Field of Science

How to test our model of competence regulation

I've been working to clarify a written explanation of how I think competence is regulated.  Now I want to add ideas about how we should test this model.  Below I've pasted the explanation, and I'm going to embellish it with information about the evidence supporting these statements, and the need for additional evidence, in italics.

Effects in rich medium:  When wildtype cells grow to high density in sBHI, cAMP levels become high in most or all cells because the phosphotransferase system senses that preferred sugars are scarce and activates adenylate cyclase. Do we know this from the phenotypes of PTS mutants and from the induction of cAMP-dependent transcription seen in microarrays?  Or have these tests only been done in MIV-treated cultures?  We do know that adding cAMP to log-phase cultures raises competence to dense-culture levels but doesn't increase the level of competence they develop when dense, and that the rise in cAMP is due to the PTS.

The elevated cAMP activates CRP, causing high transcription of sxy, but most of the sxy transcripts are not translated because their mRNA has folded into an inhibitory secondary structure.  In a small fraction of the cells in high-density cultures, this structure either doesn't form or doesn't prevent translation of sxy mRNA, perhaps because random fluctuations in the supply of purine nucleotides have slowed transcription.  The postulated connection between purine nucleotides, rate of transcription, and sxy translation is purely hypothetical at present.  This is the connection I want to find out about.

The combination of high Sxy and high cAMP then causes this subset of cells to transcribe competence genes (in the CRP-S regulon) and become competent.  However, most cells in these cultures don't express enough Sxy protein to turn on competence genes.

When cells grow in rich medium (even at high density) the PurR repressor is active and the purine-biosynthesis genes are off, presumably because of high cytoplasmic concentrations of guanine or hypoxanthine, obtained directly from the medium or by conversion of other purine precursors.  We know from microarrays  that all genes subject to repression by PurR remain off in dense cultures; if H. influenzae's PurR protein uses the same cofactors as E. coli's PurR (the two proteins have similar sequences), this must be because levels of guanine or hypoxanthine are high.

Competence levels are different in dense cultures of hypercompetent-sxy mutants and of purR mutants.  In hypercompetent-sxy mutants we postulate that the mutations that destabilize the sxy mRNA secondary structure make sxy translation insensitive to the supply of nucleotides (again, this is what I want to investigate).  This causes all cells with high cAMP to express enough Sxy protein that they become competent; in dense cultures this is most of the cells.  Even in low-density cultures, many cells with these mutations express enough Sxy to become competent. 

Speculation based on behaviour of suspect purR mutant:  The opposite effect was seen in our present purR mutant (suspect because PCR shows a intact purR gene).  In such a mutant we expect that the constitutive activation of the purine biosynthetic genes will keep levels of purine nucleotides higher than in purR+ cells.  This may reduce the fraction of cells that translate enough Sxy to become competent at high density, even though high cAMP levels are causing sxy transcription, and that's the phenotype of the suspect mutant.

In the purR- hypercompetent-sxy double mutants these effects might cancel each other out, because the sxy transcripts will still be efficiently translated regardless of the levels of purine nucleotides, but the exact effect will depend on the relative strengths of the effects of the two mutations.

The other factor I'm ignoring here is the effect of PurR on expression of rec2.  The rec2 gene has what looks like a strong binding site for PurR, but we don't know if it's real.  This is a simple problem and we ought to quickly get it solved.  I discussed strategies here and here, several months ago, but they're on hold until we have a validated purR mutant (should be soon - the RA returns tomorrow).

Effects in MIV:  When wildtype cells in low-density ('log-phase) cultures are abruptly transferred to the starvation medium MIV, the cells are suddenly cut off from the nucleotide precursors they've been getting from the culture medium.  This causes a rapid fall in the supply of purine nucleotides AMP and GMP, which are continually being consumed by transcription.  cAMP levels also rise sharply on transfer to MIV, inducing sxy transcription, and the shortage of purine nucleotides allows efficient sxy translation so that most cells become competent.  Purine nucleotides cannot be immediately synthesized from scratch ('de novo') because the genes for purine nucleotide biosynthesis have until now been repressed by PurR.  The lack of purine precursors inactivates PurR, so the de novo biosynthesis genes are turned on, at least partially replenishing the supply of AMP and GMP.

What should happen if AMP or GMP is added to the MIV?  (I think these nucleotides are readily interconverted, so only one is needed.)  Even though nucleotides must be converted to nucleosides (have their phosphates removed) for transport across the cell membrane, I hypothesize that they have a more direct (faster?) effect on nucleotide pools than do simpler base precursors such as inosine, guanine, or hypoxanthine, which require extensive biochemical processing to be converted to nucleotides.  (I hypothesize this because the simple precursors have much less effect on competence than the nucleotides.   Is there a way to test this hypothesis?)  If so, supplementing MIV with AMP or GMP prevents the development of competence in most cells because it keeps their cytoplasmic concentrations above the threshold, preventing translation of sxy mRNA.

What about mutants?  Hypercompetent-sxy mutants in MIV are expected to experience the same fall of nucleotides and rise of cAMP, and their insensitivity to nucleotide supply won't matter when the supply is already depleted. This explains why they reach the same level of competence in MIV as wildtype cells. 

What about MIV competence in a purR knockout?  The purR- cells growing in rich medium will have already made the enzymes for synthesizing purine nucleotides de novo, and our original thinking was that this pathway would maintain high enough AMP and GMP concentrations to prevent translation of sxy mRNA when the external supply of  precursors was removed.   Thus we expected these cells to respond to MIV like wildtype cells do to high density in sBHI, with only a small fraction of cells becoming competent.  But this prediction wasn't met; the purR cells became just as competent in MIV as wildtype cells (this wasn't the suspect mutant, but one that had been carefully validated).  An alternative hypothesis is that de novo synthesis of purine nucleotides in the purR mutant isn't enough to compensate for the sudden removal of the exogenous supply of precursors (i.e. the salvage pathways make a bigger contribution than the de novo pathway).  It's also possible that the de novo pathway is subject to feedback regulation that limits its contribution when salvage is active, even though the genes are fully expressed.

OK, I'd better stop and make sure I'm not relying on incompatible hypotheses:  On the one hand, I've hypothesized that the salvage pathway that takes up simple purines from the medium and converts them into AMP and GMP is less efficient than the pathway that uses AMP or GMP from the medium to produce AMP and GMP in the cytoplasm.  On the other hand, I've hypothesized that de novo synthesis of AMP and GMP (from scratch) produces substantially less cytoplasmic AMP and GMP than the salvage pathway from simple purines.  These aren't incompatible, but they might be strengthened if I learned more about the biochemical control of these pathways.  For example, might

What to do?  I can't immediately start using the purR and purH knockouts to test these ideas, because these mutants aren't ready to use yet.  Is there another way to test the hypothesis that de novo synthesis is less effective than salvage?


  1. Clem WeidenbennerJune 28, 2010 at 6:22 PM

    Is there another way to test the hypothesis that de novo synthesis is less effective than salvage?


    I know radioactive tracing is not very fashionable these days, but if you were to raise some cells with P32 labeled nucleotide in order to have DNA with P32 label (use as a salvage source) and feed C14 labeled purine for a de novo synthesis source you may be able to tease out an answer.

    Salvage would seem the route I'd want to go if it were me.

  2. Hmmm... That's a nice old-fashioned experiment. But I think those are both 'salvage'. Maybe what I'd want to do is compare both kinds of salvage (from nucleotides and from simple bases) to de novo synthesis. So feed them a C14 purine and 3H glucose or amino acids?

    But I really don't want to get deep into biochemistry if I can help it. I'm hoping that genetic approaches will be easier.

  3. Clem WeidenbennerJuly 7, 2010 at 6:43 PM

    Well I was thinking of salvage vs. de novo synthesis as the difference between recycling an uptake sequence vs building a sequence from individual nucleotides... along the lines of competence being for food acquisition instead of for sex.

    Either way, it does seem there would be lots of places for label to end up. I'll puzzle on this for a bit longer. There might be a pulse/chase way to come at this.


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