Reviewing Data and Revising Hypotheses

Hi blog, it’s been a while. I recently gathered evidence (via restriction digest) that a pair of double mutants I’ve been using (sxy-1 Δhfq and murE749 Δhfq) were swapped at some point. Since then, I’ve been carefully evaluating all the data I’ve collected and diving into the literature to try to make sense of everything. My goal was to come up with a concrete hypothesis that explains what I have seen and I think I have one. But first, we will look at the data:

If you get lost at any point, I have provided a little bit of background at the bottom of this post.

(order of the bars in the graphs is KW20, sxy-1, murE749 for any colour-blind readers out there)

First, we’ll look at murE749. We really don’t know much about why this mutant is so competent, but previous work in our lab has shown that the murE mutant expresses the same amount of Sxy protein as KW20, but has the Sxy-dependent competence genes turned on when they are typically off. (http://www.zoology.ubc.ca/~redfield/PDF/Ma&Redfield%20murE.pdf) The murE mutant also shows no competence when sxy is removed. This suggests that the effect of the murE mutation takes place somewhere downstream of Sxy in the competence-inducing pathway and its effect is dependent on the presence of Sxy. Looking at my data, it seems that removing hfq makes no significant difference to how competence murE749 is (except for, perhaps, the late-log + cAMP condition). I understand this to mean that whatever the murE mutation does, its effect supersedes the negative effect of removing hfq.

Turning to sxy-1, we see something similar. There may be a tiny reduction in competence under most conditions, but for the most part it does not seem very strong or even statistically significant. Competence is strongly induced when it is not normally in KW20 (see log phase) presumably because of higher translational efficiency of basal levels of sxy mRNA.

Finally, the most interesting results are with the single Δhfq mutant. We see a 10 fold reduction in cells lacking hfq when grown in MIV. At low density, cells with and without hfq show similar levels of competence. However, as the culture gets dense we fail to see any induction of competence in Δhfq while KW0 becomes about 50 fold more competent. The story is different when exogenous cyclic AMP is added though. The data suggests that exogenous cAMP temporarily rescues competence (log phase + cAMP).

To illustrate this further, here’s time course data of KW20 and Δhfq with and without cAMP:

This data illustrates a few things:

• Without cAMP, KW20 can become 100 fold more competent than Δhfq
• KW20 becomes more competent as cells get dense, see little to no increase for Δhfq
• Competence in both strains drops as time continues
• With exogenous cAMP, both strains become almost equally competent, perhaps KW20 slightly more than Δhfq
• Competence drops off back to no cAMP levels at about OD = 0.1 (also seen in previous set of graphs)
• Knowing all this, what conclusions can be drawn? I think it’s very likely that Hfq has a role in cAMP regulation.  

If we are to assume that Hfq in necessary to fully induce the production of intracellular cAMP, the data begins to make sense. As cells become dense and the nutrients in the medium in which they are growing become depleted, competence increases presumably due to activation of the pathway that induces cAMP production. Perhaps by removing hfq, cAMP levels fail to increase to such an environmental influence. Giving cells exogenous cAMP removes the influence of intracellular regulation, and under this condition, we see competence rescued (at least for a period of time) in the hfq knockout.

This makes me think that the influence Hfq has on competence happens before the transcription of sxy. Hfq’s function could be to:

• increase the sensitivity of the phosphotransferase system
• increase translation of adenylate cyclase (increase cAMP levels)
• reduce translation of cAMP phosphodiesterase (prevent depletion of cAMP)
• increase translation of crp (increase response to cAMP)

I don’t think that CRP is regulated by Hfq as it is in Y.pestis. If Hfq regulated CRP at the transcriptional level, it would be odd that adding exogenous cAMP would somehow remove that layer of regulation.

I would be surprised if translation of protein in the phosphotransferase system was regulated by Hfq merely because it would odd to affect sugar uptake to indirectly change cAMP levels in the cell. There may be something here I’m missing but my initial thoughts are that this should not be my focus.

So in terms of a hypothesis, I’m favouring adenylate cyclase. cAMP phosphodiesterase is another option, but I think it makes less sense. To use an analogy: if you want a room dark, it makes more sense to prevent a light switch from being turned on rather than standing there waiting to turn it off every time it comes on.

So here’s my current hypothesis:

Removing hfq causes a competence defect by reducing adenylate cyclase translation and therefore reducing intracellular cAMP levels, preventing the induction of competence.

I think this hypothesis aligns with what is seen between Δhfq/KW20. The murE mutant appears to be consistently hypercompetent whether or not cAMP is present, suggesting that it is independent of adenylate cyclase activity and it makes sense that little to no difference is seen when hfq is removed. As for sxy-1, even basal levels of sxy expression are enough to induce competence, downplaying the role of cAMP in competence regulation. Perhaps that’s why the difference between the mutant and double mutant is almost trivial.

I did want to talk about future experiments, but this post is already extremely long, so I’ll do that in a separate one.

Some Background:

About the strains

KW20 is the Rd Haemophilus influenzae strain. Δhfq is KW20 with the hfq gene removed with a spc resistance gene added. sxy-1 is the Rd strain with a point mutation that increases translation of a gene necessary for transcribing the competence regulon. This mutation increases competence by removing an inhibitory structure in the sxy mRNA and inducing higher rates of translation of the Sxy protein. murE749 has a point mutation in an enzyme required for peptidoglycan biosynthesis that increases competence by some unknown mechanism. Double mutants carry both mutations. Error bars indicate that replications have been done, typically 2 or 3 times.

About competence and cAMP

When H. influenzae is starved, the phosphotransferase system detects a lack of preferred sugar and activated adenylate cyclase (cyaA). Adenylate cyclase then catalyzes the production of intracellular cAMP. This cAMP binds to C-reactive protein (CRP), this complex then induced the transcription of CRP-dependent genes.  This set of genes involves sxy, which when induced activates the competence genes.