Hydroxyurea stalls DNA replication; does competence help cells survive?

In a post a couple of weeks ago I wrote:

Stalled replication forks:  We have been hypothesizing that one function of the competence regulon's proteins is to stabilize replication forks that have stalled because of a shortage of nucleotides.  One way to test this is to see if hydroxyurea induces either the regulon or competence, because hydroxyurea inhibits the enzyme ribonucleotide reductase, which is needed to convert NTPs to dNTPs for DNA synthesis.  I'm told that hydroxyurea is known to cause stalling of replication forks, though I haven't looked for this yet - See more at: http://rrresearch.fieldofscience.com/#sthash.IQAAtROv.dpuf

• Stalled replication forks:  We have been hypothesizing that one function of the competence regulon's proteins is to stabilize replication forks that have stalled because of a shortage of nucleotides.  One way to test this is to see if hydroxyurea induces either the regulon or competence, because hydroxyurea inhibits the enzyme ribonucleotide reductase, which is needed to convert NTPs to dNTPs for DNA synthesis. 

I've now dug up the evidence that hydrozyurea inhibits synthesis of dTPs and blocks DNA replication.  It's partly very old but solid E. coli biochemistry papers and partly new molecular biology in both E. coli and mammalian cells.  The E. coli papers have useful information about kinetics and concentrations.

Stalled replication forks:  We have been hypothesizing that one function of the competence regulon's proteins is to stabilize replication forks that have stalled because of a shortage of nucleotides.  One way to test this is to see if hydroxyurea induces either the regulon or competence, because hydroxyurea inhibits the enzyme ribonucleotide reductase, which is needed to convert NTPs to dNTPs for DNA synthesis.  I'm told that hydroxyurea is known to cause stalling of replication forks, though I haven't looked for this yet - See more at: http://rrresearch.fieldofscience.com/#sthash.IQAAtROv.dpuf

Stalled replication forks:  We have been hypothesizing that one function of the competence regulon's proteins is to stabilize replication forks that have stalled because of a shortage of nucleotides.  One way to test this is to see if hydroxyurea induces either the regulon or competence, because hydroxyurea inhibits the enzyme ribonucleotide reductase, which is needed to convert NTPs to dNTPs for DNA synthesis.  I'm told that hydroxyurea is known to cause stalling of replication forks, though I haven't looked for this yet - See more at: http://rrresearch.fieldofscience.com/#sthash.IQAAtROv.dpuf

I think the most important experimental question is not whether hydroxyurea induces competence but whether competence protects cells from the harmful effects of hydroxyurea, with or without DNA uptake.

I've only done one experiment so far, a preliminary test of the kinetics of growth inhibition (really, cell division inhibition) and killing of wildtype cells.  Over the 2 hr incubation period, 10 mM had no effect, 30 mM slowed the increase in cell numbers only for the first hour, 100 mM prevented cell division but didn't kill the cells, and 200 mM killed most cells in the second hour.   

The next step will be to measure effects of hydroxyurea on cells with different levels of competence.  Rather than artificially inducing competence, I'll mostly rely on mutants, a sxy knockout that can't turn on competence genes at all and two hypercompetent mutants, sxy-1 (moderately competent) and murE749 (very competent even during exponential growth).  But I'll also test wildtype cells with added cyclic AMP, which induces a similar  hypercompetence as the sxy-1 mutation.

I'll try two complementary ways to do these tests.  In both I will get the cells exponentially growing in rich medium and follow growth after adding different concentrations of hydroxyurea.  

Test A will use the same viable-count method I used for the first experiment, plating the cells at different times after hydroxyurea addition to see how much growth/cell division slows or stops and the extent of cell killing. Because I dilute the cells down to about 2000 cfu/ml when I add the hydroxyurea I can just plate the cultures directly, without having to dilute them further at the time of sampling.  This makes it possible to test multiple concentrations and lots of time points.

Test B will make use of the Bioscreen incubator belonging to the lab next door.  This lets me follow detailed growth curves for up to 200 samples at once.  The disadvantage is that it measures growth by changes in optical density, with a fairly narrow range (only a few doublings); it can't measure viability changes at all.

The combination of the two tests should give nicely complementary information about the effects of hydroxyurea on growth and viability.  The trickiness will be getting the initial cell concentrations for each test right - 2000 cfu/ml for Test A and about 10^8 cfu/ml for Test B.

Papers: 

Neuhard 1967. Studies on the acid-soluble nucleotide pool in Escherichia coli: IV. Effects of hydroxyurea.  Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis 145:1–6

Sinha and Snustad. 1972? Mechanism of Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli by Hydroxyurea.  J. Bacteriol. 112 :1321-1334

Davies et al. 2009 Hydroxyurea Induces Hydroxyl Radical-Mediated Cell Death in Escherichia coli.   Molecular Cell, Volume 36, Issue 5, 845-860
 (toxin-antitoxin systems involved!  Microarray data!!!)