In the previous post I described my hypothesis that the CRP-S regulon unites genes that, in different ways, help cells cope with running out of nucleotides for DNA synthesis (dNTPs). The DNA uptake genes help by getting deoxynucleotides (dNMPs) from an alternative source (DNA outside the cell) and the genes for various cytoplasmic proteins help by stabilizing the replication fork until the dNTP supply is restored.
So we really ought to try to directly test whether becoming competent helps cells survive sudden nucleotide shortage. I think I tried this a long time ago (when I was a post-doc in Ham Smith’s lab). Then I was hoping to show that DNA uptake helped cells survive transfer to MIV. I did establish a clear survival curve for cells in MIV. I also found out that adding DNA didn’t make much difference.
I tested various competence mutants we had then - these were miniTn10kan insertions that reduced competence by knocking out various genes. The only one that I remember made any difference to survival was the one we now know knocks out CRP. This is the one mutation in that set that is definitely regulatory - it prevents induction of all the CRP-S regulon genes, and all the CRP-N regulon genes too.
My hypothesis predicts that crp- cells would not turn on the genes that help them survive this crisis, and so should survive worse. BUT (as I recall) the crp mutant cells survived MIV much better than the wildtype cells! Yikes, this is the opposite of my prediction. I need to go back and reexamine this old data.
Hmmm... The old experiments had compared the number of cfu (cells capable of growing into colonies) after 100 minutes in MIV to those after about 16 hours (overnight) in MIV. I compared 6 mutants to the wildtype strain. Five of them survived better, and one much worse. But the 'much worse' strain isn't really a competence mutant at all - we now know its mutation knocks out a DNA topoisomerase that that non-specifically reduces the induction of many unrelated genes. Four of the mutants survive about 5-10-fold better, and the crp mutant survives about 100-fold better.
One concern is that my hypothesis is about short-term survival, in the emergency situation created by transfer to MIV, and so wouldn't necessarily apply to overnight survival. I haven't explicitly tested short-term survival, but the numbers of cfu at 100 minutes should be a rough indicator if this, as the cultures were all grown to about the same densities before being transferred to MIV. The old data don't show the kind of dramatic difference I would hope to see if my hypothesis is correct. Time to think about a rigorous test.
Friday fabulous flower - alotta hosta
1 day ago in The Phytophactor