Purine regulation of competence

At today's lab meeting the Research Associate presented her analysis of all the work we've done over the past >10 years on how purines affect competence. It's time to get this all sorted out. The plan is to first repeat a few basic experiments and then either set it all permanently aside, or do the additional work to clarify the effects and publish a paper.

Here are links to my previous posts about this (most recent first, oldest last):

• Overlooked evidence that purine pools regulate competence
• Does PurR regulate rec2 and does this matter?
• Our vindication is on hold...
• Make the bacteria do the work
  
• Vindication!
• Maybe PurR does repress competence genes after all
• Does PurR repress any competence genes?
• How sxy expression is regulated

Three ways we can modify purine pools:

1. By changing culture conditions: We grow cells in a purine-rich medium, sBHI, where the purine biosynthetic genes are normally kept off by the purine repressor PurR. We think that purines are probably depleted at the end of log phase (we could check for induction of purine biosynthesis genes in our old microarray time course data), and we know they are when we transfer cells into the 'MIV' starvation medium that induces competence.
   
2. By adding purine nucleotides, nucleosides or bases to BHI or MIV.
   
3. By knocking out the gene for PurR so that the biosynthetic genes are always fully on.

Several ways we can measure effects of changed purine pools:

1. We can use real-time PCR or microarrays or Northern blots to measure the amount of mRNA produced from purine biosynthetic genes, from sxy, and from Sxy-regulated genes, and from control genes that should be independent of changed purine pools (e.g. housekeeping genes, CRP-induced genes (with CRP-N site promoters)).
2. We can use gene fusions to the lacZ gene to indirectly measure the amount of transcription of sxy, rec2 and comA by assaying beta-galactosidase. We could do this for other genes too but we'd need to first construct the necessary lacZ fusions.
   
3. We can use antibodies to measure the amount of Sxy protein produced. We may have an OK antibody for pilin too.
   
4. We can measure DNA uptake using radioactive DNA.
5. We can measure transformation frequencies using genetically marked DNA. The cells used for these assays can have been competence-induced with MIV, or they can be at different stages of growth in sBHI.
   

Ways we can manipulate sxy expression:

1. We can add cyclic AMP (cAMP) to sBHI or MIV to maximally induce the sxy promoter (and all the Sxy-independent genes regulated by CRP).
2. We can use hypercompetent-mutant versions of sxy, in which the 5' end of the mRNA does not maintain the expression-inhibiting secondary structure that we suspect responds to changes in purine pools.

Regulatory effects we need to investigate: In principle, added purines might cause several different regulatory effects. First, they would prevent depletion of purine pools that normally occurs after transfer to MIV. This would activate the PurR repressor, which might normally directly repress one or more competence genes. Preventing depletion of purine pools might also directly limit sxy expression, independent of PurR.

Because we have anecdotal (not well documented) reports that adding purine nucleotides made the cells sick, I think experiments that use added purines need to carefully control for non-specific effects on growth and transcription. However, the effects of knocking out purR aren't so simple either, as the purine biosynthetic genes will then all be constitutively induced, changing the effect of transfer to MIV.

1. Does PurR directly repress any competence genes? The only candidate with a reasonably good possible PurR binding site in its promoter is rec2. We can easily use real-time PCR to measure rec2 mRNA in purR+ and purR- cells. The problem is to keep the cells' supply of purines constant, so any effects of purine pools on sxy expression are controlled. I think the best solution is to have both purR+ and purR- cells carry a mutation knocking out the last step of the purine biosynthetic pathway (purH, HI0887), so that both would be totally dependent on the external supply of purines. An independent test would be to mutate the putative PurR binding site in the rec2 promoter - we could then look for effects on rec2 mRNA and on competence.
   
2. Does the secondary structure of sxy mRMA make sxy expression sensitive to purine pools? I think our best tools for investigating this are the hypercompetence mutations in sxy. We could measure sxy mRNA, Sxy protein, and/or expression of any CRP-S genes in normal and hypercompetent cells. Ideally we would also do these experiments in a purH knockout. As a control for hypercompetence effects we could use our other set of hypercompetent mutants, with mutations in murE, but this control would be slightly compromised by our ignorance of how these mutations cause Sxy overexpression.

Simpler experiments we should do first:

1. We need to use PCR to confirm that our purR mutation is correct, because the original stock died and was recreated by transformation.
   
2. I want to repeat the experiments I described in my post about Overlooked evidence that purine pools regulate competence. Then I only tested one condition, and not carefully. The former grad student also examined this in a single sketchy time course - this showed a smaller effect. So I think I should do time courses of transformation using wildtype, purR, sxy-hypercompetent, and the double sxy/purR mutant, all growing in rich medium. If I again see that the purR knockout dramatically reduces late-log competence, and that the sxy hypercompetence mutations eliminate/reduce this effect, then I'll be confident that the previously described effect of adding purine nucleotides was not due to some general toxicity due to unbalanced growth.
3. I'll let someone else do the MIV-competence assays. We have good data showing that the purR knockout doesn't affect MIV competence, and that the purR mutant is not significantly less sensitive to addition of purine nucleotides that its purR+ parent. (However we also have good data showing that the purR knockout reduced expression of the CRP-S gene comA by about 50-fold, which is consistent with the hypothesized nucleotide pool effect of purR on sxy expression. And the knockout didn't reduce expression of rec2, consistent with the nucleotide pool effect being compensated for by loss of PurR repression of rec2.) If the hypothesis that nucleotide pools limit sxy expression is true, we predict that hypercompetence mutations in sxy will make competence in MIV much less sensitive to added purine nucleotides.
   
4. I think it will be hard to use transformation assays to detect the effect of the hypothesized repression of rec2 by PurR, because so many other genes are also needed. We would need to find conditions where the amount of Rec2 is limited by PurR and Rec2 in turn limits the transformation frequency. Hmm, I think that's likely to happen only in a hypercompetent mutant in rich medium, and then maybe only if sxy transcription is induced with cAMP. So maybe I can also do the time courses ± cAMP.