Five or six years ago we published a paper reporting that H. influenzae cells won't become competent if they're given lots of purine nucleotides.
The standard method for inducing H. influenzae to take up DNA is to start them growing in a rich medium called 'supplemented brain-heart infusion' (sBHI for short) and then transfer them abruptly to a starvation medium called MIV ("m-four"). The starvation medium lacks almost everything they normally get from the rich medium to grow, including purine and pyrimidine nucleotides, and we think the reason that cells turn on their DNA uptake genes in this medium is because they can use the DNA they take up as a source of nucleotides. If purine nucleotides are added to the starvation medium, the cells don't become competent. In the paper we were able to test expression of two competence genes and show that they didn't get turned on when the nucleotides were provided.
This only happened with purine nucleotides, not pyrimidines, and we speculated that the reason might be that competence genes were regulated by the PurR repressor. This protein is known to control expression of the genes that make purines for the cell; when purine bases are abundant in the cytoplasm they bind to PurR, enabling it to bind sites in the promotes of the purine-synthesis genes, blocking gene expression. If cells take up DNA to get nucleotides it would make sense to regulate uptake according to the amount of purines in the cell. We were encouraged in this idea by finding what looked like PurR binding sequences in the promoters of a couple of competence genes (comA and rec2). (Note that this was before we had any good idea of what Sxy does, and before we had identified the full set of competence genes.)
So a grad student knocked out the PurR gene. If our hypothesis had been right,this would have made the competence genes come on even when cells had plenty of purines. But it didn't. Furthermore the PurR mutant cells grew normally in sBHI, and developed competence on transfer to normal MIV but not on transfer to MIV with added AMP or GMP. The grad student also tested whether the comA and rec2 genes were now not repressed by nucleotides, and showed cleanly that they were.
Since then we've done a bit more analysis of his mutant. The one microarray we did showed maybe some induction of rec2. But I just went back to the grad student's notes and looked more critically at those potential binding sites for PurR in the comA and rec2 promoters - they now look very unconvincing to me. So unless I can think of a good reason to do more on this, I should stop flogging this dead horse.
Leroy Hood and the tool-driven revolution in biology
1 day ago in The Curious Wavefunction