Vindication!

About 15 years ago I hypothesized that Haemophilus influenzae takes up DNA mainly for its content of nucleotides, and predicted that the genes encoding the uptake machinery would be turned on when the cells' energy and nucleotide resources were depleted. At the time we had experimental evidence implicating an energy-depletion signal (CRP and cyclic AMP), but nothing implicating nucleotide pools.

About five years ago we reported that addition of purine nucleotides inhibited both transformation and the induction of two competence genes (comA and rec-2). This was consistent with my prediction, but because we didn't know anything about the molecular mechanism it wasn't compelling. As of last night we know a lot more about the mechanism.

Two different mechanisms are involved. First, nucleotides inhibit production of the competence activating protein Sxy, both by reducing both the amount of sxy mRNA and its ability to be translated into protein. This inhibition depends on base pairing in sxy mRNA; mutations that weaken the base pairing weaken the inhibition. Amp causes a stronger inhibition than GMP does. Second, when purines are abundant the purine repressor PurR represses one or more competence genes. Repression by PurR is stronger with GMP than with AMP, consistent with what's known about PurR's action in E. coli.

Of course this raises lots more questions (see below for my 'real number line' analogy).

What changes inside the cell when nucleotides are added to the outside? As I understand it, cells can't directly take up nucleotides because the phosphate has too strong a charge to cross the membrane, so why do nucleotides have a stronger effect than nucleosides? What molecule interacts with the sxymRNA base pairing? Does this reduce the amount of mRNA being made, or increase its breakdown? Does the molecule act at a riboswitch? Are micro-RNAs involved? Is coupling of transcription and translation involved? Does the same interaction inhibit translation of sxy mRNA, or is that a different effect? Does PurR repress transcription of the rec-2 gene? Does it repress any other competence genes? Why does adding cAMP partially counteract inhibition by GMP and, to a lesser extent, by GMP. Do the same regulatory effects occur in H. influenzae's relatives?

The real number line analogy: Scientific knowledge is like the "real number line" used in introductory math classes. In the line, every point is a number, but no matter how close together two points (or numbers) are, there are always infinitely many other points separating them. The real world similarly contains infinitely many things to discover. No matter how much we find out about something, there are always many more important things to find out. I use this analogy for beginning science students, who are often concerned that all the important discoveries have already been made.