I spent part of yesterday reading over posts from earlier this spring about how purine nucleotides and the PurR repressor might contribute to competence regulation. I then started editing an old post to make it clearer, but now I'm elevating those edits to 'New Post' status here. I'm continuing to edit them to make my thinking as clear as possible (more edits July 19).
I think it's important to consider the interdependent effects of at least two factors that affect nucleotide pools (1) the extracellular and intracellular concentrations of purine precursors, especially guanine and hypoxanthine, which control the repressing activity of the PurR repressor, and (2) the intracellular concentrations of purine nucleotides (ATP and GTP) that are available for transcription. Another issue is the cell-to-cell variation in the amounts of cAMP, sxy mRNA and Sxy protein due to random fluctuations in transcription of the sxy gene, translation of sxy mRNA and the activities of various catalytic enzymes.
Here are the paragraphs I've been working on:
So what am I hypothesizing? 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. This 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, whether by chance or because random fluctuations in the supply of purine nucleotides have slowed transcription. The combination of high Sxy and high cAMP then causes these cells to express competence genes and become competent. However, most cells in these cultures don't express enough Sxy protein to turn on competence genes.
In these cells the PurR repressor is active and the purine-biosynthesis genes are off because of high cytoplasmic concentrations of guanine and hypoxanthine, obtained directly from the medium or by conversion of other purine precursors.
Competence levels are different in dense cultures of hypercompetent-sxy mutants and of purR mutants. In hypercompetent-sxy mutants the supply of nucleotides doesn't matter because the mutations have destabilized the sxy mRNA secondary structure, so all the cells with high cAMP express enough Sxy protein to become competent when the culture becomes dense. (Even in low-density cultures, many cells with these mutations express enough Sxy to become competent.) The opposite effect is seen in the purR mutant, where the constitutive activation of the purine biosynthetic genes keeps levels of purine nucleotides relatively high, so few cells translate enough Sxy to become competent at high density, even though high cAMP levels are causing sxy transcription. These effects might cancel out in the purR- hypercompetent-sxy double mutants, because the sxy transcripts are still efficiently translated regardless of levels of purine nucleotides, but the exact effect will depend on the relative strengths of the effects of the two mutations.
Continuing the hypothesis -- 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. These nucleotides cannot be immediately synthesized from scratch ('de novo') because the genes have until now been repressed by PurR. 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. The lack of purine precursors also inactivates PurR, so the de novo biosynthesis genes are turned on, at least partially replenishing the supply of AMP and GMP.
What would happen if AMP or GMP was 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 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. Thus supplementing MIV with AMP or GMP maintains their cytoplasmic concentrations above the threshold, preventing translation of sxy mRNA and thus the development of competence in most cells.
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 the purR knockout? The purR- cells have already made the enzymes for synthesizing purine nucleotides de novo, and our original thinking was that this 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. 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.
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?
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