Gels/manuscript/PurR day

After several days of working to wrap up the CRP-S manuscript, I'm looking forward to running a few more protein gels today. The first thing I want to do is directly compare how Sxy levels change between growth conditions for any given strain. Until now I have been comparing relative Sxy levels between strains at a specific growth condition, not tracking changes within a strain. This alternate approach means I can make direct measurements of Sxy levels for one strain on one gel, instead of using normalization to make comparisons between different gels. This is a last step in getting very solid numbers for Sxy levels in H. influenzae. At the same time, I will go back and test cell samples from 2 years ago (which I mentioned having probed with a different antibody in an earlier blog post).

Last week I found that in WT cells, production of Sxy is strongly inhibited by AMP and weakly inhibited by GMP. On the other hand, hypercompetence mutants produced a lot of Sxy no matter which nucleotide was present. The surprising finding was that transformation is equally strongly repressed in all strains upon addition of GMP, while AMP has some repressing effect. This has renewed our interest in PurR and its possible role in regulating competence. A former graduate student in the lab knocked out PurR, but didn't find elevated competence. I need to go over his experiments, because if he tested transformation in non-inducing conditions, PurR repression may have been lacking, but so were the necessary inducing signals. I suspect that he and the boss thought of this and that his note book holds some interesting results, so I must go have a look. It is tempting to think that rec2 (which has s very good PurR site in its promoter) is repressed when purine pools are high or when we add AMP or GMP to culture medium. Under this model, all of the other competence machinery is induced in the presence of exogenous nucleotides, but repression of rec2 keeps transformation frequency down. Real time PCR measurements of comA and rec2 in MIV+/- GMP will tell us immediately whether this is the case.

Thus, H. influenzae potentially uses two mechanisms to regulate competence according to nucleotide pools: 1. AMP represses Sxy production in WT cells, and 2. PurR represses rec2.