State of the research: I. the new grad student's project

Before I get back into doing experiments I need to reestablish my knowledge of what everyone else in the lab is working on.  So I'm going to do a quick summary series of posts, starting with the new grad student's warm-up project.  (This isn't his real thesis project, but a smaller project he took on as a way to get a better grip on the lab's goals and techniques.)

The big-picture goal of the project is to find out how the cytoplasmic proteins DprA and ComM protect incoming DNA.  Both genes are in H. influenzae's CRP-S competence regulon, and knockouts have similar phenotypes -- DNA uptake and translocation are normal, but the transformation frequency is severely reduced.  I initially hypothesized that DprA acts by directly blocking a specific cytoplasmic nuclease that would otherwise degrade free DNA strands in the cytoplasm, but work in S. pneumoniae has indicated that it instead binds to DNA, shielding it from nucleases.  The DprA protein is ubiquitous in Bacteria (competent and not) and in the well-studied competent model systems knockouts consistently give an uptake+ transformation- phenotype.  The E.coli protein is reported to partly restore competence to an H. influenzae knockout(Smeets et al 2006).  This was interpreted as meaning that competent cells are co-opting a DprA function independent of DNA uptake, but I have two concerns with this.  First, the wildtype transformation frequency in this experiment was about 500-fold lower than it should be.  Second, E. coli has a competence regulon (though dprA isn't in it), so its DprA may not be the best example of a competence-independent protein.

We don't know anything about what ComM does (it's not involved in other competence systems, and homologs aren't known from other systems), but our hypothesis is that, like DprA, it protects DNA from degradation by cytoplasmic nucleases, either by sequestering the DNA or blocking/inactivating one or more nucleases. ComM is a member of a widely distributed family of proteins with many different functions (the AAA+-superfamily); here's the first two sentences from a recent review by Snider et al.:

The AAA+ proteins (‘ATPases associated with diverse cellular activities’) form a large and diverse superfamily found in all organisms. These proteins typically assemble into hexameric ring complexes that are involved in the energy-dependent remodeling of macromolecules.

The series of experiments we've been doing asks which nucleases DprA and ComM protect DNA from.  The basic experiment is  simple genetic test: find out whether knocking out each candidate nuclease increases transformation in a dprA or comM knockout background.  If transformation is higher when the nuclease is inactive, and the effect is much stronger when DprA or ComM is also missing, then DprA or ComM normally protect DNA from the nuclease.

I began the experiments about seven years ago by testing the ExoV nuclease encoded by the recB, recC and recD genes, because this nuclease is known to be blocked by specific phage-encoded proteins (it otherwise prevents phage reproduction by degrading phage DNA).  The results were negative, and an undergraduate (Stephanie, I think) then repeated the tests with ComM, again with negative results.  This was more than 5 years ago.  A few years ago, a different undergraduate worked on extending the results to several other candidate nucleases for which knockouts had become available (Kumar et al 2007), and now the new grad student is finishing that work.

Four different exonucleases could in principle contribute to cytoplasmic DNA degradation: ExoV, ExoI, ExoVII and RecJ.  The role of ExoVII will be tricky to investigate because its H. influenzae knockout was found to be lethal, so the grad student's goal is to compare the transformation phenotypes of dprA-knockout and comM-knockout cells with and without knockouts of exoI, recJ and exoI+recJ.

His first task was to locate or construct the various strains he needed.  By the beginning of March he had made and PCR-verified all the strains.  He was originally going to also test the exonuclease mutations in a dprA/comM double mutant, but ran into problems measuring its transformation frequency without the nuclease mutations (too low).  Of course he could still check whether the transformation frequency became much higher when one or more nuclease is knocked out, but that's relatively low priority, at least until the dprA-nuclease and comM-nuclease combinations have been tested separately.

I'll find out tomorrow how far things have progressed....