This work was undertaken with the objective of isolating and characterising the bdh gene of P. aeruginosa PAOI. Isolation of the bdh gene was initially attempted by PCR amplification and then by heterologous complementation of E. coli (LS5218) and S. meliloti (Rm11107) strains unable to catabolise 3-hydroxybutyrate. Three classes of plasmids were isolated. Class I comprised two plasmids, p5218-02 and p5218-07, isolated via complementation of LS5218, which were capable of complementing both LS5218 and Rm11107 for growth on 3-hydroxybutyrate. 3-hydroxybutyrate dehydrogenase (BDH) activity was not detected in an extract of LS5218 (p5218-02). The sole 3.6-kb EcoRI fist was partially sequenced and found to have three putative open reading frames (ORF). ORF 1 is homologous to the fusE gene of E. coli. We hypothesised that p5218-02 encodes an enzyme capable of degrading 3 hydroxybutyrate, but does not encode the bdh gene. Plasmids of class II (p30065) and class III (p30066) were isolated via complementation of Rm11107. Significant BDH activity was detected in an extract of Rm11107 (p30066), but not in Rm11107, leading to the hypothesis that p30066 carries the bdh gene.

This study was undertaken to characterize the role and pathway for assimilation of the intracellular carbon storage compound, poly-beta-hydroxybutyrate (PHB), in Rhizobium (Sinorhizobium) meliloti. Mutants unable to utilize the degradation intermediates, 3-hydroxybutyrate (HB) and/or acetoacetate (AA) were characterized. A mutant unable to utilize HB (Hbu-) while retaining the ability to utilize AA was found to be deficient in 3-hydroxybutyrate dehydrogenase (Bdh) activity. The bdhA mutant showed no symbiotic defects in association with alfalfa plants. However, when co-inoculated with the wild type, the mutant showed significantly reduced competitiveness. A more severe competition defect was observed for a PHB synthesis mutant (phaC). Both these mutants also showed reduced competitiveness when subjected to multiple cycles of subculturing through alternating carbon-rich and carbon-poor media, with the phaC mutant showing a greater loss in competitiveness. The results indicate that the ability to efficiently deposit and utilize cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability. / The gene encoding Bdh (bdhA) was isolated and sequenced. Two transcription start sites, S1 and S2 were identified but no known consensus promoter sequences were identified upstream of either start site. A sigma 54 consensus binding sequence was found to be located between S1 and S2 but no corresponding transcript was detected. Transcriptional bdhA-lacZ fusion studies indicated that gene expression was growth-phase associated. The bdhA gene from Rhizobium sp. NGR234 was also isolated and characterized and found to be highly homologous to the R. meliloti bdhA sequence. Unlike R. meliloti , NGR234 is able to accumulate PHB during symbiosis. An NGR234 bdhA mutant showed symbiotic defects on Leucaena but not on Tephrosia, Macroptilium or Vigna host plants, indicating that the phenotype was host-dependent. / Mutations that suppress the Hbu- phenotype without restoring Bdh activity were identified, indicating the existence of a Bdh-independent pathway for HB utilization. These mutations mapped to the age-1 locus, which causes enhanced growth rate on HB and AA minimal media. Introduction of plasmid-borne multiple copies of a gene encoding acetoacetyl-CoA synthetase (acsA) into the bdhA mutant also results in suppression of the Hbu- phenotype. A possible mechanism of suppression involving direct activation of HB to 3-hydroxybutyryl-CoA, followed by reduction to acetoacetyl-CoA by the NADP-acetoacetyl-CoA reductase (encoded by phaB) was investigated. A strain carrying the triple mutations, age-1::Tn5-Tp, bdhA ::Tn5 and phaB::OSmSp retained the ability to utilize HB, indicating that the bypass mechanism does not involve NADP-acetoacetyl-CoA reductase. / The phaB mutant does not accumulate PHB or utilize HB or AA. Furthermore, colonies of the phaB and phaC mutants exhibit non-mucoid phenotype on yeast extract mannitol agar. The observation that a R. meliloti exoS null mutant is also Hbu- provides further support for a link between PHB and exopolysaccharide synthesis. Since ExoS is a positive regulator of succinoglycan biosynthesis it is hypothesized that regulation of succinoglycan synthesis by ExoS requires PHB synthesis.

This research followed acetate carbon (C) uptake, metabolism, and fate through a typical modern Kraft pulp and paper mill AS system. Freshly collected mill biomass (AS floc suspensions) was placed under conditions representing four key phases of AS biotreatment: (1) initial acetate uptake by aerated starved AS; (2) ongoing acetate uptake; (3) aerobic metabolism of acetate-loaded AS in acetate-stripped effluent; and (4) anaerobic, settled biomass metabolism. Conditions mimicked the mill system as closely as possible. Acetate carbon uptake kinetics and conversion to CO2, growth products, PHB, and secreted metabolites in each of the four phases were measured. The role of PHB synthesis in the initial stripping of acetate from mill effluent and the PHB production potential of this mill AS were also investigated. / Results showed that acetate was rapidly taken up by high-affinity systems in the AS. During the initial exposure of mill-starved AS, acetate greatly stimulated AS-O2 uptake, and was quickly converted to PHB and CO 2. Upon depletion of available effluent acetate, as occurs in the downstream sections of the aeration tank, O2-uptake rates decreased and the acetate-C stored in AS-PHB was slowly released as CO2, and partly used for growth. Under secondary clarifier-like anaerobic conditions, the AS released virtually no CO2. However, substantial amounts of PHB were used for growth under anaerobic conditions and a small proportion of the original acetate C exited the cells as organic acids.

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