Imaging OmpR binding to native chromosomal loci in Escherichia coli.
Previously, an unexplained subcellular localization was reported for a functional fluorescent protein fusion to the response regulator OmpR in Escherichia coli. The pronounced regions of increased fluorescence, or foci, are dependent on OmpR phosphorylation and do not occupy fixed, easily identifiable positions, such as the poles or mid-cell. Here we show that the foci are due to OmpR-YFP (yellow fluorescent protein) fusion binding to specific sites in the chromosome. To identify positions of foci and quantify their fluorescence intensity, we used a simple system to tag virtually any chromosomal location with arrays of lacO or tetO. The brightest foci colocalize with the OmpR-regulated gene ompF, which is strongly expressed under our growth conditions. When we increased OmpR-YFP phosphorylation by stimulating the EnvZ/OmpR system with procaine, we observed a small increase in OmpR-YFP fluorescence at ompF and a significant increase at the OmpR-regulated gene ompC. This supports a model of hierarchical binding of OmpR to the ompF and ompC promoters. Our results explain the inhomogeneous distribution of OmpR-YFP fluorescence in cells and further demonstrate that for a transcription factor expressed at wild-type levels, binding to native sites in the chromosome can be imaged and quantified by fluorescence microscopy.
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018, USA.
Elizabeth A
EA
Libby
Seda
S
Ekici
Mark
M
Goulian
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D016254:Mutagenesis, Insertional; D011485:Protein Binding; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D015534:Trans-Activators; C112177:osmolarity response regulator proteins