I've now shown fairly convincingly that being competent does not enhance H. influenzae's ability to cope with hydroxyurea, which stalls replication forks by blocking the synthesis of the dNTP precursors needed for DNA synthesis. To more completely test my hypothesis that cytoplasmic competence-regulated proteins exist to mitigate the damage caused by stalled DNA replication, I'm going to test whether cells lacking the proteins DprA or RadC are more sensitive to hydroxyurea than are wildtype cells.
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We have all the mutants in the freezer:
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- dprA deletion:
- radC deletion:
- rec1 mutation: This is a very old strain, one of the original H. influenzae transformation mutants. It should be completely defective in recombinational repair, but I'm not sure how this interacts with hydroxyurea. Hydroxyurea induces recA in E. coli.
- recB/recC mutation: This is another very old strain, not a modern knockout, but it's phenotype was well studied.
I just streaked them all out so I can test them on Saturday. I'll use the same method I did for the first hydroxyurea experiments, growing them to log phase and then diluting them way down to about 2000 cfu/ml in medium with and without 50 mM hydroxyurea and following them for several hours and overnight by plating 50 µl aliquots.
While looking for any work on the effects of recA mutations on sensitivity to hydroxyurea, I discovered (i) a paper showing that hydroxyurea induces competence in Legionella pneumophila, and (ii) mention of perhaps-unpublished data showing the same thing for Streptococcus pneumoniae. The L. pneumophila authors state in their Discussion that "We currently favor the hypothesis that stalling of the DNA replication fork is the primary signal leading to competence
development."
So I should definitely also test whether hydroxyurea induces competence in H. influenzae. This will be easy: grow cells to log phase, dilute into medium containing marked DNA (MAP7, 200 ng/ml) and different concentrations of hydroxyurea (0, 5, 10, 20, 50 mM). Grow for 1-2 hr and plate ± novobiocin.
Charpentier et al. Antibiotics and UV Radiation Induce Competence for Natural Transformation in Legionella pneumophila J. Bacteriol. March 2011 vol. 193 no. 5 1114-1121.