I think our purH mutant may turn out, like our cya mutant, to be much more valuable than I had originally thought. My first graduate student made the cya mutant (defective in adenylate cyclase and thus unable to make cyclic AMP (cAMP) as her M.Sc. project. When she proposed doing this I thought only that it would let us confirm what was already known, that cAMP induces competence. But we used it to test other questions about how the regulation works, and it's also been very useful as a conditional competence mutant - it can't transform at all, but transforms fine if given cAMP.
We made the purH mutant to eliminate endogenous purine synthesis so we could test the role of the purine repressor PurR without having to consider its effects on purine nucleotide pools, and so we could manipulate purine nucleotide levels by providing precursors in the medium, without having to worry about the unknown effects of endogenous synthesis.
Now we have the mutant I'm going back through my previous posts to collect the ideas I've had about ways to use it. I've now checked its competence after induction with MIV starvation medium, and it's not significantly different from the purH+ wildtype strain (only done once so far).
1. To find out whether PurR represses rec2, we can compare spontaneous competence in the purH mutant and a purH purR mutant. We haven't constructed this double mutant yet but doing so will only take a day. We'll do this test during 'late-log' growth in rich medium, a condition where PurR is normally active. If PurR does repress rec2, we expect the double mutant to have a higher transformation frequency and higher expression of rec2 (assayed by quantitative PCR). We can also examine rec2 mRNA by quantitative PCR and by using a rec2::lacZ reporter gene strain we have in the freezer. This would be a good minor project for the post-doc, as it may lead to a publication.
2. To find out whether sxy translation is controlled by purine pools, we can manipulate purine pools and compare sxy expression (sxy mRNA assay and Sxy protein assay or mRNA assay of any CRP-S gene) in purH mutants carrying either the normal sxy gene or one of our hypercompetent mutants. If purine pools regulate translation of sxy mRNA, we expect to see the ratio of protein to mRNA increase when purine precursors are withdrawn.
3. Does purine synthesis by wildtype cells help them survive in MIV? Does it affect how competence develops at all? The one experiment I've just done says not, but this needs to be repeated in more detail.
4. Do cells in rich medium get all their purines by salvage? If so, the purH mutant should grow just as well as wildtype.
5. Does purine synthesis play any role in late-log competence? If so, the purH mutant might become more competent than wildtype cells.
6. I only today thought of this - we could also use the purH mutant to do something that's been on the back burner for more than a decade - demonstrate that H. influenzae cells actually do use the DNA they take up as a nutrient. The experiment would use a hypercompetent-sxy purH double mutant, testing whether the presence of DNA increases survival (and maybe allows growth) in medium lacking a purine precursor.
Why I'm Marching for Science
16 hours ago in Angry by Choice