Poly-3-hydroxybutyrate degradation in Rhizobium (Sinorhizobium) meliloti: Isolation and characterization of a gene encoding 3-hydroxybutyrate dehydrogenase

We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding g ene (bdhA) from Rhizobium (Sinorhizobium) meliloti, The gene has an open re ading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07), The R. meliloti Bdh protein exhibits f eatures common to members of the short-chain alcohol dehydrogenase superfam ily, bdhA is the first gene transcribed in an operon that also includes xdh A, encoding xanthine oxidase/dehydrogenase. Transcriptional start site anal ysis by primer extension identified two transcription starts, S1, a minor s tart site, was located 46 to 47 nucleotides upstream of the predicted ATG s tart codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S 1 or S2 failed to reveal the presence of any known consensus promoter seque nces, Although a sigma(54) consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R, meliloti was able to utilize 3-hydroxybutyrate as a sole carbo n source. The R. meliloti bdhA gene is able to confer upon Escherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R, me liloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects, Studies with a strain carryi ng a lacZ transcriptional fusion to bdhA demonstrated that gene expression is growth phase associated.