Organisms Prac Flashcards
transfer and freeze time shift assays to get
ancestral and evolved strains on which you can conduct fitness comparisons at 1:1 abundance
Measuring real-time evolution to make
predictions
Q1: is mutation selective or spontaneous?
- measure using phage resistance, where infection = death
Predictions for selective mutations
1) similar terminal no.
2) similar across replicates
Predictions for spontaneous mutations
1) different no. (potentially none)
2) occur at different times (early/late)
3) replicate differences
How can you elucidate observed data?
compare w models
Pseudomonas fluorescens
- abundant soil and water bacterium related to P. aeruginosa
- pyroverdine pigments
- deltamutS SBW25 isolate from sugar beet
P. aeruginosa
important human pathogen
Questions to address
1) at what rate to bacteria spontaneously acquire mutations?
2) what are the costs of a new trait and can these be minimised?
3) does environmental heterogeneity drive adaptive radiation?
DeltamutS
- higher rate of mutation (bad at DNA mismatch repair)
Protocol:
- rifampimicin
- rpoB SNP alterations prevent binding (mutation rate of AR); also hinder RNAP
rifampimicin
- target RpoB beta-subunit of RNAP
- disrupts protein synthesis
- bacterio-static/cidal
plates need to be
dry
pre-cultures
- grown w/o rifampimicin
- no prior selection for mutation
investigation into mutation rate
1) single clone culture in King’s B agar
2) single clone culture in King’s B agar + rifampimicin
+rifampimicin
- only resistant bacteria grow
; estimate spontaneous mutation rates
Determining no. of AR bacteria
- exclude susceptible cells via selection plating on KB+rifampimicin
Determine no. of initial bacteria
1) dilute for a countable density by several factors separate for each strain
countable
spot plating
spot plating
3x3 of 3X10microlites dried close to flame and cultured with lid slightly ajar
how to dilute
- vortex with a known diluent and serialise w tip ejections
- 10^-5/10^-7
Does ecological opportunity and competition promote adaptive radiation?
1) inoculate bacteria from single KB wt colony
2) grow in static/shaken tube
3) plate out culture and inoculate for 4 days
4) estimate colony diversity
adaptive radiation
- new resources/challenges/niche opportunities in an heterogeneous environment
- selects for diversity
- depends on niche positioning and preference
static tube
- microcosm of liquid medium that creates an oxygen gradient
- can be plated onto solid media
static tube morphs
1) smooth ancestor
2) wrinkly spreader at air:liquid (can collapse)
3) frizzy spreader at bottom
Describe the morphs
- distinct w relative fitness
- frequency dependent: most morphs can invade when rare
frequency dependent morph selection
- when rare, ws smothers other phenotypes
- when abundant, surface biofilm collapses
diversity observations:
1) P/A of biofilm and where
Further diversity measurements
1) vortex (breaks biofilm)
2) grow diluted samples on agar plates (40microlitres at 10-5/-6)
3) count and categorise morphotype ratios (100 from each plate)
4) calculate diversity
calculating diversity
- Gini-Simpson index
- 1 - lamba
- lambda - sigma pi^2
- pi = proportion of ith morphotype
Measuring costs of AR
1) limit access of AB
2) enzyme-AB-inactivation
3) modify/protect receptor (rpoB)
naive prediction
reversion
actual prediction
compensatory adaptation more likely
Protocol for AR
1) grow susceptible wt for 50g without antibiotic, to create susceptible evolved
2) do the same with the mutant strain
How to grow for 50 g?
- 24 hours of King’s B
- 100 fold dilution
- fresh media
- x7
To measure relative fitness, compare:
wild type susceptible and mutant resistant
To measure cost, compare:
wild type susceptible and mutant resistance
To measure compensation, compare:
evolved susceptible and evolved resistant
Relative fitness comparisons
1) against a lacZ marked ancestor
2) 30microlitres of each plus 6ml King’s B
3) oxygenic growth for 48hours, at 28 degrees C, and 200rpm
lacZ visual marker
- unmarked = white
- lactose -beta-galactosidase-> glucose + galactose
- X-gal analogue creates blue dye
Relative fitness (W) comparisons observations
- calculate density with spot plating: average number of colonies per spot
- check in R; if -ve, wrong!
Wstrain =
mustrain/mulacZ
mu =
ln(final/starting density)
Determining conc. of inoculant
(average no. colonies in spot/volume of spot) x dilution factor
Determining no of cells inoculated
conc. of inoculant x volume of inoculant
Determining initial density
(no. of cells added) / (total media + inoculant vol)
Determining final cell conc
initial conc x e^(growth rate x time)
growth rate =
1/time
Determining final cell densities
(no. of colonies counted/volume spread) x unit of conversion x dilution factor
Statistical analyses
- test significance
- combinatorial and interactive factorial ANOVA for R2 goodness of fit
How to calculate mutation frequency?
ratio of before and after in CFU/ml
Strain dynamics
- in both strains, any cells that developed spontaneous resistance early could have continued to grow and divide, producing large numbers of resistant cells
- w/o AB, resistant mutants competing against wt; lower fitness means out-competed
- ΔmutM culture accumulates mutants faster, reducing competition
What happens when you have resistant mutants and wt in the same plate?
- if antibiotics: mutants survive
- if no antibiotics: mutants outcompeted due to energy expenditure
AR mutations
- affinity
- inhibition
- what happens in the absence of A? Is it costly?
When asked “why” something happens
think about evolution. For example, high mutation rate is handy in hostile or varying environments, but bad in stable ones where it is energetically costly
Relative frequency
think about relative fitness and frequency dependence, for example social cheating from EC public goods
What happens if relative fitness is higher?
every day culture is replaced, frequency will increase
When thinking about dynamics
think about what might have happened - might something have been driven to extinction?
What can frequency dependent selection cause?
stable persistence
To get high marks
think about what might happen. What if antibiotic concentration was increased? what if it was decreased?
how to get rid of expense due to high selection pressure?
- if its chromosomal, mutate
- if its a plasmid, cure it
- evolve to express only in A presence; selected for alternation
What might increase mutation rate?
- presence of multiple stressors where the pathogen must mutate to survive
think about mutation rate as a tradeoff
it is good when you need it to survive, but bad when you don’t
fluctuating environment select for
high intrinsic mutation rates; more genetic variation per generation; more variability for natural selection
Why can you use antibiotic treatment to test for mutation rate?
- you can determine no. of resistant cells in pre-culture, because one colony on the antibiotic corresponds to one CFU single cell in the pre-culture
- this is a proxy for mutation frequency
- assumes all isolates grow at same rate, antibiotic sensitivity, and spontaneous acquisition in a null environment
Has it evolved resistance?
think about the time frame - resistance takes a long time, especially if its a large cellular process being modified. If its point chromosomes, probably not. If its TE shifts, maybe. If it is HGT (transformation, transduction, conjugation), maybe.
