I described last month how we were revisiting on old hypercompetent mutant whose causative gene was unknown. I rechecked its phenotype and prepped DNA to sequence, from both the original EMS-induced mutant strain (strain RR735) and from a 'backcross' strain where the unknown mutation had been transferred to an unmutagenized genetic background by transformation (strain R753).
Here's the phenotype again. The lower graph shows that it grows slightly slower than wildtype, and the upper graph that it has a 10-200 times higher transformation frequency in the rich medium sBHI.
The postdoc just emailed me the sequencing results. Both the original and backcross strains have the same single mutation, an amino acid substitution in the rpoD gene, which encodes the sigma-70 transcription factor.
This is a surprisingly clear result. We had expected to find many EMS-induced mutations in the original strain, and probably several mutations in the segments of DNA transferred in the backcross transformation, and were planning another series of analyses to sort out which mutation causes the phenotype. But both strains have only the one rpoD mutation, suggesting that our EMS mutagenesis wasn't nearly as heavy as we had thought. As controls we had sequenced the original and backcross strains of another hypercompetence mutant (RR749, known to have a mutation in I, and both these strains also had only the single known mutation.
A hypercompetence mutation in rpoD fits very nicely into my thinking about how competence is regulated. I'll write a separate post about this.
2 hours ago in Variety of Life