruses (prey and preditors)

Question 12

Stages of the lytic life cycle of a phage

Answer 12

Attachment
genome entry (conformational changes allow this)
replication
assembly
lysis
- Therefore, phage progeny

Question 13

Stages of the lysogenic life cycle

Answer 13

Attachment
genome entry (conformational changes allow this)
integration
daughter cells with prophages
environmental trigger
- entry to the lytic cycle from replication and assembly onwards

Question 14

Virulent phages have the …. life cycle; temperate phages undego the …. life cycle

Answer 14

1. Lytic
2. lysogenic

Question 15

How does the virion of the pahe interact with bacteria

Answer 15

In a random way, they do not seek to infect (they aren’t living entities)

When they bond, it happes through recognition of tetrapeptides, lipopolysacharides or peptidoglycans proteins and sugars

Question 16

Explain how some phages “surf their way in” to bacteria

Answer 16

Some bacteria use a flagellum structure for motility.

The flagellum is exploited by phages as their entryway, typically using curled tail fibres that wrap around a rotating flagellum

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

Question 17

Explain how the flagellotropic phage Caulobacter crescentus infect

Answer 17

1. A flexible filament extending from their heads is used to wrap around the bacterium’s rotating flagellum
2. They then spin along the flagellum towards the cell pole
3. Where the tail fibre contacts its receptor on the cell surface, the infection starts

NB: the extra fibre increases the chances of infection

Question 18

Entry of Pseudomonas phage phi6

Answer 18

Phage phi6 has a dsRNA genome that would 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:

1. Phage has spike proteins to protude from its capsid to absorb the side f a pillus in the bacteria
2. protein P6 fuses the phage lipid envelope w the bacterial outer membrane
3. this creates a nucleocapsid w exposed endopeptidase that digests a path through the peptidoglycan layer
4. using dsRNA as a template, RNA-dependant RNA polymerase (RdRPs) transcribe tje + sense RNAs that exit capsid into cytoplasm where they are directly used for translation of phage proteins, or packaged into progeny phages

Question 19

+ve vs -ve viral RNA

Answer 19

Positive-sense viral RNA is similar to mRNA and thus can be immediately translated by the host cell.

Negative-sense viral RNA is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA polymerase before translation.

Question 20

key difference between ssRNA snd dsRNA?

Answer 20

ssRNA has only one strand of RNA while dsRNA is made up of two complementary siRNA or miRNA strands. RNA or ribonucleic acid is a type of nucleic acid which is made up of ribonucleotides.

Question 21

2 ways of the lytic vs lysogenic decision

Answer 21

Individual decisions and voting – e.g. phage lambda (type of E.coli Phage)

Group decision – e.g. phage phi3T

Question 22

Individual decisions and voting

Question 23

Phage lambda life cycle

Answer 23

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

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

Cycle:

Phage, infects cell, increases CII, CII, leads to CI, CI leads to lysogeny

CII gets turned off, get lysis pathway activated because you don’t have repression of Cro

Lysogeny module = CI, Lysis module = Cro and CII

Concentration of phages in the infected cell

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

Question 24

Higher phage concentration (in phage lambda)

Answer 24

more CII synthesized – more probability of lysogeny

Question 25

Impact on metaboilic state of the host cell on phage lambda lysogeny/lysis decision

Answer 25

Starving cells are smaller, so CII concentration will be higher, favouring lysogeny when the host is starved

Question 26

two main reasons that phages decide to NOT lyse theie cells (therefore favour lysogeny) are

Answer 26

Based on concentration – too many viruses outside, undergo lysgeny to counteract (caused by gigher CII conc)

Metabolic state – if the metabolic state of the cell isnt optimal (cells are starved and therefore small), CII conc also increases and lysogeny state takes place

Question 27

Voting

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 even if the other phages follow the lysogenic cycle.

Question 28

For the lysogeny cycle to take place (voting)

Answer 28

all phages infecting the bacteria have to be synchronized in the decision of lysogeny

Question 29

Group decisions e.g. phi3T

Answer 29

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

it is taken up by neighbourinhg cells via tranport protein OOP

Low levels of arb.. = lytic
high “ = lysogenic

Question 30

Low vs high levels if arbitrium in phi3T

Answer 30

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

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

When it comes to group decisions, how do phages only communicate with their own kind?

Answer 31

Different phages use different arbitriums

Question 32

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

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

Question 33

Filamentous phages and the chronic life cycle

Answer 33

When the f1 genome enters the cell, the capsid disassembles, depositing the proteins in the cell membrane.

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.

When the f1 genome enters the cell, it does not leave the capsid outside, nor does it bring the capsid along with it into the cell - the capsid disassembles, depositing the proteins in the cell membrane for reuse later to coat progeny as they exit.

Question 34

What is the third type of phage lifecycle

Answer 34

Chronic state infections

Normally driving by viruses called Inoviridae

Inoviridae are more abundant in the environment

Question 35

Chronic state…

Answer 35

does not lead to cell death

Question 36

Most filamentous phages exit through a borrowed host secretion channel, but f1 encodes a secretion channel of its own:

Answer 36

g4p secretin

Question 37

Two mechanisms that viruses have for protection against defense mechanisms in bacteria

Answer 37

1 – Capsid – enters the cell to protect against ribonucleases

2 – ssDNA binding proteins – coats the genome, protecting them from degradation

Question 38

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

Answer 38

Outside the host cell

-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

Once the phage adsorbs to the cell, the phage and cell together are known as a virocell

Inside the host cell

-Evade host defences (lecture 2)

-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 39

How many genes are required to carry out these life cycle steps? e.g. Leviviridae Qβ

Answer 39

ssRNA genome that encodes only 4 genes

RNA genome (4212 nt) is efficient because copies of the positive-sense RNA genome function also as mRNA for translation of phage proteins – DNA synthesis is expendable!

• 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 40

The smallest DNA phage genomes are found among

Answer 40

the (tailless) Microphage.

Question 41

Microviridae φX174

Answer 41

circular ssDNA genome of 5,386 nt that encodes 11 genes

6 of the genes are used for the assembly of a capsid more complex than that of phage Qβ

In the lab, 2 of the genes are not essential for phage replication and their function is still unknown

Question 42

Temperate phages are important vectors of horizontal gene transfer among bacteria.

By doing so, they can alter and benefit the bacteria by, for example:

Answer 42

Protecting bacteria against lytic infection of other phages, by superinfection exclusion

Providing virulence factors (e.g. extracellular toxins)

Providing genes encoding for antimicrobial

Aiding with biofilm formation

Suppressing mammalian immunity against the bacterium

Question 43

Pf phages give stability to the Pseudomonas aeruginosa biofilm by:

Answer 43

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 44

Phages and mammalian immunity to bacteria

Answer 44

In the absence of phage, bacterial ligands e.g. LPS, stimulate the immune response in mammalian cells

If Pf phage is present, it is taken uo by the mammalian cells by endocytosis

This leads to the production of phage RNA that triggers specific signalling pathways (type 1 interferon) that inhibit the immune response to bacteria (inhibit TNF production and phagocytosis)