In a post last month I wondered whether the purine repressor PurR might repress competence genes, and concluded that this was probably a dead horse that I should stop flogging. But one of the grad students has some new results that suggest I was wrong.
We've known for years that adding purine nucleotides to the competence-inducing medium prevents transformation. (Oops, I just discovered that my sidebar link to the paper doesn't work any more, probably because we redid our lab web pages... ... Fixed.) I originally hypothesized that this was probably because the PurR repressor repressed competence genes, but a previous grad student knocked out the purR gene and showed that this didn't increase transformation (my hypothesis predicted it would). So I discarded the hypothesis.
Instead I hypothesized that the nucleotide effect was due to an effect of nucleotide pools on expression of the competence-gene activator Sxy. The grad student working on Sxy and CRP has now carefully examined the effect of nucleotides on sxy expression, measuring three things from each cell sample: Sxy protein, sxy mRNA, and transformation frequency. Consistent with this hypothesis, all were dramatically reduced by addition of nucleotides. So far so good.
We don't know how the purine nucleotides repress sxy, but we have a clue. I've probably mentioned before that we have mutations in the sxy gene that turn it on under conditions that normally keep it turned off. This suggested a new hypothesis, that the effect of these hypercompetence mutations is to make sxy expression independent of purine nucleotides (i.e. that the mutations negate the nucleotide effect).
The grad student has now tested this by examining the same three things (Sxy protein, sxy mRNA, and transformation frequency) from wild type and mutant cells treated with nucleotides. As predicted, addition of nucleotides to the mutants does NOT reduce sxy mRNA or Sxy protein. But, the mutants nevertheless show the same reduced transformation frequency as wild type cells.
Hmm... Sxy normally turns on all the competence genes, so why aren't the cells transforming? (Reminder: competence refers to uptake of DNA, and transformation to a genetic change caused by this DNA recombining with the cell's chromosome.) Could it be that not all of the competence genes are being turned on when Sxy and nucleotides are both present?
Back to PurR, which represses some (non-competence) genes when purines are high. The mutant results would make sense if one or more of the genes needed for transformation is repressed by PurR. We can use the purR knockout mutation to test this! All we need to do is to combine the purR mutation with one of the sxy hypercompetent mutations. If the hypothesis is correct, the transformation frequency of this double mutant should not be reduced by nucleotides.
So my job is to make the purR sxy double mutant, and the grad student will test its competence. My plan is to make (or find) some DNA of the purR mutant and transform it into one or more of the sxy hypercompetence mutants (we have five). Dead easy.
But there's a hitch. The grad student who made the purR mutant is long gone - he failed his comprehensive exams and left no forwarding address. I have his notebook but can't find his stock boxes in the fridges or freezers. Our lab freezer contains vials of what should be the purR mutant cells, but due to a freezer meltdown a couple of years ago most of these cells are probably dead. The mutant cells should be resistant to kanamycin, but my first attempt to grow cells from this stock produced no colonies on the kanamycin plate and only a few on a no-drug plate; I restreaked those yesterday and will find out today whether they are resistant. If not, I have backup plans.
Friday Fabulous Flower - Redbuds
1 day ago in The Phytophactor