Bacterial Mutation Accumulation Experiments

Background

Mutation accumulation (MA) experiments involve periodically bottlenecking a population such that evolution proceeds by close to pure genetic drift. Selection is weak under these conditions, because whatever mutations happen to be in the individual chosen to continue a line are arbitrarily fixed in further descendants. Under these conditions, all mutations, except those that are lethal or highly deleterious, will accumulate in genomes at the underlying rates at which they occur due to errors in DNA copying or unrepaired DNA damage. Fitness tends to decline, since deleterious mutations generally outnumber beneficial mutations. MA experiments can provide information about the rates of genomic deleterious mutations and their sizes that are relevant for many evolutionary hypotheses [1]. With whole-genome sequencing, they can also be used to accurately measure spontaneous mutation rates [2].

The maximum likelihood mutation rate for any category of mutations is the number observed divided by the number of elapsed generations. As more mutations are observed, estimates of these rates become more precise. Therefore, the power of these experiments also critically depends on the number of independent MA lineages that are sequenced. Generally, it is best to use a large number of lineages (≥25). With many observations, mutation rates can be subdivided to give the rates of specific types of mutations (e.g., base substitutions of different kinds) and even the rates of mutations at specific sites (e.g., deletions between specific copies of repeats).

Procedure

See this key reference for an example of a mutation experiment with E. coli [3].

Materials

For propagation:

• Agar plates (one per line per day)
  Acinetobacter: Use LB Plates.
  Deinococcus radiodurans: Use TGY Plates.

For freezing:

• Liquid growth media (for growing solid cultures for freezing)
  Acinetobacter: Use LB.
  Deinococcus radiodurans: Use TGY.
• Sterile toothpicks
• Sterile 80% glycerol

Use equivalent solid and liquid media formulations, if possible. Generally, it is better to use chemically-defined minimal media rather than rich media, because there is likely to be less variation from batch to batch that could affect mutation rates.

You will need to know how many days you need to let your plates grow to give at least medium-sized colonies of your microbe.

Setup

It is critical to start an MA experiment with a genetically homogeneous sample. To do this:

1. Revive your strain by streaking it out on an agar plate and growing colonies.
   • Transfer your colonies after an appropriate number of 24-hr periods that gives reasonably sized colonies of the ancestor.
     Acinetobacter: Grow 1 Day at 30°C.
     Deinococcus radiodurans: Grow 3 Days at 30°C.
2. Pick a single colony, streak it out on a new plate and grow colonies again.
3. From this new plate, pick a single colony and transfer it to a tube A with 10ml saline. Transfer 100µl from tube A to tube B with 10ml saline. Transfer 100µl from tube B to tube C with 10ml saline. Use 100µl from tubes B and C to inoculate an LB plate each.
4. Use a 100µl from tube A to start a culture in a tube with 10ml of medium.
   Acinetobacter: Inoculate into 10mL of LB.
   Deinococcus radiodurans: Inoculate into 10mL TGY.
5. Freeze the liquid culture (900ul culture with 300ul 80% glycerol). This will be your original stock for all characterization of the MA line.
6. Pick colonies at random from your agar plates and streak each on its own agar plates. Each colony initiates an independent MA line. Pick N colonies. Number each MA line: 1 to X.
   • The first day of growth on individual plates is actually the T= 1 to T=2 transfer. T=0 to T=1 was the first day of growth where all lines were colonies on the same plate or plates.

Daily transfers

Transfer your colonies every 24 hrs (or however many days each growth cycle to get colonies takes). The timing should be as precisely the same as possible at each transfer. Generally, aim for the time when the plates are placed in the incubator to be +/– 1 hr of the previous transfer's. .
Acinetobacter: Grow for 24 hours.
Deinococcus radiodurans: Grow for 72 hours.

You must have a procedure for picking completely random colonies to not bias toward large or small colonies. One common strategy is to make parallel streaks that cover an entire plate using swipes of a single toothpick. After growth, you then pick the past colony in the last streak that grew each time. Generally, you should just barely touch the edge of the last colony with the toothpick when transferring.

Note: In cases of emergency, you can leave the plates at 4°C for a day or two at most, but significant deviations from a well-controlled transfer pattern are not advised. As part of the experimental design, you need to know the number of generations of growth at each cycle and that the environmental conditions that might affect mutation rates were kept constant.

Archiving samples

Each growth cycle from single-cell streaked on a plate to colony is about 20-25 generations. You should save frozen liquid cultures at least every 10–40 transfers, depending on the species.

Every 20 transfers (20 days) for A. baylyi

Every 10 transfers (30 days) for D. radiodurans

To do this, drop the first toothpick (the one used to pick up the colony and make the first streaks) into a test tube with a few milliliters of your liquid growth medium (LB for AB and TGY for DR). Grow shaking in an incubator at an appropriate temperature (30C for both strains).

After a 24 hr incubation, add 1mL culture and 250mL 80% glycerol to a cryovial. Label cryovial with strain+number, date, number of transfers and experiment.

References

1. Halligan, D.L. & Keightley, P.D. Spontaneous mutation accumulation studies in evolutionary genetics. Annu. Rev. Ecol. Evol. Syst. 40, 151-172 (2009).
2. Kondrashov, F.A. & Kondrashov, A.S. Measurements of spontaneous rates of mutations in the recent past and the near future. Phil. Trans. Roy. Soc. London B. 365, 1169-1176 (2010).
3. Kibota, T.T. & Lynch, M. Estimate of the genomic mutation rate deleterious to overall fitness in E. coli. Nature 381, 694-696 (1996).