Field of Science

Expression of DNA uptake genes in rich medium - a puzzle

I've been working on the toxin/antitoxin paper.  Right now I'm going through the RNA-seq data for the antitoxin knockout (again!), looking for hints of how unopposed toxin expression prevents DNA uptake.  The two graphs below show mRNA levels of the mutant compared to wildtype cells at the same stage of competence induction (upper panel, 30 min in MIV; lower panel, 100 min in MIV). The green bars are expression in the mutant (unopposed toxin) and the grey bars show the range of expression in wildtype cells. (In the upper panel the grey bars are centered on the mean expression at this time point.)

Conclusion:  Expression of competence genes is normal or near-normal at 30 min (when competence-gene transcription normally peaks), but is substantially lower than wildtype at 100 min (when DNA uptake and transformation peak).

Can this reduction explain the absolute competence defect of the mutant?  I think not.

Some other informative comparisons:  

1.  Compare the antitoxin knockout (∆toxA) to the toxin knockout (∆toxT) and the toxin/antitoxin double knockout (∆toxTA):  At 30 min, competence genes in all three knockout mutant have very similar transcription levels (more similar to each other than to KW20).  But ∆toxT and ∆toxTA have normal competence.  At 100 min some ∆toxA operons are a bit lower than in ∆toxTA (comABCDE is at about 65% and pilABCD is at about 50%).

2  Compare the antitoxin knockout to a hfq knockout:  The hfq knockout (∆hfq) is the only mutant we tested whose competence is reduced but not eliminated; it's MIV-induced transformation frequency is about 10% of the wildtype level.  At 30 min it's competence-gene expression levels are mostly higher than those of ∆toxA, which has no detectable transformation (∆toxA TF is 3-4 orders of magnitude lower than ∆hfq).  At 100 min its expression is overall a bit lower than ∆toxA.

3. Compare the antitoxin knockout to wildtype cells in 'late log': Here's where it gets weird.  We've known for a long time that competence rises when cell growth slows as cultures get dense (peaking around OD = 1-2).  Our old microarray experiments showed that expression of competence genes increases then too; in the paper we said that expression levels increased about 4-20 fold, but we didn't present any data. So I decided to compare wildtype expression levels in late log with ∆toxA expression levels at 100 min of induction.  

But I was surprised to see that, in our RNA-seq data, competence-gene expression levels in rich medium don't increase as the culture gets dense.  In the graph below, each cluster of blue bars is a DNA-uptake gene, with three replicate bars at OD=0.02 (true log phase, light blue), OD=0.6 (end of log phase, medium blue) and OD=1.0 (dark blue).  In most cased the dark blue bars are not noticeably higher than the other bars, indicating that the gene is not induced at all when cell density increases.

My first response was to try to find the original microarray data, to see how big an induction we actually saw.  It's probably buried somewhere in my computer (not with the array manuscript files), but I can't find it.  So instead I looked in my notebooks for any problems with the wildtype samples used for the RNA-seq analysis, and here I think I found the explanation.  Along with each sample we prepared for the RNA analysis, we froze one tube of cells that could be checked later for competence or other issues (e.g. contamination).   In May 2015 we had noticed the unexpectedly low expression levels of these samples, so we thawed out OD=1.0 samples and transformed them.  They were about 100-fold less competent than they should have been, which is consistent with their low gene expression.  This comparison is still useful, because even with this nearly undetectable induction the cells did become at least 10-fold more competent that the ∆toxA cells do after MIV induction.

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