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

Transcription of the ferric citrate transport system of Escherichia co il K-12 is repressed by Fe2+-Fur and activated by ferric citrate. Ferr ic 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-depende nt 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-b inding site. Overexpressed Feel formed inclusion bodies which were sol ubilized and purified in active form using a mild detergent. Fecl, in conjunction with RNA polymerase core enzyme, directed transcription fr om the fecA promoter in an in vitro run-off transcription assay. Furth ermore, Feel retarded the electrophoretic mobility of a specific 75 bp DNA fragment located upstream of fecA. An in vivo competition experim ent between the fecA promoters of wild-type and mutant strains identif ied the nucleotide positions 2747, 2749, 2751 and 2753, located within the 75 bp fragment, as important for Feel-induced transcription. Mobi lity band shift of fecA promoter DNA caused by cell lysates required g rowth of cells in the presence of ferric citrate and expression of Fec A, Fecl and FecR. These data support the previous assignment of Feel, based on sequence homologies, to a new subfamily of eubacterial RNA po lymerase sigma(70) factors that respond to extra-cytoplasmic stimuli a nd regulate extracytoplasmic functions.