So this morning I sat down with the post-docs to pick their brains about the above questions. The conversation kept moving on to the larger issue of how nucleotides, nucleosides and bases affect expression of competence genes, but for now I want to only focus on the PurR repressor and the rec2 gene. My hope is that we can do the necessary experiments to answer the above questions (and write a paper on the results), and that this will give us a clearer framework in which to address the other questions.
But even this goal looks more complicated than I expected (foolish me - I should know better by now). Here 's what we think we know:
The rec2 promoter has a sequence strongly matching the PurR repressor binding site characterized for E. coli. Such sites are also found in all of H. influenzae's purine biosynthesis genes, so we're confident that H. influenzae PurR binds to the same sequence as its homolog. This location of this sequence in the promoter predicts that PurR binding will prevent transcription, as expected for its known repressor function. This thus predicts that transcription of rec2 is repressed by PurR.
Either of the purines guanine and hypoxanthine can act efficiently as corepressors of E. coli PurR (they bind to different sites in the protein). The H. influenzae PurR protein is very similar (55% identical amino acids, shorter by only one amino acid) so we can confidently expect it to have the same corepressors. This then predicts that rec2 is repressed when guanine or hypoxanthine are present at significant concentrations.
The Rec2 protein is needed for the second stage of DNA uptake - passing the DNA across the inner membrane, from the periplasm into the cytoplasm, but not for transport of DNA across the outer membrane. So repressing rec2 is expected to reduce the amount of DNA recombining with the chromosome, and thus the transformation frequency, but not the amount taken up into the periplasm (what we measure when we give cells radioactive DNA).
H. influenzae cells are normally grown in a rich medium that should have lots of purines, so we expect the genes regulated by PurR to be off, and our microarray analysis confirms this. The PurR repressor is expected to be inactive in cells made competent by starvation with MIV medium because it contains no purines, and this is also confirmed by our microarrays; the purine biosynthesis genes turn on within 10 minutes in MIV.
The rec2 promoter also has a strong binding site for the activator protein CRP; this site is of the 'CRP-S' type typical of competence gene promoters, whose induction requires the co-activator protein Sxy. CRP and Sxy are also active in MIV, and this induces transcription of all genes with CRP-S promoters. Thus rec2 appears to have two regulatory sites, one for the CRP/Sxy activator and one for the PurR repressor, and both these effects are expected to increase its transcription in MIV. I want to find out the extent to which PurR derepression contributes to this induction.
One way to test this is to look at induction in the absence of CRP or Sxy. We did that in our original microarray analysis; the effects on rec2 transcription are a bit smaller than those on the other CRP-S genes, consistent with PurR having some effect, but the difference may not be significant.
A reciprocal approach is to look at the effect of knocking out the purR gene. This is potentially much more informative, because the purR- cells are still fully transformable so we can look for effects of purine supplementation on DNA uptake and transformation with and without PurR.
Experiments comparing a purR knockout to purR+ cells showed little or no effect on MIV-induced transformation frequencies. Adding AMP or GMP (at 0.5 mM, tenfold less than previously used) reduced transformation about 1000-fold regardless of PurR. Introducing hypercompetence mutations (sxy-1 or sxy-2) to the purR+ cells reduced the effect of AMP by about 100-fold and of GMP by 10-20-fold. Removing PurR from these mutants sometimes further decreased this effect by about 3-fold, but this may not be significant.
Other experiments, done by a former PhD student and a former undergraduate student, examined the effects of supplementing MIV with PurR corepressors on competence and on rec2 and comA transcription. The competence assays showed no effect on transformation with 5 mM guanine, a slight decrease with 5 mM adenine, and a slight but apparently significant increase with a mixture of uracil and hypoxanthine (5 mM each). The transcription assays found no significant effect of 1 mM guanine or hypoxanthine on expression of lacZ fusions to either rec2 or comA (a control with no PurR site). Purine nucleosides (inosine, adenosine, guanosine) and nucleotides (AMP, GMP), all at 5 mM, did reduce expression of the fusions by 5-10-fold.
None of this strongly supports the hypothesis that PurR has a significant effect on rec2 transcription. However the results with sxy+ cells are likely to be complicated by the effects of nucleotides on the sxy expression needed for induction of the CRP-S genes, including rec2. The results with the hypercompetent sxy-1 and sxy-2 mutants should be independent of this effect, as we know that these cells make large amounts of Sxy protein even when nucleotides have been added. But we still see a strong effect of AMP and GMP that is only slightly (if at all) relieved by knocking out purR.
What additional experiments should be done? I'll have to think more.
Other tasks for the soon-to-be-departing Co-op tech
11 hours ago in RRResearch