Field of Science

Experimental design good, results discouraging

Well, I tested whether competence helps cells survive treatment with hydroxyurea, and the answer appears to be 'No'.

First a reminder of why I did this experiment: For years I've been hypothesizing that the function of at least some cytoplasmic genes in the H. influenzae competence regulon is to stabilize replication forks that have stalled because of a shortage of nucleotides.  Because the simple chemical hydroxyurea specifically inhibits the enzyme ribonucleotide reductase, which is needed to convert NTPs to dNTPs for DNA synthesis, the most important experimental question is whether competence protects cells from the harmful effects of hydroxyurea, with or without DNA uptake. - See more in this post.

What I did:  Cells with different levels of competence, in exponential growth in rich medium, were transferred to the same medium with and without 50 mM hydroxyurea, and growth and survival were followed by plating and by measurement of OD600.

What I observed:  Over a 3 hr period where DNA replication was arrested by hydroxyurea, cultures that were constitutively or partially competent did not exhibit increased growth or survival.

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The cells:
  1. K: Wildtype strain KW20.  Not competent in exponential growth but inducible by starvation.
  2. KC: Wildtype strain KW20 with 1 mM cAMP added 45 min earlier to induce moderate competence
  3. 5: Mutant strain RR563.  Has a hypercompetence mutation in sxy so is moderaately competent in exponential growth.  Similar transformation frequency to KC.
  4. 6: Mutant strain RR648.  Has a knockout of sxy so cannot become competent at all.
  5. 7: Mutant strain RR749.  Has a hypercompetence mutation in murE.  Competence is fully induced in exponential growth.
The results:  

Cell growth:  These cells were diluted at t = 0 into medium ± hydroxyurea, and their growth was followed by measuring the turbidity of the culture. Cells with arrested replication are expected to continue growing but to cease division (the cells form filaments), and that's what these cells did - growth was slowed only slightly by 50 mM hydroxyurea.

Cell division:  The same initial cultures were diluted 1:50,000 into medium ± hydroxyurea and their numbers were followed by plating and counting colonies.  Now we see that hydroxyurea did arrest cell division; the cells with hydroxyurea doubled only once or twice in the time that the control cells doubled more than seven times. 

Cell survival:  This is the same data as the above graph, with the addition of cfu counts after the very dilute cultures continued incubating overnight.  Ignore the '300 min' label on the X-axis; this was really after another 16 hr of incubation.  Cells in some of the hydroxyurea cultures divided a few more times, one culture kept the same cfu, and the cfu of the cells with cAMP decreased about 10-fold, probably due to the cAMP's general perturbation of gene expression.  There's no correlation with level of competence - the most competent cells increased only a bit more than the cells unable to become competent.  (The cells in the control cultures grew overnight to the expected 10^9 cfu/ml.)

Complications and plans:  One weakness of this experiment is that many (perhaps most) of the cells in a competent culture are not transformable, so many may not be expressing the cytoplasmic proteins that I hypothesize are protective.  This could reduce the sensitivity of the experiment by a lot.  

One way to clarify this would be to also assess survival of the transformable cells, by adding novR transforming DNA to the cultures and plating cells on novobiocin plates as well as plain plates.  This will make the experiment more complicated, largely because I'd have to work with less dilute cultures and do some dilutions for all the cells on plain plates.On the other hand, having the results of the experiment I've just done will let me streamline the plating, partly making up for the extra work and the uncertainty of survival and transformation frequency on the nov plates.  This strategy won't work for cultures that aren't competent at all (there will be no novR transformants), so I'd leave out the KW20 and RR648 cultures.  But it should work nicely for KW20+cAMP, and for RR563 and RR749. 

Could I also leave out most or all of the no-hydroxyurea controls?  Do I expect the number of transformants to parallel the total numbers of cellsIn the absence of hydroxyurea?  Perhaps not, since new competent cells will continue to become transformed over the time of the experiment.  So I'd better retain these controls.

Unexpected discovery: Cells grow faster when they're very very dilute.  The control cells in the top graph (blue lines) appear to be growing exponentially as expected; the log-scale lines are straight until the second-last time point.  The doubling time is about 35 minutes, which is typical for our cultures.  But when the cells were at a very low density in the same medium (second graph), they grew with a doubling time of about 24 minutes, faster than I've ever seen!  So I should do a separate experiment, following change in cfu of wildtype cells from from very dilute to more dense.  

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