Good news Band news
Sorry, dear blog, for neglecting you for another 7 days. It's been a most productive week, which included: Some nice protein data, an unfortunate revelation about "replicating" data, an NSERC application written, and sleepless nights.
For the past few weeks, I have been having a tough time quantifying relative Sxy levels between cells in different growth conditions. I have been plagued by poor blots and poor protein stability. Here, though, repetition of experiments and long days has remedied most of the problems.
The major dissapointment and frustration came from repeating an experiment to test whether knocking out purR relieves purine nucleotide repression of transformation in Haemophilus. Background: when cells are transferred to MIV containing AMP or GMP, the cells do not transform. Two weeks ago I found that cells with mutations "sxy-1" and "sxy-2" (which result in up-regulation of the sxy gene) are less sensitive to AMP repression, but are repressed by GMP to the same extent as WT cells. Quantifying Sxy protein showed that there is lots of Sxy in sxy-1 and sxy-2 cells, so it is not limiting to DNA uptake/transformation. This suggested that another regulator, quite possibly PurR, repressed transformation in the presence of purine nucleotides.
Last week, I tested whether cells lacking purR are sensitive to GMP repression (we hypothesized that they would not be repressed). I only tested the double mutants (purR-, sxy-1/purR-, and sxy-2/purR-) for sensitivity to nucleotides, and compared the results to my earlier transformation data from WT, sxy-1, and sxy-2. At the same time, I changed the concentration of nucleotides from 1mM to 0.5mM because cell growth was inhibited by the presence of nucleotides and I hopped to aleviate this, while preserving the regulatory effect, by decreasing the nucleotide concentration. The purR- results were identical to WT, whereas the sxy-1 and sxy-2 strains were 100x more competent in GMP if they lacked purR. Because of the consistency between the WT and purR- strain, I mistakingly concluded that the two independent experiments were directly comperable.
Late last week I repeated the experiments. The results for sxy-1/purR- and sxy-2/purR- strains were identical to the previous experiment, however WT, purR-, sxy-1, and sxy-2 cells all demostrated higher transformation frequencies, effectively eliminating the purR effect I thought I had detected earlier.
The bioinformatic evidence that a competence gene, rec2, is PurR repressed is very strong. Thus, we cannot yet abandon a role for PurR in regulating competence. In the next few days I will again test whether PurR represses competence, but this time I plan to use guanosine and hypoxanthine in the hopes that they will be less toxic to cells than are GMP, and especially, AMP, and that these nucleosides will have a similar tranformation-repressing effect on cells. First, though, I must confirm that two former lab members working on nucleotide supplementation had similar resuts with nucleosides: one has reported that guanosine has a strong repressing effect.