Field of Science

Not the birthday present I would have preferred

I seem to have now thoroughly disproved one of my favourite hypotheses, that cytoplasmic genes in the competence regulon act to help cells survive depletion of pools of deoxyribonucleotides (dNTPs).

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.

The second graph (below) shows the transformation results, which should reflect the growth and survival of the competent cells in each culture. (The dashed line and red arrow indicate an 'upper-limit' data point where no transformants were seen.)  For wildtype cells + cAMP (blue lines) hydroxyurea had identical effects on competent and non-competent cells.  For the fully hypercompetent strain RR749 (purple) the competent cells were slightly more affected.  For the partially hypercompetent strain RR563 (green), transformants were reduced about 5-fold by hydroxyurea, and for RR563 + cAMP (red) transformation was decreased 10-fold at 90 min and undetectable at 230 min (dashed line and red arrow).

I left the cultures shaking overnight and plated them again the next day (graphs below).  I'm only showing this for completeness; it doesn't really add anything to the conclusions.  All the no-hydroxyurea cultures were at about 10^9 cfu/ml, and the hydroxyurea cultures were between 10^4 and 10^7 cfu/ml.  The transformation frequencies of the hydroxyurea cultures were the same as (wildtype + cAMP) or about 10-fold lower than their untreated counterparts.

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.

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