In Vivo and in Vitro Footprinting of a Light-regulated Promoter in the Cyanobacterium Fremyella Diplosiphon

Certain filamentous cyanobacteria, such as Fremyella diplosiphon, modulate the components of their light-harvesting complexes, the phycobilisomes, and undergo complex morphological changes in response to the wavelength of incident light, or light quality. The operon encoding the subunits of phycoerythrin, cpeBA, is transcriptionally activated in green light and is expressed at very low levels in red light. To begin elucidating the signal transduction pathway between the detection of specific light wavelengths and changes in gene expression, we have used in vivo footprinting to show that a protein is bound to the region upstream of the cpeBA transcription start site in both red and green light: two guanosine residues at -55 and -65 bp are protected from dimethyl sulfate modification in vivo. Using DNA mobility shift gel electrophoresis, we have shown that partially purified extracts of F. diplosiphon from both red and green light contain DNA-binding activity specific for the cpeBA promoter region. Using in vitro footprinting with dimethyl sulfate and DNase I, we have defined a binding site for this putative transcription factor, designated PepB (phycoerythrin promoter-binding protein), that extends from -67 to -45 bp on the upper strand and from -62 to -45 bp on the bottom strand, relative to the transcription start site. The binding site includes two hexameric direct repeats separated by 4 bp, TTGTTAN4TTGTTA. We conclude from these results that PepB is bound to the region upstream of the cpeBA promoter in F. diplosiphon in both red and green light. Therefore, additional factors or protein modifications must be required to allow light-regulated transcription of this operon.