TRANSCRIPTIONAL REGULATION OF FERRIC CITRATE TRANSPORT IN ESCHERICHIA-COLI K-12 - FECL BELONGS TO A NEW SUBFAMILY OF SIGMA(70)-TYPE FACTORS THAT RESPOND TO EXTRACYTOPLASMIC STIMULI

Transcription of the ferric citrate transport system of Escherichia coli K-12 is repressed by Fe2+-Fur and activated by ferric citrate. Ferric citrate does not have to enter the cytoplasm; it initiates a signal transduction mechanism by binding to the outer membrane receptor FecA. Presumably, a conformational change is transmitted in a TonB-dependent manner to the FecR protein. FecR activates Fecl, and Fecl activates transcription of the fecABCDE transport genes. In this communication. Fecl was isolated after cloning fed downstream of an ideal ribosome-binding site. Overexpressed Feel formed inclusion bodies which were solubilized and purified in active form using a mild detergent. Fecl, in conjunction with RNA polymerase core enzyme, directed transcription from the fecA promoter in an in vitro run-off transcription assay. Furthermore, Feel retarded the electrophoretic mobility of a specific 75 bp DNA fragment located upstream of fecA. An in vivo competition experiment between the fecA promoters of wild-type and mutant strains identified the nucleotide positions 2747, 2749, 2751 and 2753, located within the 75 bp fragment, as important for Feel-induced transcription. Mobility band shift of fecA promoter DNA caused by cell lysates required growth of cells in the presence of ferric citrate and expression of FecA, Fecl and FecR. These data support the previous assignment of Feel, based on sequence homologies, to a new subfamily of eubacterial RNA polymerase sigma(70) factors that respond to extra-cytoplasmic stimuli and regulate extracytoplasmic functions.