A NOVEL REGULATORY SWITCH MEDIATED BY THE FNR-LIKE PROTEIN OF LACTOBACILLUS-CASEI

FNR (regulator for fumarate and nitrate reduction) and CRP (cAMP recep tor protein) are global regulators which regulate the transcription of overlapping modulons of target genes in response to anaerobiosis and carbon source in Escherichia coil, An ORF, designated fin because it e ncodes an FNR-like protein of the FNR-CRP family, has been found in La ctobacillus casei, The product of the flp coding region (FLP) was over produced in E. coil, purified and crystallized, FLP is a homodimeric p rotein in which each subunit can form an intramolecular disulphide bon d. The isolated protein also contains nonstoichiometric amounts of Cu and Zn. Although the DNA recognition helix of FLP resembles that of FN R, the flp gene failed to complement the anaerobic respiratory deficie ncy of an fnr mutant when expressed in E, coil and it neither activate d nor interfered with transcription from FNR-or CRP-dependent promoter s in E. coil. Site-specific DNA binding by oxidized FLP (the form cont aining intrasubunit disulphide bonds) was abolished by reduction. The interconversion between disulphide and dithiol forms thus provides the basis for a novel redox-mediated transcriptional switch. Two non-iden tical FLP-binding sites, distinct from FNR-and CRP-binding sites, were identified in the melR region of E. coil by gel-retardation analysis. A further eight FLP-binding sites were selected from a random library . A synthetic oligonucleotide conforming to a putative FLP site consen sus, CA/CTGA-N-4-TCAG/TG (the most significant bases are underlined), was retarded by FLP. Functional tests showed that FLP represses the ae robic transcription of a semi-synthetic promoter in E, coil, A C5S var iant of FLP lacking the ability to form intramolecular disulphide bond s was unable to bind to FLP sites and failed to repress transcription in vivo.