Field of Science

Overlooked evidence that purine pools regulate competence

The research associate has prodded me into looking back at old experiments that used our knockout of the purR gene, and I've just realize that one of them provides strong evidence that purine pools regulate competence.

PurR is the purine repressor - it keeps expression of the genes needed to synthesize purines off unless purine pools are depleted. We initially knocked it out because we'd found that adding purines to starvation medium prevented induction of competence genes, and we thought that the competence genes might be directly repressed by PurR. If that were true then knocking out purR should increase competence under otherwise non-inducing conditions, but it didn't.

We now think that the purine effect we saw arises mainly because purine pools control the translation of the sxy mRNA. (PurR may also repress one competence gene, rec2; it has what looks like a strong PurR binding site in its promoter, but so far we don't have good evidence of repression.) The graph below shows how transformation frequencies are changed by introducing the purR knockout into wildtype cells and into five different strains with mutations in sxy that we expect to make sxy expression less sensitive to purine pools. The cells are not being starved, but are in fairly dense cultures in rich medium, a condition where competence is weakly induced. I forget what I was expecting at the time I did this experiment - I think I constructed the strains for a grad student who planned to look at levels of sxy mRNA or protein.

The blue bars are the transformation frequencies of purR+ cells, and the pink and green bars the transformation frequencies of two independent purR- isolates. The left most group is sxy+ cells, and the five other groups are different sxy hypercompetent mutants.

Let's assume that the only direct effect of the purR knockout is on the purine genes. Because there is no repressor in this mutant, these genes are transcribed at their maximum rate all the time, even when cells are growing in rich medium containing an abundant supply of purines. Thus the purine pools in the purR- cells are never depleted, even when the cells are transferred to the competence-inducing starvation medium.

The first thing to notice about the results is that the sxy+ purR- cells transform 100-fold worse than the wildtype parent. Thus preventing purine pools from becoming depleted dramatically reduces the induction of competence. So we really need to now directly compare the expression of competence genes in these two strains. I predict that they will all be down, except maybe rec2. We have done one microarray comparing purR+ to purR- cells; I wonder if the data is still accessible now that our expensive GeneSpring license has expired?

The second thing to notice is that the sxy mutant strains are much less sensitive to the effect of the purR knockout. Remember that these are not sxy knockouts; all but the first (RR699) have wild-type Sxy amino acid sequences. Rather these strains carry mutations that weaken the secondary structure of sxy mRNA, a structure that we have shown to directly limit the amount of Sxy protein produced from the mRNA. So, this new result shows that cells with a weakened sxy secondary structure are insensitive to the state of purine pools!

This was a quick-and-dirty experiment. I did it once and then froze the cells away. Now it's time to thaw them out and do some careful experiments of both transformation frequencies and gene expression. If we can also clarify the rec2 situation we'll have a nice paper.

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