seminar 3: Harrison

Question 1

what are mobile genetic elements?

Answer 1

sets of genes that have a transmission advantage over rest of genome
- can be horizontally transmitted

Question 2

give points about the bacterial virus phage

Answer 2

• drive bacterial mortality
• used in C cycling
• form local adaptations and maintain diversity
• kill about 40% bacteria in oceans every day

Question 3

what is phage therapy?

Answer 3

used by humans to tackle bacterial infections

Question 4

what are prophages?

Answer 4

phages that can jump into the chromosome so are powerful mutualists

• attack and kill other bacteria
• can carry virulence traits
• often carry important toxins

Question 5

what do plasmids and ICEs do?

Answer 5

dont exit the cell
- manipulate cellular host to form bridges/pilli that fuse to cell membrane of another cell to send DNA to infect new cell

Question 6

what type of gene transfer do plasmids use and what do they often carry lots of?

Answer 6

horizontal

lots of bacterial genes from different hosts/chromosomes

Question 7

what are satellite/nested elements? (4)

Answer 7

• jump between genomes/host via bigger elements
• package into other viruses
• hitchhike on pilli
• can spread through entire microbial community

Question 8

what are the 2 main ways microbes and their mobile genetic elements evolve?

Answer 8

• plasmid preference

- simultaneous interactions shaping evolutionary outcomes

Question 9

give 3 reasons why plasmids are considered costly

Answer 9

• metabolic burden
• disruptive (produce own regulatory proteins)
• epistasis (genes may have bad relationships with other genes)

Question 10

what was the study bacteria and its plasmid +size?

Answer 10

P.fluorescens

large plasmid 1/10th size genome containing mercury resistance operon

Question 11

what is the relationship between the plasmid and bacteria dependent on?

Answer 11

context- the environment

Question 12

how can it be considered a parasitic relationship?

Answer 12

when contains the plasmid but no mercury there is a 50% reduction in fitness and reduced growth rate

Question 13

when mercury is added how does the relationship between plasmid and bacteria alter?

Answer 13

becomes mutualistic and benefits of resistance outweigh costs

Question 14

what was the brief procedure to begin with when looking at evolution along the parasitism-mutualism continuum? (3)

Answer 14

• 6 gradings of mercury
• tracked plasmid frequency
• 450 bacterial generations
  sequence evolved clones

Question 15

what was found for the parallel evolution at 2 loci? (3)

Answer 15

• no mutation in the plasmid
• same gene targeted across and within treatments
• 3/4 population evolved a mutation in Gac S/A

Question 16

how was a knockout strain created and how does this compare to the wild type?

Answer 16

removing genes from ancestral bacteria

cost removed in knock out strain

Question 17

what do Gac S/A form and how do they work together?

Answer 17

2 component regulator of extracellular protein production

- control downstream regulation of other genes

Question 18

how does the ancestral strain compare to the evolved strain in terms of parallel transcriptomic response to plasmid?

Answer 18

ancestral
- 1/5th bacterial genome upregulated

evolved
- gene expression profile eventually returns to ancestral state as response regulators targeted

Question 19

briefly explain the steps of the Gac system (5)

Answer 19

1. GacS receives signals
2. Gac A activated
3. sRNAs produced that inhibit RNA binding proteins rsmA
4. rsmA grabs mRNA but is triggered to release it so is translated
5. extracellular proteins produced

Question 20

what can sometimes be introduced to the Gac system from plasmids and what does this result in?

Answer 20

plasmid carries encoded rsmA

• causes bad copy of the binding protein to be produced
• likely non functional proteins created
• likely excess extracellular proteins produced that the cell does not need to aid in plasmid transfer

Question 21

how do cells tackle the plasmid causing excess protein production?

Answer 21

knocks out Gac S/A genes with stop codons and frame shifts

• prevents repression of binding proteins
• reduces extreme upregulation of these proteins

Question 22

what constantly occurs across the parasitism mutualism continuum?

Answer 22

compensatory evolution

• plasmids benefits
• evolution and mutations act to reduce costs of plasmid
• so resistance gene likely maintained

Question 23

what are bacteria phage interactions seen as?

Answer 23

antagonistic coevolution

Question 24

what was the experimental set up for studying how competing selection pressures alter evolution of outcome? (4)

Answer 24

• set up with bacteria only, bacteria and plasmid only, bacteria and phage only, bacteria, plasmid and phage
• 150 generation batch transfer
• grow bacteria on agar with milk
• zones of clearing around bacteria with functioning Gac

Question 25

what do the enzymes that the bacteria excrete do?

Answer 25

degrade proteins to gain metabolites

Question 26

with the plasmid and bacteria only what frequency of Gac colonies was reached? - how did this differ when the phage was involved?

Answer 26

high | - low, no fixation

Question 27

what was it discovered that phages constrain?

Answer 27

plasmid Gac A/S compensatory evolution

Question 28

what was discovered when doing a streak assay with phage and bacterial colonies on agar plate?

Answer 28

- bacterial colonies that can grow and pass through the line of phage is resistance - bacteria that doesn't spread pas is susceptible

Question 29

what did plasmids alter to do with phages?

Answer 29

alter bacteria phage coevolution

Question 30

are bacteria that evolved with or without the plasmid less susceptible?

Answer 30

without

Question 31

what are phages with plasmid population?

Answer 31

less infective

Question 32

if the host is very resistant what will evolution drive?

Answer 32

higher rates of infectivity in coevolving populations

Question 33

what has plasmid presence restricted?

Answer 33

evolution of resistance in bacteria and infectivity in phage

Question 34

what did plasmid with phage fighting populations evolve? and when is the rate of these higher?

Answer 34

mucoid bacterial culture phenotype | - higher with plasmid and phage together compared to in absence of plasmid

Question 35

when sequencing clones from each population of treatments - were genes shared across all treatments? - what showed up as predicted? - what does the fact it is antagonistic coevolution mean? - what does changing LPS genes do? - what did bacteria with plasmid and phage show?

Answer 35

- none across all treatments - repeated compensatory mutations in Gac A/S genes - drives faster evolution and more LPS genes targeted - hides bacteria from the phage as phages recognise these - different targets of mucoid and no Gac mutations

Question 36

what was the summary result for: a) bacteria and phage b) bacteria, phage, plasmid c) bacteria, plasmid

Answer 36

a) strong phage resistance via LPS b) weak phage resistance via mucoidy and no compensatory mutations c) bacteria evolve compensatory mutations

Question 37

what is the protein causing mucoid phenotype? and what does it do?

Answer 37

alginate | slows access of phages to cell surface

Question 38

what does loss of Gac A/S to the system mean and what is this known as? when is this of particularly high cost?

Answer 38

loss of phage defence antagonistic pleiotropy - when in presence of phages

Question 39

what alters the outcome for pairwise relationships?

Answer 39

selection from multiple symbionts

Question 40

what is key to understand for understanding horizontal gene transfer?

Answer 40

mobile genetic elements in terms of evolving symbionts