Field of Science

Successful time course of competence development

Yesterday I did the failed time course again.  The goal was still to replicate the earlier quick-and-dirty experiment that had suggested that knocking out the purine repressor prevented competence development in late-log cultures.  This time the cells grew better, and the results are clear.

I had four strains:  KW20 is wildtype, RR3005 has the purR knockout, RR699 has the sxy-1 hypercompetence mutation that we think should make competence induction less dependent on depletion of nucleotides, and RR1345 has both the purR and sxy-1 mutations.  The graph below shows that the wildtype and sxy-1 strains grew at similar rates, and the two strains with the purR mutation grew a bit slower, perhaps because they were wasting resources on synthesizing nucleotides.  (All four cultures stopped growing at about half the density they should reach with the best medium.)
The next graph shows the transformation frequencies of the four cultures at the same times.  The wildtype cells (blue diamonds) showed the usual pattern, with very low transformation frequencies when cells were growing exponentially (first time point), and 1000-fold higher transformation when the culture became dense.  The purR mutant (blue circles) also started out very low, but its transformation frequency remained low throughout growth, about 200-fold lower than its wildtype parent.

The sxy-1 mutant (green squares) also behaved normally.  Its log-phase transformation frequency was >1000-fold higher than the wildtype strain, and it became about 50-fold more competent when the culture got dense.  (Its final competence and that of the wildtype strain were both a bit lower than I normally see - I suspect this is due to the lower growth in the poorer medium.)  The transformation frequencies of the purR sxy-1 double mutant (green triangles) were lower, but only about 3-9-fold.

So this experiment confirms both observations from the quick-and-dirty one.  First, the purR mutation does prevent the competence development that normally occurs when cultures get dense.  Since this mutation's major effect is to keep the purine biosynthesis pathway maximally active even in exponential growth, this suggests that running short of purines (purine nucleotides?) is the signal that normally induces competence when cultures get dense.  The microarray analysis showed that wildtype cells at high density still have enough of the purine precursors hypoxanthine and inosine to keep PurR in repressing mode.

Second, the sxy-1 mutation makes cells much less sensitive to the competence-inhibiting effect of the purR mutation.  The mutation causes hypercompetence by weakening the secondary structure of sxy mRNA, so this new result supports our hypothesis that the function of the secondary structure is to sense depletion of nucleotide (purine) pools.  When the stem is weakened by mutation, it behaves as if nucleotides are depleted even when they're not, causing many cells make enough Sxy protein to become competent even in log phase.  Some of the other sxy hypercompetent mutations have stronger effects (sxy-2 and maybe sxy-3), so I need to check if they are even less sensitive to the purR mutation.

I should also make a purR double mutant with the other kind of hypercompetence mutation.  We know that some point mutations in murE, a gene responsible for one of the steps in cell wall synthesis, cause even stronger hypercompetence than mutations in sxy.  But we have no idea how these mutations do this - we've ruled out most of the obvious explanations.  (I would have thought I'd posted previously about this set of mutants, but I can't find anything by searching for 'peptidoglycan' or 'murE' or 'cell wall', so maybe I haven't.  I'd better do a separate post about them.)

We already know that the mRNA secondary structure limits translation of the sxy mRNA into Sxy protein.  In my mind, the simplest way for the secondary structure to sense depletion of nucleotide pools is the following:  (1) Depleted pools slows the rate of mRNA elongation; (2) Because the two parts of the main stem are separated by ~100 nucleotides (I forget the actual number), slower elongation delays the formation of the secondary structure.  (3) Because the ribosome binding site and start codon are in the region between these parts, this delay makes them more accessible and increases the initiation of translation. (4) once translation has started, the secondary structure can't form.

I would really like to complete the story by showing that the rate of transcription determines the efficiency of translation.


4 comments:

  1. Clem WeidenbennerApril 5, 2010 at 12:49 PM

    Rosie:
    How much variation might one expect between a couple of different wild type strains? Are there any other purR knockout strains? Is the purR knockout from the KW20 strain?

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  2. Hi Clem,

    Independently isolated H. influenzae strains typically are 2-3% divergent at sequences that can be aligned, and have 10-20% of their genomes composed of sequences so different that they can't be aligned. The latter feature is typical for other bacteria too.

    As far as I know, ours is the only H. influenzae purR mutant, and yes, it's in the KW20 background.

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  3. Clem WeidenbennerApril 7, 2010 at 4:35 PM

    Rosie:
    Thanks for the update. I have to admit not working with bacteria for quite a few years (ok, decades). So how hard would it be to transfer the purR knockout to another wild type strain? Background effects can be very significant in higher plants so it makes me wonder how H. influenzae or other competent bacteria would handle it.

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  4. That's a good idea. The purR gene was knocked out by inserting a kanamycin-resistance cassette, so we can easily transfer the mutation into one of the clinical strains we've been using.

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