Field of Science

Reclaiming my explainer energy (from MOOCing back to blogging)

My Useful Genetics MOOC (massive open online course) isn't really over yet, but almost all the work is done so I'm finally able to think about research.  And it's high time, because we have two research visitors in the lab for the next few months, the post-doc and I have two reviews to write, and I need to write a grant proposal for a Sept. 15 deadline.

I'd better start with the proposal.  We've had 5 (or is it 6?) unsuccessful tries to get CIHR funding for work on the mechanism of DNA uptake so, even though one of our visitors brings expertise that would help with this, I've given up hope of ever getting serious funding for this work.  We then made one try to get funding for a new project, aiming to predict recombination in natural populations.  This was solidly rejected.  So now I'm going to go back to our funded strength, the regulation of competence.
We have several papers in this area recently, and lots of directions to investigate.
Here I've pasted directions onto our standard diagram of competence regulation - all the blue boxes are regulatory problems in serious need of investigation.  I won't necessarily include them all in the proposal, and there are probably other problems I've forgotten about, but here are the basics:
  1. Fructose:  Competence is regulated by cAMP levels which are elevated by the H. influenzae phosphotransferase system (PTS) in response to depletion of the sugars that the PTS would otherwise transport.  In H. influenzae the only such sugar is fructose.  Regulation by fructose levels in the host environment makes no regulatory sense.
  2. Sxy-CRP interactions: We've done a lot of work on these, and by we I mean the former RA (she has a great new job in a high-powered lab across the street) and former grad student/postdoc (he has a great new job at the University of Regina).  But we still don't know what's going on.
  3. Hfq:  Hfq is important for the activities of small regulatory RNAs. Knocking out Hfq reduces competence 10-fold, by an unknown mechanism that's independent of the purine-repression effect described below.
  4. Toxin-antitoxin system: One pair of genes regulated by competence turns out to encode a probable toxin-antitoxin system, with the antitoxin preventing the toxin from preventing DNA uptake and transformation.  We have no idea  whether this futile combination has any biological function.
  5. Regulation by purines: We have a nice new paper out about the purine regulation, showing that it acts by stabilizing the sxy mRNA secondary structure that blocks translation.  But we have only very indirect evidence of how it does this.
  6. Effect of PRPP on sxy mRNA translation:  The indirect evidene suggests that purines may regulate sxy mRNA translatability by changing the intracellular level of the intermediate PRPP (phosphoribosyl pyrophosphate).  We probably can't investigate this biochemically, because PRPP is not something you can buy (probably very unstable?).  But maybe we can investigate its role genetically, by making more mutants.
  7. 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.
  8. murE mutants: Point mutations in the cell wall biosynthesis gene murE cause constitutive expression of the competence regulon.  We have no idea why.


  1. You are right, hydroxyurea is the stuff to use to induce replication fork stalling. Since you inhibit RNR, it is also reversible by washing your culture. I'm not really sure if bacterial RNR is affected by HU, though. I'm only aware of HU being used in eukaryotes.
    Alternatively, you could try camptothecin that induces fork stalling by TOP1 inhibition. This is also reversible, but again I don't know about effectivity in bacteria.
    An approach that will work in all organisms is cross-linking of DNA by Mitomycin C, but that is definitely irreversible.
    In all of those cases you will also have to think about repair pathways, since fork stalling will induce homologous recombination.

  2. We know from experiments I did 20 years ago that crosslinking by mitomycin C doesn't induce competence. Now I think it's important that the fork stalling be due to nucleotide scarcity, so HU is just what we want.

    So I just did a Google Scholar search for 'hydroxyurea Escherichia coli' and found two nice papers from 1967 and 1972 that showed excellent inhibition, and the range of test conditions to use. Now I just need to check that these conditions aren't toxic to H. influenzae.


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