Imaging OmpR localization in Escherichia coli.
We have used a fusion of GFP to the response regulator OmpR to image the spatial distribution of OmpR in live cells of Escherichia coli. We observed foci of increased OmpR-GFP fluorescence that appear to be due to interactions with the histidine kinase EnvZ. We also observed colocalization of OmpR-GFP with clusters of plasmids carrying OmpR binding sites, which enabled us to develop a simple method for imaging the binding of OmpR to DNA in live cells. We used the peak fluorescence intensity within cells to quantify the extent of OmpR-GFP localization either due to interactions with EnvZ or due to binding DNA. With these assays we compared the effects of osmolarity and procaine, both of which are believed to modulate EnvZ activity. Our results suggest that, at least under our growth conditions, procaine activates EnvZ-OmpR signalling whereas osmolarity has, at best, a weak effect on the EnvZ-OmpR system.
Department of Physics, University of Pennsylvania, Philadelphia, 19104, USA.
Eric
E
Batchelor
Mark
M
Goulian
D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D009097:Multienzyme Complexes / Q000032:analysis* / Q000378:metabolism; D009994:Osmolar Concentration; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011343:Procaine / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction; D015534:Trans-Activators / Q000032:analysis* / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D049452:Green Fluorescent Proteins; D011343:Procaine; C057843:envZ protein, E coli