Here are my initially thoughts on the murE mutant. I talked to the post doc for a while and have a little handle on it now.  We came up with two reasonablish and non mutual exclusive models for why a murE mutant could be hyper competent

Model 1) Transformation frequency increases the most after growing as cells that are growing as quickly as possible are transfered to the starvation media MIV. Perhaps this is because the cells are perceiving a signal that tells them “hey, you are not replicating anymore” and this signal is part of cell wall biosynthesis. In a murE mutant growth is not affected, but maybe the cells are getting that signal and for some reason it is only being heard by the competence regulon. What are other physiologies that could be affected by a signal that says that growth has abruptly stopped?

Model 2) MurE is the step in cell wall biosynthesis directly before the recycling pathway. If the mutation makes murE super awesome or really bad at its job this the recycling pathway pools will be affected accordingly.

Model 3) There is a transcription factor in the recycling pathway called AmpR(called that because mutations in it causes ampicillin resistance). In E. coli and Haemophilus AmpR is caled GcvA. I’ll refer to it as GcvA from now on. A brief look into the literature revealed it’s something I need to investigate fully.

EDIT: I had wrongly copied down that the transcription factor was called GcrA, not GcvA. These are two different, but related proteins, corrected. Thanks Rosie!

This pseudomonas paper will need to be read too.

Some short thoughts and assignments

– Does DNA bind to cell walls? Old paper says so but I need to find out the modern opinion. Read the Baccilus literature to see what their deal is

– There are some bacteria without cell walls, but with functional and conserved biosynthesis genes including murE. Chlamydia Papers to read.

Some first step methods to start investigating:

– If a murE mutant is complemented with WT murE(or the other way around), what happens? Does the effect go away? This is important because it will tell us if the mutation is dominant – which could mean several things like being awesome at de novo synthesis or making some strange x factor by product, or if it’s recessive and masked by a functional copy

– Clone murE into e. coli, or make an allele specific mutation of E. coli’s murE. Does it affect E.colis transformation? This question will be key, and will shape the direction of investigation.

Thats it for now. Tomorrow I’ll start gathering lab notebooks

Spare thought about rec2: Phage recombination is drastically lowered in a KO mutant. Is there a build up of phage in the periplasm? There’s a method for destroying the outer membrane and washing off everything in the periplasm. This would only be plausible if rec2 cells infected with the phage at the right temperature without needing to recombine also had lower plaques forming units(or maybe not? some reason its specific to recombination but not just infection?)

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2 Comments (+add yours?)

  1. Rosie Redfield
    May 21, 2011 @ 12:16:56

    1. The ampR homolog is called gcvA, not gdrA (error caused by my bad printing?).

    2. I think Caixia spent a lot of time testing murE749 complementation by murE+, though I can’t remember the conclusion. For some reason this experiment wasn’t straightforward.

    3. Phage HP1 plaques just fine on rec2 mutants; it just doesn’t make recombinants.

    Reply

    • dnogas
      May 22, 2011 @ 12:23:19

      Thanks Rosie. I found some slides of Caixias expirments in JW’s binders. The results were roughly

      MurE+/pmurE+= low competence
      MurE+/pmurE749= high competence
      MurE749/pmurE+= low competence
      MurE749/pmurE749= high competence

      The results confused me so I asked Josh, they used an over expression plasmid which explains the different results depending on which copy was on the plasmid and which was on the chromosome

      Reply

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