We are currently trying to measure mutation rates to rifampicin resistance in Pseudomonas stutzeri. We already have encouraging results showing how a particular genotypic change increases mutation rates to streptomycin resistance and to goal is to calculate mutation rates an additional phenotype so we could begin to think that the genotypic change generally increases mutation rates. For other reasons, we wanted to measure the mutation rates to rifampicin resistance in a streptomycin resistant background strain.
Since P. stutzeri is competent for natural transformation, we transformed a rif and strep sensitive strain (rifS and strepS hereafter) using genomic DNA from a rif and strep resistant strain (rifR and strepR). No problem yet as we got plenty of strepR colonies back.
The next step is to use a fluctuation test to calculate mutation rates to rifR using cultures started from a single strepR colony. The basic idea of a fluctuation test is to grow many independent cultures starting from very low cell densities (typically ~1000 cells, in order to insure that there are no rifR colonies at the start), and then plate the entirety of these cultures under selective conditions once the cultures have grown to appreciable densities. If there are no rifR cells at the beginning of growth , you can use the distribution of rifR colonies that appear across the independent cultures in order to calculate mutation rates.
The picture below describes what you might expect from a typical fluctuation test.
When you plate out samples at time 0, there should be no rifR colonies (which I'm showing as blue circles if present). After growth, in this case 24 hours, rifR cells will have arisen by spontaneous mutation INDEPENDENTLY in each culture. As you can see from the picture, the expectation is that some cultures will not contain any rifR cells, some will have a few, and some will contain many rifR colonies. Those with many colonies are called "jackpots"and may appear as confluent lawns, hence the totality of blue in the picture. There is a distribution of rifR cells across independent cultures because mutations that lead to rifampicin resistance will occur at different points of the growth curve in each culture. A single mutation that arises during the first cell division after starting the experiment will lead to jackpots because these rifR cells have the chance to divide many times before plating. A rifR cell that arises during the last division before plating will only be represented by one colony because it hasn't had the time or resources to divide and proliferate. You can use this distribution to actually figure out mutation rates. Here is Stan Maloy's explanation of the fluctuation test.
Back to our P. stutzeri experiments...when we plated out the fluctuation test for our (what we thought) strepR rifS isolate, we got this result back from the time zero plate.
There was a lawn of rifR bacteria, which is statistically higher than zero (not really, but go with me on this). Even though the mutation rate to rifR is relatively high, something was fishy here because there should have been no colonies. We went back to the original plate of strepR transformants, and streaked additional isolates to both strep and rif plates. Even though the strain which we had originally transformed was rifS, I was surprised when roughly half of the strepR transformants were also rifR.
What could explain this result? The mutations that give rise to the strepR phenotype usually occur within a gene called rpsL. This gene codes for one of the many proteins that make up bacterial ribosomes, and indeed, the mechanism of action for streptomycin is inhibition of translation. The first thing I did was investigate the genomic context, what genes surround rpsL, within the one previously sequenced P. stutzeri genome. Here's what I found at the Pseudomonas genome database (which is extremely handy BTW).
All annotated genes in this section of the P. stutzeri genome are represented as red boxes. rpsL is the red box surrounded by a very dark line. The numbers on the bottom of the picture display the position of each gene in the P. stutzeri genome (rpsL is roughly at position 888,000 out of ~4,000,000). Here's where it gets interesting. Mutations that lead to rifampicin resistance typically occur in a gene called rpoB. This codes for a subunit of RNA polymerase, which makes sense (again) because the mechanism of action for rifampicin is to inhibit transcription. As I described above, we originally made the strepR strain by transforming a rifS strepS strain with genomic DNA from a rifR strepR strain. rpoB and rpsL are only about 5000 bp apart and I originally had no clue that rpoB and rpsL were genomic neighbors. Since, during natural transformation, genomic fragments sized 5Kb and above can be recombined into the recipient genome I'm guessing that a substantial fraction of the cells recombined both strepR and rifR mutations during this step from the donor genomic DNA. Since the exact positions of recombination are essentially random, cells that remained rifS after transformation must have recombined smaller fragments that only contained the strepR mutation. Here's a quick picture of what I think happened.
Rifampicin resistance is colored red, streptomycin resistance is colored blue. Cells that are rifR strepR are colored purple. While there were likely rifR strepS cells generated after transformation, these won't grow on the strepR plate so that's why there aren't red colonies. Mystery solved...and this maybe something I can utilize in future experiments although as of right now I have no idea how. If we would have originally picked a rifS strepR colony for the fluctuation test, I wouldn't have realized any of this. Sometimes science is random.
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