MEGAPLASMID AND CHROMOSOMAL LOCI FOR THE PHB DEGRADATION PATHWAY IN RHIZOBIUM (SINORHIZOBIUM) MELILOTI

Chromosomal and megaplasmid loci that affect the poly-3-hydroxybutrate (PHB) degradation pathway in Rhizobium meliloti were identified. A cl one that restores the ability of certain R. meliloti mutants with defi ned deletions in megaplasmid pRmeSU47b to use 3-hydroxybutyrate or ace toacetate as the sole carbon source was isolated from a cosmid library of R. meliloti genomic DNA. Tn5 insertion mutagenesis, followed by me rodiploid complementation analysis, demonstrated that the locus consis ts of at least four transcriptional units, bhdA-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-hydroxybutyra te as the sole carbon source while retaining the ability to use acetat e (thus ensuring an intact glyoxylate cycle and gluconeogenic pathway) . These mutants fell into four classes, as determined by replicon mobi lization experiments and genetic linkage in phage transduction; one cl ass 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.