Interaction of a ∆hfq mutation with the rpoD mutation

Here's the last part of the summary of what our senior co-op technician has been doing.  The last set of experiments tested the interaction between a new ∆hfq mutation we've been studying, which reduces competence) and the rpoD mutation (which increases competence).

The Hfq protein binds small regulatory RNAs, helping them to form base-pairs with the mRNAs that they regulate.  In other bacteria we know that this base pairing can either reduce the mRNA expression (usually mediated by RNase E degradation) or increase it (by reducing the effect of otherwise-inhibitory mRNA secondary structure).  Our ∆hfq mutation reduces competence in MIV-induced cells by about 10-fold, suggesting that it increases the translatability of sxy mRNA.

The technician tested whether this effect is still seen in cells with the rpoD mutation.  She first had to construct the double-mutant strain.  This was relatively easy because the ∆hfq mutation is 'marked' with a SpcR cassette, and the honours student who's studying this mutation had already made chromosomal DNA.  So she used his chromosomal DNA to transform strain RR753 (rpoD mutant) to spectinomycin resistance.

She then did a competence time course, following development of competence in rich medium in four strains.  In the graph below we see that the ∆hfq mutant (green line) develops competence later than the wildtype cells (KW20, blue line) and to a lower final level.  This mutation also reduces the competence of the rpoD mutant (compare red and purple lines), although not as severely.

So we conclude that, whatever Hfq is doing to promote competence, it's still at least partly needed by the rpoD mutant.

The honours student has been analyzing the interactions of the ∆hfq mutant with other factors and other hypercompetence mutations.  I'll do a separate post pulling this together, unless he does it on his blog first.

Effects of cAMP and AMP on competence development by the rpoD mutant

This is a continuation of yesterday's post on the phenotype of our hypercompetent rpoD mutant strain RR753.  Yesterday we wrote about its behaviour under 'normal  growth conditions, and now we're going to consider two new factors, cyclic AMP (cAMP), which induces competence under what are otherwise non-inducing conditions, and AMP, which inhibits competence development under what are normally inducing conditions.

First the effect of adding cAMP: We tested this by adding 1 mM cAMP to cells growing exponentially at an OD600 of 0.1, and measuring transformation 60 min. later.  At this growth stage, normal cells do not transform detectably, but addition of cAMP turns on sxy transcription.  Some of the resulting sxy mRNA is translated and the Sxy protein acts with CRP to stimulate transcription of genes encoding the DNA uptake machinery.  In the tech's experiment, cAMP addition raised the transformation frequency about 500-fold, from 1-3 x 10^-8 (just at the detection limit) to 6.5-8 x 10^-6.  The rpoD mutant is somewhat transformable even with out cAMP (01-3 x 10^-6), and cAMP addition raised this about 100-fold, to ~2 x 10^-4.

So we conclude that the rpoD mutant does not bypass the need for cAMP in competence induction. This rules out the boring hypothesis that changing Sigma 70 activity perturbs cellular metabolism, causing an elevation of baseline cAMP levels that in turn causes the mutant's increased competence. Instead it's consistent with our interesting hypothesis that the rpoD mutation likely changes one or more events after the sxy transcription is stimulated by cAMP and CRP.

Next, the effect of adding AMP:  Maximal competence is normally induced by transferring exponentially growing cells from rich medium to a 'starvation' medium called 'MIV' and incubating them for 100 min.   Previous work has shown that adding purine nucleotides or nucleosides (usually 1 mM AMP) to the MIV prevents normal competence development by reducing the translation of sxy mRNA (Sinha et al. 2013).  The next experiments tested whether AMP has the same effect in the rpoD mutant.

Both wildtype and rpoD mutant cells have high transformation frequencies after incubation in MIV. In these experiments the rpoD cells had slightly higher transformation frequencies (about 4 x 10^-3) than the wildtype cells (about 1 x 10^-3).  Adding AMP to the MIV used to induce competence reduced the transformation of wildtype cells more severely than seen in previous work, about 5000-fold (from 1.7 x 10^-3 to 3.4 x 10^-7) and at least 10,000-fold (from 3 x 10^-4 to 3 x 10^-8).  The AMP also reduced the viability of the cells by several fold. (Both replicates gave no transformants at all with added AMP, so these estimates are upper limits.)

Added AMP also reduced the transformability of the rpoD mutant.  The first replicate gave no transformants (the plated cells were too dilute) indicating that transformability as reduced at least 1000-fold, and the second replicate showed a ~6700-fold reduction.

These numbers are all lower than previous results, but the conclusion is clear that adding AMP to the MIV medium strongly inhibits the development of competence in the rpoD mutant.  We don't know how the added AMP caused the reduction in competence, but, based on other evidence from analysis of purine-biosynthesis mutants, I've hypothesized that the key factor is a decrease in the concentration of another metabolite (PPRPP), which maybe interacts with sxy mRNA.  Production of Sxy by the rpoD mutant is still sensitive to this effect, so... (I don't know what).

Phenotype of the rpoD mutant

This mutation causes H. influenzae cells to become competent prematurely, and to reach levels of competence in rich medium that are about 100 times higher than normal cells.  The mutation causes a single amino acid substitution in domain 3 of the 'housekeeping' transcription factor called 'sigma 70'.  rpoD is an essential genes, needed for transcription of most housekeeping genes.  Since the mutant strain (named RR753) shows only a very slight decrease in exponential growth rate we think the mutation causes only a very minor change in the protein's function

My earlier post this morning said I had never explained my hypothesis about how this mutation causes increased competence, but actually I did, briefly here.  Here's the key sentences from that post:

My hypothesis is that the mutation's effect on transcription of sxy mRNA increases competence by increasing sxy translation.   I've long hypothesized that slowing elongation or increasing pausing in the 100 nt segment of sxy mRNA that forms its regulatory secondary structure will promote sxy translation by increasing the ribosome's access to the sxy ribosome-binding site and start codon. 

Then I listed some low-tech phenotypic analyses that the senior of our two co-op technicians could do:

• Is RR753 sensitive to the inhibition of competence by added purines?
• What's the effect of an hfq deletion in this background?
• How does this strain respond to added cAMP?
• How does it respond to the standard competence-inducing MIV treatment?
• Does the mutation increase competence of a sxy mutant (sxy6) that has an extra-stable secondary structure?
• Does it further increase log-phase competence of the sxy hypercompetence mutants, which have weakened sxy mRNA secondary structures?

She's now done all but the last of these, and we're considering what she should do next.  So here we're going to summarize what she's found and what we think it means.

Her first experiments gave a better characterization of RR753's growth rate.  She's done both Bioscreen growth curves (high-precision analysis of exponential growth) and manual ones (lower precision but better for cultures at low and high densities).

First the Bioscreen results:  Here growth of RR753 (red line) is compared to wildtype cells (KW20) and two other hypercompetence mutants, RR563 (sxy-1) and RR749 (murE).  This clearly shows the slightly slower exponential growth of the rpoD mutant.

The Bioscreen analysis measures OD600, so it doesn't tell us about the actual numbers of viable cells. The tech has also done a number of manual growth curves, mostly as control parts of experiments examining other variables.  These agree with the Bioscreen results in showing usually a slightly slower exponential growth rate, and no obvious differences in later survival.  

Her next time courses replicated my earlier measures of transformation frequency.  It looks like the rpoD mutant differs from the other hypercompetence mutants (in sxy and murE) in having very low competence at very low cell density, perhaps as low as that of wildtype cells.  The other mutants at 100-1000-fold more competent than wildtype cells at very low cell density. The rpoD mutant may also become highly competent at lower cell densities than wildtype cells, but may not be any different than the other hypercompetent mutants - these data are hard to interpret.

What could be the significance of having very low competence at very low cell densities?  I'd been assuming that the moderate competence of the sxy-1 hypercompetent mutants at low cell density reflected a baseline level of sxy transcription and an increased efficiency of translation.  If the rpoD mutation acts as I've hypothesized, it should have the same effect.  Provided the cells are in real exponential growth, the cell density shouldn't matter.  Might the rpoD mutant have two different 'exponential' growth phases, one at very low cell density and another at moderately low cell density?

Is this an important issue?  It would be quite a bit of work to investigate carefully, so let's set it aside for now.

The next experiments analyzed the effects of adding cAMP (known to stimulate transcription of sxy) and AMP (known to reduce translation of sxy mRNA).  I'll leave these for the next post.

What should our 'senior' co-op tech do next?

We have two co-op (undergraduate) technicians at present (paid from the last of our leftover CIHR funds).  Each is with us for 8 months; one started in May and the other in September, so there's a 4 month overlap.

The senior one has done almost all of the work preparing the samples for the RNA-seq project, and lately she's also been doing competence time courses to characterize the phenotype of our hypercompetent rpoD mutant.  She's looked at growth conditions, at the effects of added cAMP (competence up) and added AMP (competence down), and the effects of knocking out the small-RNA regulator Hfq (down).  Writing this post makes me realize that we haven't summarized her results anywhere, so I'll sit down with her and pull it all together this morning (I think that will be another post).

Now we need to decide whether there are still more rpoD phenotype assays she should do, or whether she should move on to another project for her last two months.  Since I hypothesize that the rpoD mutation causes competence by slowing sxy transcription and increasing its mRNA translatability, she could assay the effects of of the rpoD mutation in combination with our various sxy mutations that affect its mRNA translatability.  But this would be very much a fishing expedition, lots of work but probably no new insights (because it's not testing any specific hypotheses).

Hmmm, looking back, I discover that I've never written a blog post clearly explaining the general hypothesis about how the rpoD mutation causes hypercompetence.  I think it's time I did that.

So long since I've posted...

But here's an update on the RNA-seq saga.

We've been struggling to get evidence that our RNA preps are of good enough quality for library preparation and sequencing.  Because the Ribo-zero kits that we use to remove the rRNA from the samples are so expensive, we wanted to be sure the final mRNA concentrations and integrity would be good enough to make the equally expensive sequencing worthwhile.

Unfortunately my attempts at using a Bioanalyzer have been very frustrating (and it's not cheap - several $ per sample), with odd smeary/spiky patterns and poor recognition of the size standards. This is partly because the concentrations are so low and the 'pico-RNA kits are very fussy, and partly because it's just a fussy procedure and easy (for me) to make mistakes.

We sought advice from the person who will be preparing the sequencing libraries for us.  She said that samples that appeared bad on Bioanalyzer results often gave excellent sequencing results, and suggested that we have her prep an initial 8 libraries.  She would then pass these on to the person who does the sequencing, who could just run her initial quality checks on the libraries and tell us whether there were suitable for sequencing.  So that's what we did, and the news was good - these initial libraries look OK.

Now it's time to do the Ribo-zero and final clean-up on the rest of the samples, so they can be handed over for eventual library prep and sequencing.  There's a two-month backlog so we likely will get the results as a Christmas present.

Our sample processing had been a bit scattershot, so now I needed to go through the sample information and make sure we have the right samples, and that the numbers add up.  They do, so I think we're all set to quickly complete the remaining Ribo-zero treatments and use RNeasy-Min-elute columns to clean up each RNA sample after Ribo-zero.  Then the remaining 64 samples can be handed over.  (The 'we' here is our excellent UBC-Co-op program technician.)

We have accidentally wasted two of our Ribo-zero treatments on duplicate samples, and I was thinking that this meant that we'd have to buy another 6-sample kit ($650).  But now I think we can get by without this, either by not treating two samples (the G6 samples in the table above) or by using slightly less of the reagent for a subset of samples.

We had been using only 1 µg of total RNA for Ribo-zero treatment, but for these samples we'll sue all the DNase-treated RNA we have (typically several µg).  In some cases the volume of sample will be too high (max 28 µl for Ribo-zero), but singe the samples are in H2O we can just evaporate them a bit to decrease the volume.