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  • Poly-ββββ-hydroxybutyrate and glycogen metabolism in Rhizobium leguminosarum

    E.M.Lodwig1, K. Findlay2, A.J.Downie2, P.S.Poole1 1School of AMS, University of Reading, Reading, RG6 6AJ, UK; 2John Innes Centre, Norwich, NR4 7UH, UK

    Correspondence e mail: [email protected]; Poster available at http://www.ams.rdg.ac.uk/microbiology/posters.htm

    Introduction

    The role of the carbon storage compounds poly-β-hydroxybutyrate and glycogen in bacteroids is not fully understood. Both compounds are synthesised in bacteroids however the overall pool sizes vary considerably. Furthermore, mutation of the biosynthetic pathways has been shown to effect the efficiency of symbiotic nitrogen fixation in beans (Cevallos, et al., 1996; Marroqui et al., 2001). Therefore the pathways appear to be involved in the regulation of bacteroid metabolism. Here we have constructed single mutants in phaC (PHB synthase) and glgA (glycogen synthase), and a double mutant, to assess their impact on the R. leguminosarum bv. viciae symbiosis.

    Summary

    Both single mutants in phaC (RU1328) and glgA (RU1448) nodulate peas and fix nitrogen. However, the efficiency of at least the RU1328 symbiosis is not consistent. Both single mutants appear to alter the carbon balance in infected plant cells therefore we suspect carbon metabolism of the plant has a strong influence on the effect of the mutations. The mutant in both genes (RU1478) nodulates peas but forms bacteroids that senesce prematurely and appear to give a Fix- phenotype (nitrogenase activity is not yet available). Together these data suggest that at least one pathway is required for nitrogen fixation but that carbon metabolism via either pathway is relatively plastic.

    References Cevallos, et al. (1996). Journal of Bacteriology 178: 1646-1654. Marroqui, et al. (2001). Journal of Bacteriology 183: 854-864.

    Acknowledgements Sylvia Marroqui for supplying plasmids and cosmids. Biotechnology and Biological Sciences Research Council (UK) for funding.

    Mutant construction

    Mutants were constructed in R. leguminosarum strain A34 (Strr).

    The mutant in phaC was constructed by disrupting the gene with an omega cassette (Spcr). The mutant (RU1328) was confirmed by Southern blot. PHB was not detectable in free living cells of RU1328 grown on fructose (A34 - 65.0 µg mg protein-1).

    The glycogen synthase mutant was constructed by disrupting glgA with transposon TnB60 (Kanr). The mutant (RU1448) was confirmed by Southern blot. Glycogen was not detectable in free living cells of RU1448 grown on sucrose (A34 - 70.16 µg mg protein-1).

    The double mutant (RU1478) was constructed by recombining glgA::TnB60 into RU1328.

    Nodule and bacteroid ultrastructure

    Nodules were sectioned and studied under light microscope and TEM.

    PHB - There was a decrease in the amount of starch accumulation in interzone II-III in RU1328 nodules (Fig 1). There were no visible differences between the bacteroids of A34 and RU1328.

    GLG - There was a dramatic increase in the amount of starch accumulation throughout RU1448 nodules (Fig 2). There were no visible differences between the bacteroids of A34 and RU1448.

    PHB - GLG - Preliminary studies show RU1478 nodulates peas but the bacteroids formed senesce prematurely (Fig 3) and consequently there is a dramatically reduced zone of functional bacteroids within the nodule (nitrogenase activity not yet available to determine Fix phenotype).

    Symbiotic performance of single mutants

    Pea (Pisum sativum L. cv. Avola) seeds were inoculated with the wild type and single mutants to assess the nitrogenase activity (acetylene reduction) at flowering and the dry weight of mature plants (approximately 7 weeks growth).

    PHB - We have conducted two large scale greenhouse trials with the PHB synthase mutant. In the first experiment there was a decrease in nitrogenase activity (acetylene reduction) of 50% in RU1328 (A34 - 5.93 ± 0.59; RU1328 - 2.99 ± 0.17 µmoles ethylene plant-1hr-1. P