Field of Science

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 (∆toxinantitoxin) 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.

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