Monday, June 1, 2015

Things worth mentioning about competence genes in competence mutants

I've turned my focus on trying to figure out how/why the various competence mutants we have alter regular competence. To do this, I'm comparing expression of competence genes between strains.

A quick message about normalization: 
DESeq2 (the R package I used to normalize the count data) normalizes data to allow, for instance, a comparison between gene A in condition X and gene A in condition Y. It does not normalize in a way that makes comparisons between gene A and gene B possible because it does not take into account differences in gene length (longer genes are expected to have more reads).

To make comparisons between genes possible, instead of asking "how much expression of gene A is there?" I ask "how much expression of gene A is there compared to gene A expression in KW20 M0?" I did this by dividing normalized count values by the normalized count in KW20 M0. In essence, KW20 M0 is treated as a baseline and measurements are deviations from that baseline.

Here is some data (error bars are standard error):

The first set of plots shows KW20 competence gene expression. As you can see, most competence genes are ~10-100x induced in M2 relative to M0. Compare this to the sxy or crp knockout where very little induction is seen. Interestingly, HI0365 seems to be particularly down in these knockouts.

The toxin and double toxin/antitoxin knockout strains behave similarly to KW20 (except for the toxin/antitoxin genes, of course) and peak at pretty much the same levels in M2 as KW20. Competence expression in the double knockout (taxx) seems to be a bit lower than KW20 in M3 though. Overall, nothing unexpected.

Knocking out hfq appears to cause slower induction of competence (compare M1 to data above) and definitely does not lead to full induction of the competence regulon when comparing to KW20 M2. Presumably, this failure to completely turn on the competence regulon leads to the 10x comptence defect seen in this strain.

This one is confounding. There's definitely high expression of the toxin/antitoxin pair when the antitoxin is knocked out (at all timepoints!), suggesting that the toxin is self-promoting. This behaviour is seen in other toxin/antitoxin pairs as well. This strain was shown to not be able to uptake DNA, but competence gene expression is comparable to the hfq knockout strain (which is only mildly less competent). The only competence gene that is consistently down in this strain is HI1631 which pretty much nothing is known about (except that it may have some restriction enzyme like function... at least it has a motif that suggests that). Interesting to note though, the 3-enzyme restriction modification system hsdR, hsdM, and hsdS is hugely upregulated in the toxin/antoxin double knockout. This is odd because you'd think removing both the toxin and antitoxin should have no effect whatsoever on the transcriptome (or that you would see the same thing if you just removed the toxin...?)

Note, these two have BHI samples only. Strikingly, rpoD and sxy-1 behave very similarly. Sxy-1 is hypercompetent because of the weakened 5' stem mRNA structure which leads to more Sxy protein and expression of competence genes. Perhaps the rpoD point mutation works in a similar way to increase sxy translatability.

Finally, we have the infamous murE point mutation. Competence gene expression increases a bit from B1 to B2. Apparently competence genes are expressed more in murE in sBHI than KW20 in MIV. At the very least, it's possible to say that murE is hypercompetent because something is causing aggressive expression of competence genes (and not some change of membrane permeability, for example).


Rosie Redfield said...

I'm just starting to go through all this. I'm interested in the few cases where gene expression goes down below the KW20-M0 baseline, but the graphs appear to be cut off at about a 2-fold decrease.

Rosie Redfield said...

About hsdR. It might be relevant to DNA uptake, since it includes a DNA-translocation motor that can pull thousands of bp of DNA (see

This paper also says: " genetic elements, including phage and conjugative transposons and plasmids, have developed an extensive range of anti-restriction measures
to overcome the RM barrier". Toxin-antitoxin systems are also thought to be protective measures evolved by transposons and plasmids, so maybe the toxin's real job is blocking translocation by the hsdR system, and a side effect is that it blocks DNA uptake!

That might explain why the antitoxin knockout completely blocks DNA uptake and transformation but only modestly reduces expression of competence genes.

Is hsdR expression only up in the double (toxin-antitoxin) knockout, not in the toxin knockout?

Lauri said...

Wow - sxy1 and rpoD are strikingly similar, even without the only 1 replicate for rpoD. Do they have similar hyper competence in the time courses? The work we did with the beta-gal fusions suggested that rpoD upregulated sxy transcription. we didn't see evidence of increased translation but I don't think we could rule it out either. I have to look again to see how sxy transcript levels at in rpoD753 compare to sxy transcript levels in KW20 and also to sxy transcript levels in sxy-1.