- Does PurR regulate rec2 expression?
- Do nucleotide pools regulate Sxy expression?
One good condition is early log phase growth in rich medium: cAMP is low, purines are high regardless of PurR activity, and Sxy is as off as it gets. Another is early log phase growth in rich medium with added cAMP: purines are still high, but Sxy expression is intermediate. Under these conditions, seeing higher rec2 expression in the purR mutant would be evidence that PurR normally represses rec2.
Starvation in MIV is not so good a condition: exogenous purines are gone, but the amount of endogenous purines may differ between PurR+ and PurR-. This could reduce Sxy expression in the purR mutant and thus activation of the CSP-S-controlled rec2 promoter. So seeing no change in rec2 expression would not be good evidence that PurR doesn't repress rec2. However, seeing that the purR knockout increased rec2 expression would be good evidence of repression, but might underestimate its magnitude. We could also just test whether supplementing MIV with either of PurR's co-repressors, guanine and hypoxanthine, affects rec2 expression - our preliminary experiments suggest that they don't affect transformation frequencies.
We could also test the hypercompetent sxy mutants, because we think that their Sxy expression in MIV may be less affected by nucleotide pools. Here, seeing that the purR knockout didn't change rec2 expression would be stronger evidence that PurR doesn't repress rec2. Analysis in MIV will be cleanest once we have a purH mutation to eliminate endogenous purines. In this background, seeing no effect of the purR knockout on rec2 expression would be unambiguous evidence that PurR doesn't repress rec2.
For question 2, we can measure Sxy protein expression directly with Western blots or with Laura's lacZ fusions, measure mRNA levels of Sxy-regulated genes with realtime PCR, measure DNA uptake, or measure transformation frequencies. One of Laura's fusions eliminates the sxy mRNA secondary structure, and this may be very useful as a control. We want to manipulate the cell's supply of nucleotides for transcription, which we can do by knocking out purH (eliminating endogenous synthesis of purine nucleotides), by knocking out purR (making endogenous synthesis of purine nucleotides constitutive) and by providing exogenous nucleotides, nucleosides or bases. We don't know how big the effects of the latter two interventions would be.) There's also a defined medium for H. influenzae, which can be supplemented with different amounts of inosine or other purine precursors. Hypercompetent sxy mutations may change the sensitivity of Sxy expression to nucleotides. If we're measuring transformation frequencies we also need to control for PurR-mediated effects on rec2 expression.
What culture conditions and genetic combinations should we compare, and what should we measure? Although not knowing whether PurR represses rec2 will initially limit interpretation of the results, it makes sense to initially measure the transformation frequencies of the culture samples that are collected for mRNA and protein measurements. We may want to leave the lacZ fusion analysis for later, as a corroboration.
We don't yet have a purH mutant, so we can't knock out endogenous production of purine nucleotides. However we can figure out how big a contribution endogenous synthesis makes in wildtype cells under different conditions, by comparing old microarray data on expression of PurR-regulated genes during growth in sBHI with the expression induced by transfer to MIV. We also have one array comparing PurR+ and PurR- cells, grown in sBHI + cAMP to an OD of about 0.5, so we can compare fully-derepressed to repressed expression.
LATER: OK, I've gone through the Imagene files of one of our microarray time courses (9 slides) and our one purR+/purR- microarray, manually pulled out all the genes with 'pur' and 'pyr' names into a new Excel file (by sorting by gene name), and examined their expression ratios throughout growth in sBHI and after transfer to MIV. The result is very clear: genes regulated by PurR are not induced at all in sBHI, even when the culture gets close to stationary phase. The genes are strongly induced by transfer to MIV and by the knockout of purR. (I can't show graphs here because I've forgotten how to force Excel to give me sensible axes. I'll post it tomorrow.)
This means that our purR knockout cells should have much higher expression of the purine biosynthetic genes at the time they're transferred to MIV, and much higher expression of them in late log. Furthermore, if PurR does regulate rec2, rec2 should normally be PurR-repressed in late log, with repression only released in MIV and in the purR knockout.