I've now examined the effects of knocking out the small RNA-regulating protein Hfq under a wide range of conditions, testing our hypothesis that it regulates competence by helping unfold the translation-inhibiting stem of sxy mRNA.
In my previous experiment I found that the hfq knockout (∆hfq) causes a ten-fold decrease in transformation, both during growth in rich medium and after transfer to the starvation medium MIV. This time I also tested the mutation in combination with either of two hypercompetence-causing mutations (sxy-1 and murE749), and under culture conditions. The reasoning was that if ∆hfq's transformation defect is due to a defect in sxy translation, it should be reduced or eliminated by the sxy-1 mutation, which we know destabilizes the RNA stem. Seeing a similar effect of the murE749 mutation might suggest that this mutation also acts by destabilizing an RNA pairing structure, perhaps the same sxy mRNA stem.
Here are the results.
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Starting from the bottom up: In the competence-inducing medium MIV we see the same ~10-fold defect in the wildtype background but no defect in the sxy-1 or murE749 backgrounds. This supports the above hypothesis and suggests that the murE749 mutation also acts by disrupting RNA pairing.
We think that transfer to MIV medium causes two events that together cause expression of the competence genes: (i) cAMP levels go up, and (ii) the mRNA stem no longer blocks translation of sxy mRNA into Sxy protein. Simply adding cAMP to log-phase cells induces only a low level of competence, since the mRNA stem continues to block its translation. This predicts that adding cAMP to hfq mutants will give 10-fold lower competence, but instead we see that competence is nearly normal in the wildtype background and fully normal in the sxy-1 or murE749 backgrounds. This suggests that ∆hfq's competence defect is not duo to a defect in destabilizing the sxy mRNA stem, but instead to an effect on intracellular cAMP levels.
In late-log cells (in rich medium) we think that the low-level competence normally observed is due to a spontaneous increase in cAMP levels, not to destabilization of the sxy mRNA stem. But the experiment saw a larger-than-expected defect in the wildtype background, a ~10-fold defect in the sxy-1 background, and no defect in the murE749 background. I don't know what to make of this.
The final condition was 'overnight cultures' - cultures that grew to maximum density and remained at 37°C on the roller wheel until morning. The hfq+ and ∆hfq cultures in the wildtype background gave no transformants at all, but both hypercompetent backgrounds showed much stronger competence defects than under other conditions (>100-fold). However this could be an artefact of the cessation of growth on expression of the novobiocin resistance allele.
Overall, what should we conclude? I find the cAMP results to be the most compelling; they strongly suggest that our hypothesis is wrong; Hfq does not contribute to the translatability of sxy mRNA.![]()
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In my previous experiment I found that the hfq knockout (∆hfq) causes a ten-fold decrease in transformation, both during growth in rich medium and after transfer to the starvation medium MIV. This time I also tested the mutation in combination with either of two hypercompetence-causing mutations (sxy-1 and murE749), and under culture conditions. The reasoning was that if ∆hfq's transformation defect is due to a defect in sxy translation, it should be reduced or eliminated by the sxy-1 mutation, which we know destabilizes the RNA stem. Seeing a similar effect of the murE749 mutation might suggest that this mutation also acts by destabilizing an RNA pairing structure, perhaps the same sxy mRNA stem.
Here are the results.
We think that transfer to MIV medium causes two events that together cause expression of the competence genes: (i) cAMP levels go up, and (ii) the mRNA stem no longer blocks translation of sxy mRNA into Sxy protein. Simply adding cAMP to log-phase cells induces only a low level of competence, since the mRNA stem continues to block its translation. This predicts that adding cAMP to hfq mutants will give 10-fold lower competence, but instead we see that competence is nearly normal in the wildtype background and fully normal in the sxy-1 or murE749 backgrounds. This suggests that ∆hfq's competence defect is not duo to a defect in destabilizing the sxy mRNA stem, but instead to an effect on intracellular cAMP levels.
In late-log cells (in rich medium) we think that the low-level competence normally observed is due to a spontaneous increase in cAMP levels, not to destabilization of the sxy mRNA stem. But the experiment saw a larger-than-expected defect in the wildtype background, a ~10-fold defect in the sxy-1 background, and no defect in the murE749 background. I don't know what to make of this.
The final condition was 'overnight cultures' - cultures that grew to maximum density and remained at 37°C on the roller wheel until morning. The hfq+ and ∆hfq cultures in the wildtype background gave no transformants at all, but both hypercompetent backgrounds showed much stronger competence defects than under other conditions (>100-fold). However this could be an artefact of the cessation of growth on expression of the novobiocin resistance allele.
Overall, what should we conclude? I find the cAMP results to be the most compelling; they strongly suggest that our hypothesis is wrong; Hfq does not contribute to the translatability of sxy mRNA.
