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.
That's interesting stuff. I would have sworn this scenario was right for sRNA/Hfq interaction with sxy mRNA, especially considering this is what occurs in V. cholerae. However, that sRNA is induced by chitin.
ReplyDeleteIn the Log + cAMP experiment there still appears to be a 5-fold defect between hfq+ and hfq mutant in the WT background. Also, do you know how the amount of cAMP added in that experiment compares to the amount of cAMP induced by moving cells to MIV media? This might be completely wrong, but could it be possible that much more cAMP is added to the system as compared to when cells are in MIV? This would result in more sxy mRNA correct? Could this mask the translation defect slightly in the hfq mutant simply because there is more sxy transcript available to be translated. If I understand correctly the secondary structure inhibition is not 100%.
However, I agree that the in the Late-log experiment, the results in the sxy-1 background are confusing. But, because Hfq is required for multiple things, could not having it in late-log be deleterious to the cell in ways that affect transformation independent of whether Sxy protein is made?
Good luck. I work on sRNAs so I just like an sRNA regulation explanation on principle. :)
-Estevan Santana
Munson Lab Grad Student
Hi Estevan,
ReplyDeleteMeasuring intracellular cAMP is difficult, especially in very small cells like those of H. influenzae, and of course cAMP is affecting other cellular processes.
This was a fairly crude survey, and I think the next step is to do careful time course comparing hfq+ and hfq- growth and competence development in the wildtype and sxy-1 backgrounds.