Hierarchical and co-operative binding of OmpR to a fusion construct containing the ompC and ompF upstream regulatory sequences of Escherichia coli.
BACKGROUND
OmpR is a transcription factor that regulates the expression of the porin genes ompF and ompC in Escherichia coli. The phosphorylation state of OmpR, directed by the osmosensor EnvZ, determines its ability to bind to the upstream regulatory regions of these genes, a total of 14 phospho-OmpR binding sites. While it has been possible to study the stoichiometry and hierarchy of the OmpR-DNA interaction in the upstream regions of ompF and ompC, their disunited location on the bacterial chromosome has made it difficult to compare the individual binding affinities of respective sites.


RESULTS
Using 1,10-phenanthroline-Cu+ footprinting on a fused construct containing both the ompF and ompC upstream regulatory sequences, and gel shift experiments on oligomers corresponding to individual sites, we have established a comparative hierarchy for OmpR binding, as F1, C1 > F2, F3 > C2 > C3. In addition, the binding patterns reveal an apparent co-operative relationship between OmpR molecules bound at several upstream motifs. Densitometric analyses of the footprinted regions provide support for these observations. Mutational analysis of this construct reveals that the alteration of a conserved cytidine in the F1 motif (-86) causes a loss of OmpR affinity and disrupts hierarchical OmpR-binding in the entire ompF region.


CONCLUSIONS
The present results provide a unique view of the OmpR interaction with the two respective promoters, ompF and ompC, and an insight into the question of how the expression of ompF and ompC are reciprocally regulated by medium osmolarity.
Bergstrom
L C
LC
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA. inouye@rwja.umdnj.edu
Qin
L
L
Harlocker
S L
SL
Egger
L A
LA
Inouye
M
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D003720:Densitometry; D004586:Electrophoresis / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000737:chemistry / Q000494:pharmacology; D018272:Porins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C113724:OmpC protein; C109237:OmpF protein; D010618:Phenanthrolines; D018272:Porins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; C038556:bis(1,10-phenanthroline)copper(1+) ion