LRP IS A DIRECT REPRESSOR OF THE DAD OPERON IN ESCHERICHIA-COLI

Expression of the degradative D-amino acid dehydrogenase (dad) operon is known to be increased when Escherichia coli is grown in the presenc e of D- or L-alanine. Alanine is thought to act as an inducer to block the action of a postulated repressor. This operon is also believed to be regulated by catabolite repression. We have used in vivo and in vi tro experiments that show that the dad repressor is the leucine-respon sive regulatory protein (Lrp), dad expression in a dad-lacZ operon fus ion strain was increased four- to sevenfold when cells were grown in m inimal medium containing alanine or leucine. A strain lacking Lrp had high-level constitutive dad expression. Gel retardation and footprinti ng studies revealed that Lrp binds in vitro to multiple sites over a l arge area in the dad promoter region. This binding was reduced by alan ine or leucine. In vitro transcription assays, using a plasmid templat e and primer extension analysis, identified three major dad transcript s (Tr1, Tr2, and Tr3). The formation of these transcripts was differen tially regulated by cyclic AMP-cyclic AMP receptor protein complex, an d each was strongly repressed by Lrp. Alanine or leucine completely (f or Tr1 and Tr2) or partially (for Tr3) reversed Lrp inhibition. Site-d irected mutagenesis of an Lrp binding site strongly reduced Lrp bindin g and prevented Lrp repression of dad transcription in vivo and in vit ro. Taken together, these results strongly suggest that Lrp and alanin e or leucine act directly to repress and induce, respectively, transcr iption of the dad operon.