Megaplasmid and chromosomal loci for the PHB degradation pathway in Rhizobium (Sinorhizobium) meliloti

Chromosomal and megaplasmid loci that affect the poly-3-hydroxybutyrate (PHB) degradation pathway in Rhizobium meliloti were identified. A clone that restores the ability of certain R. meliloti mutants with defined deletions in megaplasmid pRmeSU47b to use 3-hydroxybutyrate or acetoacetate as the sole carbon source was isolated from a cosmid library of R. meliloti genomic DNA. Tn5 insertion mutagenesis, followed by merodiploid complementation analysis, demonstrated that the locus consists of at least four transcriptional units, bhbA-D. We also identified loci involved in 3-hydroxybutyrate and/or acetoacetate utilization by screening for mutants that had lost the ability to use 3-hydroxybutyrate as the sole carbon source while retaining the ability to use acetate (thus ensuring an intact glyoxylate cycle and gluconeogenic pathway). These mutants fell into four classes, as determined by replicon mobilization experiments and genetic linkage in phage transduction; one class corresponded to the bhb locus on pRmeSU47b, two classes mapped to different regions on the chromosome and the fourth, bdhA, represented by a single mutant, mapped to another pRmeSU47b locus, near bacA. The bdhA mutant is deficient in 3-hydroxybutrate dehydrogenase activity.