Why do ~1% of cells become competent in sBHI?

(Some rethinking and editing done June 15.)

So now we know that purines aren't depleted in sBHI, even when the culture gets dense.  At least, they're not depleted enough to inactivate PurR.  But we also know that about 1% of the cells in dense cultures become competent, and that transcription of all of the CRP-S-regulated genes is increased.  (Not necessarily increased in all cells, and maybe fully increased in 1% of the cells.)

If we ignore our less-than-compelling data about added nucleotides and effects of knocking out purR, the simplest interpretation would be that this competence is caused by rising levels of cAMP, due to depletion of preferred sugars (fructose?) from the medium.  Maybe the only-1% activation would be because stochastic fluctuations in the metabolic signals cause only 1% of cells to activate sxy transcription.  Or maybe sxy mRNA in only 1% of the cells doesn't form its translation-preventing secondary structure.

If the only-1% activation is caused by stochastic fluctuations in cAMP levels (just below and just above some threshold), then adding lots of cAMP should overcome this and make all cells competent.  But adding 1 mM cAMP in early log just causes the 1% competence to happen sooner (~ 45 minutes after cAMP addition), and this competence doesn't increase when the culture with cAMP reaches the cell density that normally induces 1% competence.    (This analysis should be repeated - it was done ages ago and not that well.)

After showing that H. influenzae's only PTS sugar uptake system is fructose-specific, and that the PTS system regulates cAMP levels, a former grad student tested the effect of 0.5% fructose on development of competence in sBHI.  Surprisingly, she found no effect; she also found only a probably-insignificant 10-fold effect in MIV. She also constructed a lacZ-based cAMP reporter gene, and used it to detect changes in intracellular cAMP during growth and competence induction.  She found that cAMP levels went up several-fold in late log, and went up similarly when early-log cells were transferred to MIV.  This supports the above hypothesis that cAMP levels are not what limits late-log competence to 1% of cells (i.e. cAMP levels are plenty high, and something else is preventing most cells from becoming competent).

This suggest that the lack of full competence induction in late-log sBHI cultures might instead be due to borderline effects (of nucleotide pools?) on translatability of sxy mRNA, at a time when sxy transcription has been fully induced by high cAMP..  That would be consistent with the phenotype of hypercompetent-sxy mutant cells, whose sxy mRNA is always translatable - they show ~1% competence in early log and full competence in late log or after addition of cAMP. (We would then attribute the only-1% competence in early log to borderline effects (of nucleotide pool depletion?)on sxy transcription (threshold concentrations of cAMP?).

This post was motivated by my finding that purR- cells are much less competent in late log than purR+ cells (not yet replicated), and that this effect is much weaker in hypercompetent-sxy mutants.  The microarray analysis (the previous post) shows that the wildtype competence induction isn't due to an increase in the activity of the PurR-repressed genes in late log, so any difference between wildtype and purR mutant should be because these genes are on in the mutant but off in wildtype cells.  And as the main thing these genes do is produce purine nucleotides, the simplest interpretation is that having a better supply of purine nucleotides reduces production of Sxy protein in late log, and thus that the supply of purine nucleotides affects sxy expression.  The difference in the hypercompetent-sxy mutants would then suggest that the secondary structure of sxy mRNA is responsible for sxy's sensitivity to purine nucleotide availability.

(One issue I need to keep in mind is the difference between a modest effect on all cells and a dramatic effect on a few cells.  Recent single-cell observations from other labs are showing much more dramatic cell-to-cell differences than most microbiologists have been assuming.)

So what am I hypothesizing?  When wildtype cells grow to high density in sBHI, cAMP levels become high in most or all cells.  This cAMP causes high transcription of sxy, but most of the transcripts are not translated because their mRNA has folded into an inhibitory secondary structure.  In a small fraction of the cells, the supply of purine nucleotides simultaneously falls low enough to prevent this structure from forming during transcription, thus allowing their sxy transcripts to be efficiently translated; the combination of Sxy and cAMP then causes these cells to express competence genes and become competent.  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 Sxy protein and become competent when the culture becomes dense (only some in early log).  In the purR mutant, the levels of purine nucleotides rarely fall below the threshold, so very few cells become competent in late log even though high cAMP levels are causing sxy transcription.  And in the purR- hypercompetent-sxy double mutants, the cAMP-induced sxy transcripts are efficiently translated regardless of levels of purine nucleotides.

Continuing the hypothesis -- effects in MIV:  When wildtype log-phase ('early-log') cells are abruptly transferred to MIV, cAMP levels rise sharply at the same time as the cells are suddenly cut off from the nucleotide precursors they've been getting from the culture medium.  The high cAMP induces sxy transcription and the lack of purine precursors induces the purine regulon and causes a sudden fall in the supply of purine nucleotides (because the enzymes to synthesize them from scratch ('de novo') have yet to be made).  This drop in purine nucleotides allows efficient sxy translation, so most of the cells become competent.  Hypercompetent-sxy mutants are expected to experience the same fall of nucleotides and rise of cAMP, and they reach the same level of competence.  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 prediction was that withdrawal of the external supply of precursors wouldn't increase translation of sxy mRNA (i.e. these cells would respond to MIV like wildtype cells do to high density in sBHI).  But this prediction wasn't met; the purR cells responded to MIV like wildtype cells do.  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 (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. 

Experiments to do?  OK, I think I've argued myself right back to where I was a couple of weeks ago, but on a slightly more solid footing.  What does the above hypothesis predict that we can test?  (1.)  First I need to replicate the late log competence results, testing the purR+ and purR- cells in both wildtype and hypercompetent cells.  (2.)  The late-log competence effect of the purR knockout should go away in a purR purH double mutant.  The purR+ purH single mutant should also become competent normally in both sBHI and MIV.  So, if the late-log effects are reproducible, I think we can show that they are due to changes in purine nucleotide pools that are sensed by the sxy mRNA secondary structure.

But can we show that this effect is also responsible for Sxy production in MIV?  The effects of adding purine nucleotides, nucleosides and precursors are not straightforward and I'm going to assume that this is because of complications in the various salvage pathways.  So I want to find a less unnatural way to manipulate nucleotide levels in the cell.  But constitutively expressing the PurR-repressed genes didn't do it.  What else could we do?  Might another purine-supply gene be limiting at the time of transfer to MIV, one that isn't repressed by PurR?  Could we prevent cells from using purine salvage in sBHI?  They might then grow slowly and not respond to transfer to MIV.