A novel regulatory switch mediated by the FNR-like protein of Lactobacillus casei.

FNR (regulator for fumarate and nitrate reduction) and CRP (cAMP receptor protein) are global regulators which regulate the transcription of overlapping modulons of target genes in response to anaerobiosis and carbon source in Escherichia coli. An ORF, designated flp because it encodes an FNR-like protein of the FNR-CRP family, has been found in Lactobacillus casei. The product of the flp coding region (FLP) was overproduced in E. coli, purified and crystallized. FLP is a homodimeric protein in which each subunit can form an intramolecular disulphide bond. The isolated protein also contains non-stoichiometric amounts of Cu and Zn. Although the DNA recognition helix of FLP resembles that of FNR, the flp gene failed to complement the anaerobic respiratory deficiency of an fnr mutant when expressed in E. coli and it neither activated nor interfered with transcription from FNR- or CRP-dependent promoters in E. coli. Site-specific DNA binding by oxidized FLP (the form containing 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-identical FLP-binding sites, distinct from FNR- and CRP-binding sites, were identified in the meIR region of E. coli 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 consensus, CA/CTGA-N4-TCAG/TG (the most significant bases are underlined), was retarded by FLP. Functional tests showed that FLP represses the aerobic transcription of a semi-synthetic promoter in E. coli. A C5S variant of FLP lacking the ability to form intramolecular disulphide bonds was unable to bind to FLP sites and failed to repress transcription in vivo.