Organization and expression of the Escherichia coli K-12 dad operon encoding the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase.

摘要

A fragment of the Escherichia coli K-12 chromosome complementing the D-amino acid dehydrogenase and catabolic alanine racemase deficiency of a dad operon deletion mutant was cloned in a mini-Mu plasmid. The dadA and dadX genes were localized to a 3.5-kb part of the plasmid insert. The nucleotide sequence of this fragment revealed two open reading frames encoding 432- and 356-amino-acid-long proteins. We show here that they correspond to the dadA and dadX genes. The dadA gene can encode only the smaller of the two subunits of D-amino acid dehydrogenase. A computer search revealed the presence of a flavin adenine dinucleotide-binding motif in the N-terminal domain of the deduced DadA protein sequence. This is in agreement with biochemical data showing that the D-amino acid dehydrogenase contains flavin adenine dinucleotide in its active center. The predicted dadX gene product appeared to be 85% identical to a dadB-encoded catabolic alanine racemase of Salmonella typhimurium. The organization of the dadA and dadX genes confirmed our previous conclusion based on the genetic data (J. Wild, J. Hennig, M. Lobocka, W. Walczak, and T. Kłopotowski, Mol. Gen. Genet. 198:315-322, 1985) that these genes form an operon. The main transcription start points of the dad operon were determined by primer extension. They are preceded by a putative sigma 70 promoter sequence and two cyclic AMP-cyclic AMP receptor protein (cAMP-CRP) binding sites, one of higher and one of lower affinity to CRP. We propose that the high-affinity site, centered 59.5 bp upstream of the main transcription start point, plays a role in cAMP-CRP-mediated activation of dad operon expression in the absence of glucose.