Characterization of BkdR-DNA binding in the expression of the bkd operon of Pseudomonas putida

Abstract

The bkd operon of Pseudomonas putida consists of the structural genes encoding the components of the inducible branched-chain ketoacid dehydrogenase. BkdR, a positive regulator of the bkd operon and a homolog of Lrp of Escherichia coli is encoded by a structural gene adjacent to, and divergently transcribed from, the bkd operon of P. putida. BkdR was purified from E. coli containing bkdR cloned into pCYTEXP1, an expression vector. The molecular weight of BkdR obtained by gel filtration indicates that BkdR is a tetramer, and the abundance of BkdR in P. putida was estimated to be about 25 to 40 copies of the tetramer per cell. BkdR bound specifically to the region between bkdR and bkdA1, the latter being the first gene of the bkd operon. One BkdR-DNA complex was observed in gel mobility shift patterns. Approximately 100 bp was protected from the action of DNase I by BkdR, and the addition of L-branched-chain amino acids enhanced the appearance of hypersensitive sites in the protected region. There are four potential BkdR-DNA binding sequences in this region based on similarity to Lrp-binding consensus sequences. Like many other transcriptional activators, BkdR regulates expression of its structural gene. DNAs from several gram-negative bacteria hybridized to a probe containing bkdR, indicating the presence of bkdR-like genes in these organisms.