Last week's experiment tested whether cultures with higher levels of competence were less affected by hydroxyurea, which inhibits synthesis of dNTPs. It found no correlation, but the conclusions were weakened by presence of many non-competent cells in the cultures. So in this new experiment I also measured the numbers of surviving cells that had become transformed to novobiocin resistance by marked DNA I added to the cultures. Because these cells must have been competent to become transformed, their survival should specifically show how hydroxyurea affects competent cells.
The results show that the frequency of transformed cells was not increased by hydroxyurea treatment, in fact it was lowered in 2 of the 4 cultures and unchanged in the other 2.
I tested 4 cultures, each with and without 50 mM hydroxyurea:
- 'Kc' is wildtype cells with competence partially induced by 0.2 mM cyclic AMP (1/5 the dose I used previously). The expected transformation frequency (TF) in log phase is 10^-5 - 10^-4.
- '5c' is the hypercompetent strain RR563, fully induced by addition of 0.2 mM cAMP. Expected TF is 10^-3 - 10^-2.
- '5' is the hypercompetent strain RR563. Expected TF is 10^-5 - 10^-4 in exponential growth, higher in a dense culture.
- '7' is the very hypercompetent strain RR749. Expected TF is ~ 10^-3.
The first graph shows total cells (cfu) over 4 hr of incubation with (open symbols) and without (solid symbols) hydroxyurea. The cells are at different densities, all more dense than in the previous experiment, because I also needed to plate for transformants. With no hydroxyurea the cells grew exponentially as expected (RR749 doubling time 25 min, 5c slower because of the cAMP, and Kc and 5 slowing down as they became dense. Growth of the two relatively dense low-competence cultures (Kc and 5) was only transiently slowed by hydroxyurea, whereas growth of the two low-density maximally competent cultures stopped and cell numbers fell.
I can think of some caveats, but they're quite weak. For example, it's possible that hydroxyurea prevents competent cells from becoming transformed, or causes them to become unable to take up DNA. There may also have been a confounding effect of cell density - the two relatively dense cultures were much less affected by the hydroxyurea.
But overall, the obvious conclusion is that being competent does not help cells survive or grow when dNTP pools are depleted by hydroxyurea treatment. So I wonder what the cytoplasmic genes in the competence regulon contribute. It's certainly possible that they've been selected for their recombination-enhancing effects, as everyone else assumes, but this depends on the assumption that recombination is the funciton of DNA uptake, which I still think very unlikely.