I ran into a colleague at the coffee pot today, and asked his expert advice about ways to investigate whether RNA polymerase pauses or stalls when transcribing the sxy gene.
He said that pausing is quite easy to show using a commercially-available E. coli in vitro system (as I had hoped it would be), but that showing that a H. influenzae sequence causes pausing in this system would only be significant if we first showed that the H. influenzae sxy gene was regulated in vivo in E. coli as it is in H. influenzae. The alternative is to use a H. influenzae in vitro system, but we would have to purify the components ourselves, which is well beyond both our abilities and our real interests.
We might be able to show the regulation in E. coli, if the sxy gene wasn't so toxic to E. coli (see Making lemonade). Well, we could perhaps work with a truncated gene, subject to the same transcriptional controls but not producing Sxy protein... Hey! In fact, one of the very first sxy plasmids I made would be just the thing! The plasmid is named pDBJ90 (the name I think is the initials and year of the boyfriend of the student who made it); it contains only the 5' half of the sxy coding region but all of the upstream sequences that affect its transcription. And it's in a high-copy vector. And as far as I know the insert is stable, though we've learned that we should always check the sequence. I don't know whether our Sxy antibody will recognize the truncated protein it should produce.
What would the experiment be? Grow the E. coli cells with the plasmid in minimal medium with added purines and pyrimidines. Add cAMP to induce the sxy promoter, and transfer half the culture to the same medium with no purines or pyrimidines. Measure the amounts of sxy mRNA and protein at several time points for each half of the culture. If the ratio of protein to RNA is higher when the purines and pyrimidines are absent, then we can do the in vitro experiment.
The colleague also suggested an entirely new way to look at the relationship between sxy transcription and sxy translation. He reminded me that mutations in the genes for RNA polymerase can make the polymerase resistant to the antibiotic rifampicin, and told me that these mutations can affect the efficiency of transcription in ways that might change sxy expression. So here's the experiment plan:
Starting with wildtype (rifS) H. influenzae cells, select cells that are resistant to rifampicin. I think these are quite easy to select; we could try several different rifampicin concentrations. Pool all the colonies that grow up on rifampicin plates, and select hypercompetent ones by transforming the pooled cells with novR DNA while they are in log-phase growth in sBHI. If we get any hypercompetent cells, check whether the hypercompetence is caused by the rifR mutation, by using their DNA to transform fresh cells to rifR. If yes, we've shown that mutations in RNA polymerase can cause hypercompetence.
We do already have one RNA polymerase mutation that affects competence induction. It's an insertion that doesn't change the sequence of RNA polymerase but probably reduces the amount of polymerase in the cell, and it decreases competence rather than increasing it. I can make up a just-so-story that fits this mutant into our current model, but it's just handwaving.
How can you trust non-gardeners?
12 hours ago in The Phytophactor