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, UKCorrespondence e mail: e.m.lodwig@reading.ac.uk; 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) nodulatepeas 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.

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

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

Mutant construction

Mutants were constructed in R. leguminosarumstrain 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 RU1478nodulates peas but the bacteroids formed senesce prematurely (Fig 3) and consequently there is a dramatically reduced zone of functionalbacteroids 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<0.001, n=36) and a decrease of 40% in biomass of peas nodulated by RU1328 compared to A34 (Fig 4). In the second experiment there were no differences (data not shown). These experiments differed only in the time of planting (May and July respectively) which would influence plant carbon metabolism.

GLG - There was no significant difference in the nitrogenase activity (A34 - 3.70 ± 0.60; RU1448 -3.38 ± 0.51 µmoles ethylene plant-1hr-1, n=8) or dry weight (A34 - 1.05 ± 0.10 (n=11); RU1448 -1.01 ± 0.08 (n=13) grams) of peas nodulated by RU1448 compared to A34 (data from one experiment).

Fig 2. Section through a pea nodule infected by AA34 and B RU1448. Note the high amounts of starch (S) accumulated at periphery of infected plant cells in B are absent in A. Bar 2µm.

A B

S

Fig 3. Section through a pea nodule infected by AA34 and B RU1478. Note extensive deterioration of bacteroid membranes (BM) and vacuolisation of bacteroids in B . Bar 1µm.

A B

BM

Fig 1. Section of a pea nodule infected by AA34 and B RU1328. Starch (S) accumulation at periphery of infected plant cells in A is dramatically reduced in B. Bar 100µm.

A

B

SS

Fig 4. Pea plants nodulated by WT & PHB mutant at 7 weeks growth. Dry weights: A34 -2.65 ± 0.13 (n=42); RU1328 - 1.674 ± 0.09 (n=45) grams.

A34 RU1328