Matt Gethers/CRI, Thailand/Project Overview
The OhrR/Ohr Regulatory System
When stressed by organic hydroperoxides, Pseudomonas aerugenosa responds by allowing OhrR (Organic Hydroperoxide Reductase Repressor) to be reduced. The reduction induces a conformational change that causes the repressor to fall off the operon and allows for transcription of Ohr. Ohr, in turn, reduces the organic hydroperoxide.
Mayuree is examining the regulatory network surrounding Ohr/OhrR. In addition to OhrR, she believes GpxR may have a role in regulation of Ohr. It seems that overproduction of GpxR downregulates Ohr (I think this is an hypothesis at this point). Overproduction of GpxR also causes pseudomonas to become black. Mayuree thinks this phenotype is due to an effect of GpxR on HmgA,HmgB, or HmgC (enzymes in the homogentisate pathway). Downregulating the activity of these enzymes would cause the buildup of homogentisate, the reduced (or oxidized, I don't remember) form of which is black.
Before further delving into the pathway, Mayuree would like to purify HmgR (Homogentisate Repressor). I'm placing the purification strategy here.
Generate HmgR Mutants
In addition to purifying HmgR for in vitro experiments, I'll also generate some HmgR mutants for characterization. Mayuree explained some cool strategies for generating knock-outs and said that I'll be using a multiple cloning strategy because the AT-rich pseudomonas genome makes PCRs painful.