Completing the toxin/antitoxin project

The honours undergraduate has finished her work on the toxin/antitoxin genes, and has submitted and successfully defended an extraordinarily good thesis.  The thesis is written as a polished scientific paper, which is ready to submit except for some gaps in the data.  My job now is to fill these gaps.

She began by confirming the mutant phenotypes. We've already shown (Sinha et al. 2012) that the toxin knockout prevents DNA uptake as well as transformation. Under competence-inducing conditions the antitoxin knockout prevents transformation, and the toxin knockout and double knockout both have normal transformation.  This is consistent with our hypothesis that the Toxin protein does something that prevents competence development, and the Antitoxin protein prevents this.

Her next step was a phylogenetic analysis of the toxin and antitoxin genes.  This work is complete. She identified three groups with the gene pair, and used synteny analysis to show that the pair entered these groups by independent horizontal transfer events.

Because there is no outgroup she was unable to root the tree, so the order of transfer events is not established.

Actinobacillus and Haemophilus species are all members of the Pasteurellaceae, and A. pleuropneumoniae has a competence system very like that of H. influenzae.  Her next project was to knock out the A. pleuropneumoniae toxin and antitoxin homologs, so see if they affect competence in the same way the H. influenzae homologs do.  She used PCR to amplify and clone the genes with flanking DNA, and then used inverse-PCR to create linearized versions of this plasmid lacking either or both of the genes.  (I'd better diagram this out because I'm going to have to do some similar work.)

She then ligated SpcR cassettes into the linear PCR products, creating circular plasmids with the SpcR cassette replacing each (or both) genes, and transformed these back into the A. pleuropneumoniae chromosome, selecting for the cassette.

Once she'd made her knockout mutants she checked their competence phenotypes, and found that all were normal.  She would have concluded that the A. pleuropneumoniae toxin does not block competence development, but in the interim she had discovered that the GenBank record for the toxin gene was incorrectly annotated, missing the last 5 amino acids because these overlap with the start of the antitoxin gene.  This meant that the antitoxin knockout she had created was also missing the end of the toxin gene, and therefore might have actually been a double knockout (if the terminal toxin amino acids are important for its function).

So one of my jobs is to redo the antitoxin mutagenesis, using a new primer that preserves all of the toxin gene, and check the growth and transformation phenotypes of this new correct mutant.  If I find that it has a transformation defect I'll also check its DNA uptake.

 She then examined the growth properties of the H. influenzae and A. pleuropneumoniae mutants.  The A. pleuropneumoniae mutants all grew normally, but the H. influenzae antitoxin knockout grew significantly slower in log phase than the other H. influenzae strains.  This suggests that the H. influenzae toxin, when unopposed by antitoxin, interferes with cell growth even under conditions where the cells are not competent.  The toxin knockout also grew a bit slower.

She then examined the RNAseq data for the wildtype and knockout strains.  Each culture was sampled and sequenced at four time points: T0 is log phase in rich medium, just before transfer to the competence inducing medium MIV, and T1, T2 and T3 are 10, 30 and 100 minutes after transfer to MIV.  

Here we discovered another problem – two of the ‘antitoxin-knockout’ cultures used for these analyses were incorrect - they were instead knockouts of a different competence gene, comN.  So we have three replicates of the toxin and double knockout strains, but only one correct replicate of the antitoxin knockout strain.  The two missing replicates have been recultured and their RNA preps remade, and they are now being sequenced.  My contribution here will be to help the summer student finish the analysis of this data.  (By 'help' I mean pester him with questions and requests for explanations...)

Analyzing the data she had showed that log phase expression of the toxin mRNA increases dramatically when the antitoxin is missing - this is consistent with characterization of related toxin/antitoxin pairs, where the antitoxin protein represses transcription of the toxin/antitoxin operon.  She found lots of other interesting (confusing/perplexing) effects.  Since most of these are based on the single antitoxin-kockout replicate, we'll examine them more thoroughly once we have all three replicates.