Poly-3-Hydroxybutyrate Degradation in Rhizobium (Sinorhizobium) meliloti : Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase

Abstract

We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading 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 features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 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 carbon source. The R. meliloti bdhA gene is able to confer uponEscherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying alacZ transcriptional fusion to bdhAdemonstrated that gene expression is growth phase associated.

Identification and characterization of genes, encoding the 3‐hydroxybutyrate dehydrogenase and a putative lipase, in an avirulent spontaneous Legionella pneumophila serogroup 6 mutant

“…It is known for other microorganisms that BdhA catalyses the first steps of PHB granule degradation (28). In this study, the MS1 strain indeed showed a higher accumulation of PHB granules than its parental strain suggesting a role bdhA in the metabolism of this important source of energy.…”

Sinorhizobium melilotistrain 1021bioSandbdhAgene transcriptions are both affected by biotin available in defined medium

“…BioS transcription was also at a maximum in the presence of nanomolar amounts of biotin [7,8]. To our knowledge the bdhA gene, which is co‐transcribed with the xdhA gene [9], is the second identified S. meliloti locus which is regulated by biotin on a transcriptional level and not linked to biotin synthesis.…”

“…A comparison of the bioS and bdhA 5′ regions containing the identified promoters of both genes revealed the presence of common elements in both DNA regions [8,9]. One GNGCATTTGCNC motif consisting of 10 nucleotides (eight identical in both promoters) was detected immediately upstream of the −10 regions in both promoters.…”

“…In order to test whether biotin might be involved in the regulation of PHB degradation we mobilized a bdhA‐lacZ fusion into S. meliloti strain 1021 and tested bdhA‐lacZ transcription in the absence and presence of added biotin. Because bdhA expression was reported to be at a maximum in stationary growth phase cultures [9], we measured bdhA‐lacZ activity only in stationary growth phase cultures. In the presence of biotin bdhA‐lacZ activity in S. meliloti strain 1021 was in general 4‐ to 5‐fold higher compared to cultures which had been growing in the absence of added biotin.…”

“…Another possible mechanism involves bioS , a biotin‐inducible gene in S. meliloti that affects competitive growth [7,8]. A third mechanism might involve the bdhA gene encoding the production of 3‐hydroxybutyrate dehydrogenase enzyme, which is central to PHB degradation in S. meliloti [9]. This hypothesis is supported by the work of Encarnacion et al [10] showing a significant accumulation of PHB in Rhizobium etli cells in the absence of added biotin.…”