Isolation and characterization of mutants of Rhizobium meliloti unable to synthesize poly-3-hydroxybutyrate (PHB)

The isolation and characterization of four mutants of Rhizobium meliloti unable to synthesize poly-β-hydroxybutyrate (PHB) are reported. The mutants were independently obtained via Tn5 transposon mutagenesis and exhibited physiological and cytomorphological characteristics similar to those of the parental strain, as well as overlapping DNA profiles. These were assessed at both the plasmid and total genome level, using for the latter the sensitive technique of pulsed-field gel electrophoresis in a contour-clamped homogeneous electric field. With respect to the parental PHB+ strain, the loss of PHB-synthesizing ability in the four mutants was demonstrated by gas chromatography, transmission electron microscopy, and enzymatic tests. Localization studies of Tn5 insertion showed that the PHB− phenotype had, in all mutants, a transposon insertion in the same region, although not in the same position. The symbiotic traits (nodule-inducing ability on Medicago sativa and acetylene-reducing activity of nodules) of the mutants did not differ significantly from those of the parental R. meliloti.Key words: Rhizobium meliloti, poly-β-hydroxybutyrate (PHB), PHB synthase.