Field of Science

Grant proposal's done! What experiment shall I do?

I clicked 'Submit' on my grant proposal last night; my immediate teaching responsibilities are light, and there's nothing else big on my plate, so now I get to start doing experiments again!

I think the most fun thing to do will be to join the sabbatical visitor and the co-op tech in doing mutant hunts for hypercompetent strains.  They're mutagenizing the rpoD gene and screening for new mutations that cause hypercompetence, and I can use the same methods on the murE gene.

This old post describes what we know about the relationship between murE and competence.  Well, what we used to know, because now we have new RNA-seq data that will tell us how transcription changes.  Basically, we have four independent mutants that all cause very similar extreme-hypercompetence phenotypes.  murE749 is the main one we've studied.  Some lacZ-fusion analyses indicate that it acts by causing overexpression of genes in the competence regulon (we looked at two genes) and one low-quality microarray appeared to confirm this and (maybe?) show some overexpression of sxy (the regulatory protein that controls expression of the competence regulon).

We assume that the other murE mutations act the same way.  But we have absolutely no idea how the mutations cause the phenotype.  MurE is an essential cytoplasmic enzyme in the pathway that synthesizes the cell wall.  The mutants all grow normally (though we haven't done a BioScreen run), and are not unusually sensitive to any simple test of cell-wall function.
One big part of the puzzle is how the mutations change the protein. The diagram above shows that three of the mutations change a poorly conserved amino acid (at position 435); these changes wouldn't be expected to have any serious impact on the enzyme's catalytic function.  So how do they have such a big impact on cometence?

On the other hand, the mutation in murE751 changes the strongly conserved leucine at position 361 to a very different amino acid (serine).  Leucine is hydrophobic but serine is polar, so they make very different interactions with their surroundings.  Because this leucine is highly conserved we think it must play an important role in the enzyme's catalytic function.  This would explain how the mutation can have a big effect on competence, but leaves us instead wondering why it doesn't have a big effect on cell growth.

I need to do several things:
  1. Update my reading to find out what's been learned about MurE function since we published our paper way back in 2000.
  2. Dig into the new RNA-seq data to see what it tells us about RNA changes in the murE749 mutant.  This will require finally learning some R and/or getting help from other lab members.
  3. Isolate new murE mutations that also cause hypercompetence.
Lots of fun!

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