Field of Science

Does expression of the toxA operon depend on ToxT as well as ToxA?

Short answer:  Yes, but not in the way we expected.

First, here's a diagram showing the toxTA operon and the mutants we're examining:

The grey bars show the extents of the deletions.  The ∆toxT and ∆toxTA mutants have a SpcR/strR cassette inserted at the deletion point, but the ∆toxA mutant has only a short 'scar' sequence at the deletion point.

A few months ago I wrote a post about evidence that ToxA prevents transcription of the toxTA operon from an unexpected internal promoter.  Here's a better version of the graph I showed there (note that transcription is going from right to left):

It looks like there are two promoters.  The CRP-S promoter is competence-induced so inactive in the M0 samples and maximally active in the M2 samples.  The unexpected internal promoter (labeled 'toxTA' in red) is not active in wildtype but highly active in all the ∆toxA samples.  Really I should consistently refer to this as the 'putative' internal promoter, but I won't bother for this post.

The line heights in the above graphs are hard to interpret because of the log scale, so here's a linear scale view.

These expression patterns appear to show normal repression of the 'toxTA" internal promoter by ToxA, and release from repression in the ∆toxA mutant.  The internal location of the ToxA-repressed promoter is unexpected, but its repression by ToxA fits the pattern observed for the well-studied homologs of this type of toxin/antitoxin system.

In these homologs, transcription of the operon is actually repressed by a toxin-antitoxin complex, not by antitoxin alone.  So we would expect to see that repression of the internal toxTA promoter is also released by knockouts of toxT or of both genes.  But that's not what we see.

We can't check the effect of a toxT knockout on transcription from the internal promoter, because the internal promoter is deleted in our toxT mutants.  But we can check the effect of deleting both toxT and ToxA, because the deletion in our double mutant starts well downstream of the internal promoter.

Surprisingly, transcription from the internal promoter is not increased in the double mutant.  The first figure shows coverage for wildtype, ∆toxA and ∆∆toxTA at time point M0.  We see that the CRP-S promoter is inactive in all three strains, and the internal promoter is very active only in ∆toxA (purple).  It's actually less active in the double knockout (green) than in KW20 (blue).

This figure shows transcription of the same strains at time point M2, when the CRP-S promoter is most active.  Now we see transcription from the CRP-S promoter in all strains, although definitely weaker in ∆∆toxTA.  We again see strong transcription from the internal promoter in ∆toxA (purple), but very little in ∆∆toxTA.

Could some sort of artefact be responsible for the apparent lack of transcription from the internal promoter in the double knockout?  One likely candidate is read-mapping artefacts caused by the presence of toxTA deletions and insertions in the mutant reads but not in the wildtype genome sequence they are being aligned to.

So we could check for these effects, one of the former honours students took a a set of mutant-specific reference sequences for the toxTA region, and separately aligned each set of mutant reads to its corresponding mutant reference sequence, and explained to me how to examine the reads and coverages using the Integrated Genome Viewer (IGV).

For all the M0 (log phase growth) and M2 samples (max CRP-S induction) I noted the number of reads covering position 400 (at the toxT start codon, ~ 35 bp downstream from the CRP-S promoter, and covering position 500 (about 75 bp downstream from the internal promoter).  (I didn't bother analyzing M1 and M3 samples.)  I normalized each coverage value by the number of reads in the sample, and calculated the mean coverage over the three replicate samples for each time point.

Here's a linear-scale graph:

And here's the corresponding log-scale graph:

And here's the conclusions:

This rules out read-alignment artefacts as an explanation for the apparently low transcription from the internal promoter in the toxTA mutant, and from the CRP-S promoter in the toxT mutant.

I'm now going to go back and generate the data for M1 and M3.  Then I'll update this post.  For now, ignore the notes below.

OK, the data for the M1 and M3 time points don't change the conclusions at all.

So, questions we still don't know the answers to:

Why is competence-induced transcription from the CRP-S promoter down modestly in all the toxTA mutants?  (Compare actual mean M2 values: wildtype: 830, ∆toxA: 513, ∆toxT: 213; ∆∆toxTA: 173.)
In wildtype cells in log phase, neither antitoxin (ToxA) or or toxin (ToxT) are likely to be present.  In wildtype cells at M2, both proteins are likely to be accumulating.  We don't know whether there will be more of one than the other - usually the toxin is more stable, and the antitoxin is unstable unless it is bound to toxin.  We don't know what the HI0660 toxin's 'toxic' activity is, and we don't know of any other expected activity for the HI0659 antitoxin except binding toxin and repressing toxTA transcription.
The lower transcript levels in the mutants suggests that both ToxA and ToxT contribute positively to transcription from the CRP-S promotor, or to the stability of the resulting transcripts.  
Could toxin in wildtype cells, and unopposed toxin in ∆toxA, be somehow stabilizing transcripts?  Doesn't make much sense to me.
Why is log-phase transcription from the putative internal promoter way up in ∆toxA but way down in ∆∆toxTA?  (Compare actual M0 values: wildtype: 134; ∆toxA: 1713; ∆∆toxTA: 170.)
Taken at face value, this would seem to mean that ToxT actively stimulates toxTA transcription, or stabilizes another transcription factor.
I hope someone else has some ideas!

Learning to use the NCBI Gene Expression Omnibus

As part of our workup for the toxin/antitoxin manuscript, I want to find expression data for the homologs of the Haemophilus influenzae toxin and antitoxin genes.  The former post-doc recommends that I use NCBI's Gene Expression Omnibus ('GEO') for this.

I'll need to learn how to search the GEO for specific accession data and data from specific taxa.

I'll also need to find out the specific identifiers for the genes I'm interested in, in the species I'm interested in.  I think I can use BLAST searches (queried with the H. influenzae sequences) to find the species and links to the DNA sequences of the homologs, and then I can look at the gene records to find the strain and gene identifiers.

Then I need to check if anyone has reported doing gene-expression studies on this strain or species (ideally the same strain, but I think/hope the gene identifiers will be consistent across strains).  These reports should contain the GEO accession numbers for the data.

Then I can ask GEO for the data for this study.  But I don't know what format the data will be in, nor how hard it will be to find the information about the genes I'm interested in.

The best situation would be if GEO has done its own analysis on the data and made this available as a curated 'GEO Dataset' or a 'GEO Profile'.  I could then search the GEO Profile to see their analysis of the particular genes I want to learn about.

Here's a phylogenetic tree showing the relationships between the concatenated toxin/antitoxin sequences in the species they're found in.  (I should also have a tree showing the relationships of the species themselves, but I haven't drawn that yet.)

So I'll start by searching BLAST with the amino acid sequence of HI0660, the H. influenzae toxin, and limiting the search to Streptococcus species.  Because I want to find the genes, not the genomes, I'll do a BLASTP search against Streptococcus protein sequences in the database.  OK, this gets me species and accession numbers for the (mostly 'hypothetical') proteins, but not usually strain identifiers or gene names.  

Will I be able to use a gene accession number to search GEO?  Apparently yes!

Now let's try searching GEO for the gene accession number of a gene I'm sure it has in at least one Profile:  The H. influenzae CRP gene:

But GEO does find it if I search for it by its gene name: 'HI0957'.  

I think the red bars are expression levels in the six samples of the study.  Yes, and clicking on them gets me the actual data from the study.  Now I can see the Y-axis and discover that, although the red bars look dramatically different, the differences are actually quite small (range 6300-6300).

So this control search tells me that I need to know gene names (or whatever identifiers are being used) for the genes whose expression I want to learn about.  Hmmm...  I wonder if GEO provides any help for this issue.

I also tried starting with a TBLASTN search, which will get me the position in the genome where the homolog is encoded.  I can then look at the genome sequence, find the position, and see if the gene has a name.  The Streptococcus pneumoniae homolog of HI0660 (the H. influenzae toxin) is SP_1143, but searching GEO for this finds nothing