During free-living reproductive growth, Sinorhizobium meliloti accumulates poly-beta-hydroxybutyrate (PHB) and glycogen, and produces and excretes exopolysaccharides and beta-1,2-glucan. In previous investigations, PHB-minus mutants of S. meliloti 41 were obtained and studied; and the genes for PHB biosynthesis, phaAB and phaC, were described. In this work, the role of an open reading frame (orf) upstream of phaAB is studied. This orf is designated aniA because the gene was found to be expressed during anaerobic growth. Under low oxygen conditions, glycogen decreases and the production of extracellular polymeric substances (EPS) is partially repressed. When the aniA mutant is incubated under oxygen-limiting conditions, the only significant change observed is an overproduction of EPS. Subsequent in planta tests showed that although the mutant strain produced abundant nodules, only very low acetylene-reduction activity was detected, indicating that nitrogen fixation was not adequately supported by endogenous substrates.

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Genes encoding fiketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORFl were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB-Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB-strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORFl with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.

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.

In the present paper we report the exclusive microbial preparation of polyhydroxyalkanoates (PHA) containing 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB) as comonomers through the use of unexpensive carbon sources such as whey from dairy industry. Polymers were produced by growing H. pseudoflava DSM 1034 in minimal medium supplemented with sucrose, lactose or whey without any co-substrate added. The chemical and physical properties of the polymers were fully characterized by GPC, DSC, TGA analyses and the composition by GC and (1)H NMR examinations to especially confirm the content of different monomeric units. The presence of 4HB units into PHA samples is particularly aimed in thermoplastic applications where greater flexibility is required and conventional rigid PHAs tend to fail. Usually the insertion of 4HB into chain backbone consisting of 3-hydroxyalkanoates requires expensive carbon sources mostly of petrochemical origin. According to our study the production of P(3HB-co-3HV-co-4HB) terpolymer can be obtained directly by the use of lactose or waste raw materials such as cheese whey as carbon sources. Although the amount of 4HB in the produced terpolymers was usually low and not exceeding 10% of the total molar composition, a PHA containing 18.4% of 4HB units was produced in 1 step fermentation process from this structurally unrelated carbon sources. The crystallinity of the terpolymer is basically to be markedly affected with respect to that of conventional PHAs, thus obtaining a comparatively less rigid material and easier to be processed.