What do the toxin and antitoxin gene products do?

Now that I'm finally close to finishing my benchwork task for the Honours student's manuscript, I've gone back to thinking about the results and implications of our RNA-seq analysis.

When the Honours student wrote the manuscript (actually her Honours thesis, but in excellent manuscript format), we had only incomplete RNA-seq results - specifically we had only one replicate of the critical antitoxin mutant.  The other two replicates were in the pipeline at the time, and the full dataset was analyzed subsequently by the other Honours student when he stayed on for the summer.

I'm going to just summarize the results now, and come back to them later.

Basic points:

1. The antitoxin knockout mutant has normal RNA levels for all the genes that regulate the competence regulon (crp and sxy, which encode the transcription enhancers CRP and Sxy, and cya, which encodes the adenylate synthase that synthesizes the essential cyclic AMP cofactor for CRP.
2. Consistent with this, the expression levels of the competence regulon genes are not very different than in wildtype cells.  A few genes are down by 40-50%, but most are near-normal, with error bars that overlap the range of wildtype expression (see his complicated green figure below - compare the heights of the bright-green bars with the spans of the grey shaded areas, which represent the normal expression levels at the bright-green timepoint).  
3. The double knockout (∆toxin ∆antitoxin) transforms normally, so the competence defect of the antitoxin mutant is due to competence-blocking activity of the toxin.
4. The transformation defect of the antitoxin knockout is much more extreme than these expression levels would predict.  We see few or no transformants (transformation frequencies less than 10^-8), whereas wildtype cells give transformation frequencies higher than 10^-3.  
5. The antitoxin mutant also has an extreme DNA uptake defect, so the transformation defect is not caused by defective recombination machinery. 
6. The summer student also did an RNA-seq analysis of the hfq knockout mutant he had worked on for his Honours project. This mutant has a more severe reduction in expression of all the competence-induced genes, but a much less severe defect in transformation (only about ten-fold lower than wildtype cells).  Thus the antitoxin mutant's competence defect is unlikely to be due to modestly lower expression of one or more key competence genes.
7. In the antitoxin mutant the toxin mRNA is overexpressed during exponential growth.  This is consistent with the roles of related antitoxin's in other systems, where it acts as a repressor of transcription of the toxin-antitoxin operon.
8. The antitoxin knockout cells have a normal doubling time in exponential growth, and survive competence induction and stationary phase just as well, so the toxin protein must not be toxic for growth or survival.

Where does all this leave us?  One possibility is that the toxin directly blocks DNA uptake, by some mechanism we are completely ignorant of.  But related toxins are known to act by cutting mRNAs on the ribosome, so it's possible that the RNA-seq results are misleading in that they detect all RNAs, including ones that have been cut.

Luckily the summer student wrote an R script to compare coverage patterns between wildtype and mutant cells, and generated lovely graphics showing the effect of the antitoxin knockout on coverage of segments containing competence-induced genes.  Just as an example, here's his comparison of expression of the pilABCD operon in wildtype (purple) and hypercompetent (green) cells.

He's generated data for all the competence-induced genes in the antitoxin knockout, so I'll check these to see if there are any alterations in transcript profiles that might indicate the action of a mRNA-cleaving toxin.

Toxin/antitoxin knockout updates, and bonus DNA uptake results

My last post was all about failure, so it's high time I updated things with some successes.

Constructing an Actinobacillus pleuropneumoniae antitoxin gene knockout:  At the last report, I had what I thought were four independent knockout mutants, but my attempts to PCR- amplify the genomic segment containing the knockout were not working.

I eventually switched to using a different thermostable polymerase (NEB's standard OneTaq) rather then the fancier Q5 polymerase I had been using.  Eureka - the PCRs all worked perfectly, giving strong bands of approximately the expected sizes.

...then I let everything sit around for a month while I dealt with other things...

Now I'm finally following up.  The first step is to digest these PCR products with a few other enzymes that should cut in either the genomic segments or the inserted SpecR cassette.  I've made rough predictions of the expected fragment sizes, which are all different for the ∆A mutant, wildtype cells, and the two mutants made by the Honours student (∆T and ∆TA).

The next step will be to do more PCR amplifications.  My original amplifications used the F and R primers that amplify a 2.6 kb segment containing the toxin and antitoxin genes (~300 bp each).  Now I'll use the F primer with the S-R reverse primer for the SpecR canssette, and the R primer with the S-F forward primer for the cassette.

If these both give the expected fragments then I'll (probably) send the PCR amplicons for each mutant to be sequenced.

If the sequencing confirms that the knocked-out genes are gone but the remaining gene is intact, then I'll give a sigh of relief.

Determining the competence phenotype of the Actinobacillus pleuropneumoniae antitoxin gene knockout:  My first test of the transformability of my first two ∆antitoxin mutants showed transformation defects, but in later tests they transformed within the range of the wildtype control.  But there was a lot of experiment-to-experiment variation in transformation levels (see graph below), so I'd like to do it one more time, to get clean publishable data.

Bonus DNA uptake results:  Just before Christmas the grad student finished his DNA preps of H. influenzae chromosomal DNA fragments that had been recovered after being taken up into the periplasm of competent H. influenzae.  He sent these to the former post-doc for sequencing, and the post-doc has now sent us some lovely preliminary results.  

The grad student had used DNA preps that had been sheared to two different size ranges.  We expected the genome coverage of the long fragments (mean length ~6 kb) to be fairly uniform, since almost all of them should contain at least one instance of the preferred uptake sequence motif.  These 'USS' motifs are distributed fairly evenly around the chromosome, with a mean spacing of about 1 kb.  We do see this, but with enough anomalies to keep things interesting.  And we expected coverage by the short fragments (mean length ~0.25 kb) to be much more strongly dependent on chromosomal position, since many such fragments would not include a USS.  And we do see this, again with interesting anomalies.