After epic faffing around with various defined media*, I finally have clean results confirming that wildtype H. influenzae can grow without added inosine but our new purH mutant can't. (Although, given how much variation I've seen in growth in different versions of these media, I still need to confirm this result at least once more.) So now it's time for some serious thinking about what experiments to do.
One goal is to find out whether the rec2 gene is indeed repressed by the PurR repressor. In the purH mutant, PurR doesn't affect synthesis of purine nucleotides, and thus shouldn't affect competence in other ways. Once we have a properly confirmed purR mutant (we're still waiting for the sequences), we'll test the purH mutant and the purR purH double mutant for competence. We should probably do this both under conditions where expression of other competence genes is as high as it can be, and where expression of other competence genes is limiting. So first we should test competence during growth in rich medium and after full induction by MIV; we could also add cAMP, test hypercompetence mutants, test defined medium. We also should do more-or-less direct tests of rec2 expression levels with and without PurR. Doing these in the purH mutant background will eliminate the proposed regulatory effects of purine nucleotide synthesis.
And now I think I can also start using defined media and the purH knockout to investigate the proposed regulatory effects of purine nucleotide synthesis. The defined media for H. influenzae typically contain lots of inosine because, many years ago, it was discovered that cells transferred from a defined medium to starvation medium didn't become very competent unless the defined medium had contained lots of inosine. But nobody had followed this up as a component of competence regulation. But I can, now that I have cells growing well in defined medium with and without inosine (and no other purine source).
If I transfer wildtype cells from cMMA ± inosine to MIV, do they become competent? Do cells become competent at all when growing in cMMA ± inosine? How competent are hypercompetent mutants growing in cMMA ± inosine? I guess the first step is to do a time course of growth and competence of wildtype cells in cMMB ± inosine, including transfer to MIV.
Better go pour some plates.
* and discovering, when I went to put our new 500 mg bottle of glutathione away, that we already had a 10 g bottle that I'd somehow overlooked.
A new kind of problem
12 hours ago in RRResearch