Carbon and nitrogen metabolism of free-living Frankia spp. and of Frankia-alnus symbioses

The research reported in this thesis deals with the symbiosis of <em>Frankia</em> spp. and <em>Alnus glutinosa. Frankia</em> spp. are actinomycetes giving rise to the formation of nitrogen-fixing nodules on the roots of a number of non-leguminous plants. In these nodules <em>Frankia</em> spp. live within the plant cells and obtain all sources of carbon and energy from the plant, giving fixed nitrogen in exchange.<br/>To answer the question what compounds <em>Frankia</em> spp. obtain from the plant, insight into the metabolism of this microorganism is required. To obtain this insight, researches have been made with the free-living <em>Frankia</em> AvcI1, isolated from root nodules of <em>Alnus viridis</em> ssp <em>crispa</em> by Baker <em>et al.</em> (1979). <em></em> This organism has been isolated and cultivated on complex media. In order to obtain insight into the C- and N-source requirements of <em>Frankia</em> AvcI1, a simple and well-defined growth medium is needed. The composition of this medium and the C- and N-metabolism of <em>Frankia</em> AvcI1 are the subjects of Chapter II of this thesis.<br/>In Chapter II.1 it is shown that <em>Frankia</em> AvcI1 is able to grow on a medium containing Tween 80 (an oleate ester of polyethyleneglycol sorbitan) as sole C-source and either glutamic acid or NH <sub>4</sub> Cl as sole N-source. The growth yield of <em>Frankia</em> AvcI1 on various media is reported. It is shown that the doubling time of <em>Frankia</em> AvcI1 growing on QMOD/Tween medium is about 2 days, which is slow, even for an actinomycete. When growing with Casamino acids as nitrogen source, <em>Frankia</em> AvcI1 selectively takes up glutamic acid and aspartic acid, leaving the concentrations of <em></em> the other detectable amino acids unchanged. The mechanism of this selection is still unclear.<br/>Chapter II.2 contains further data on the C-sources utilized by <em>Frankia</em> AvcI1 It is shown that <em>Frankia</em> AvcI1 can utilize as C-source also other Tweens, <em>viz.</em> Tweens 20, 40, 60 and 85, and in addition several fatty acids, viz. acetic, propionic, butyric, valeric, caproic, caprylic, capric, palmitic and stearic acids. No growth of <em>Frankia</em> AvcI1 was observed on media containing triglycerides as C-sources.<br/>The dependence of the growth yield on the nature of the carbon source and the concentration of NH <sub>4</sub><sup>+</sup> in the media is shown. Utilization of NH <sub>4</sub><sup>+</sup> as nitrogen source of <em>Frankia</em> AvcI1 growing in the Tween/NH <sub>4</sub><sup>+</sup> medium is confirmed by incorporation experiments with <sup>15</sup> N-NH <sub>4</sub> Cl.<br/>The results reported in Chapter II.3 show that <em>Frankia</em> AvcI1 does not take up glucose from a medium containing both glucose and Tween 80. In agreement with this observation, it is demonstrated that the activities of isocitrate lyase and malate synthase in cells grown on the QMOD/Tween medium (containing both glucose and Tween 80) are of the same order of<br/>magnitude as in cells grown on the Tween/NH <sub>4</sub><sup>+</sup> medium (containing Tween 80 as sole C-source).<br/>Organisms growing on fatty acids, and degrading these compounds to acetyl-CoA, start gluconeogenesis with the action of the glyoxylate cycle (Kornberg and Krebs, 1957) leading to the conversion of 2 acetyl-CoA molecules to 1 molecule of succinate. The presence of the glyoxylate cycle enzymes isocitrate lyase and malate synthase in <em>Frankia</em> AvcI1 cells grown with Tween 80 as C-source is therefore not surprising. The succinate formed in the glyoxylate cycle can be converted to phosphoenolpyruvate by the subsequent action of the enzymes succinate dehydrogenase, fumarase, malic enzyme and pyruvate orthophosphate dikinase, which are found in cell-free extracts of <em>Frankia</em> AvcI1 From phosphoenolpyruvate, gluconeogenesis can continue with the action of phosphoglycerate kinase and 3-phosphoglyceraldehyde dehydrogenase.<br/>For energy generation, <em>Frankia</em> AvcI1 can oxidize the acetyl-CoA derived from fatty acid breakdown in the citric acid cycle, although the low activity of the enzyme succinyl-CoA synthetase leaves room for the presumption that the citric acid cycle is not very operative in <em>Frankia</em> AvcI1 This is not impossible since the oxidation of fatty acids yields many reducing equivalents.<br/>The data contained in Chapter II.4 show that <em>Frankia</em> AvcI1 does not take up succinate from a medium containing acetate plus succinate or succinate alone. In accordance, no repressing effect of succinate on the activities of the glyoxylate cycle enzymes was observed, whereas in cells grown on propionate these enzymes were not found, indicating that they are hot constitutive in <em>Frankia</em> AvcI1<br/><em>Frankia</em> AvcI1 is able to utilize several amino acids as sole nitrogen source, <em>viz</em> . alanine, γ-aminobutyric acid, aspartic acid, glutamic acid, glycine, leucine, phenylalanine, serine, threonine, tyrosine and valine.<br/>No differences in C- and N-source requirements were observed for the three <em>Frankia</em> strains AvcI1, CPI1 (isolated by Callaham <em>et al.,</em> 1978, from <em>Comptonia peregrina</em> root nodules) and AgSp+1 (isolated by Quispel and Burggraaf, 1981, from <em>Alnus glutinosa</em> spore-(+) type root nodules). These three strains were shown to utilize either Tween 80 or acetate, but no ethanol, lactate, glucose or succinate as sole C-source, and either NH <sub>4</sub> Cl, Casamino acids, aspartic acid or alanine as N-source.<br/>From the data given in Chapter II it will be clear that <em>Frankia</em> AvcI1 is able to grow on a well-defined medium, which is a prerequisite for obtaining a clear insight into the physiology of the organism. The alcoholic root extract (Quispel and Tak, 1978) or the soybean lecithin (Lalonde and Calvert, 1979), added to the medium as growth factors, can be replaced by Tween 80 or fatty acids which are utilized as carbon source, while NH <sub>4</sub> Cl or amino acids can be utilized as nitrogen source.<br/>It is unknown whether <em>Frankia</em> spp. grow with the same growth rate on media with different constituents. The growth yields presented in Chapter II are usually small as compared to the amount of carbon present in the media. This suggests that either an additional component of the medium is present in limiting concentrations or that <em>Frankia</em> spp. can only grow for a limited time after inoculation.<br/>The only group of compounds known so far to be utilized as C-source by free-living <em>Frankia</em> AvcI1 are fatty acids, either free or esterified. The question what carbon source <em>Frankia</em> spp. living symbiotically in the nodule obtain from the plant, still remains to be answered. Based on the data obtained for free-living <em>Frankia</em> spp. it is not unlikely that some fatty acid functions as C-source under such conditions. Other possible candidates for this function are plant lipids like the alcoholic root extract (Quispel and Tak, 1978) or the soybean lecithin (Lalonde and Calvert, 1979). It is unlikely that sugars like glucose or dicarboxylic acids like succinate are playing this role, unless the ability of <em>Frankia</em> spp. to take up one or more of these compounds should alter during the transition from the free-living to the symbiotic stage.<br/>The cultivation of free-living <em>Frankia</em> spp. is a powerful tool in discovering symbiotic interactions of the endophyte and the host. In Chapter II.4 of this thesis it is shown that by replacing acetate as C-source of the medium by propionate, the activities of the glyoxylate cycle enzymes are repressed. Other authors (Tjepkema, Ormerod and Torrey, 1980 and 1981, Gauthier, Diem and Dommergues, 1981) reported a medium in which free-living <em>Frankia</em> spp. show vesicle formation and N <sub>2</sub> -ase activity. The present knowledge thus enables one to influence regulation in freeliving <em>Frankia</em> spp., which is important in studying the symbiotic interactions mentioned above.<br/>In Chapter III attention is paid to the assimilation of the ammonia produced by the endophyte living symbiotically in the root nodules of <em>Alnus glutinosa</em> grown in the greenhouse from seeds collected in Wageningen. In Chapter III.1 the composition of the pool of free amino acids in root nodules and the xylem tissue of stems is reported. It is shown that citrulline is the predominant free amino acid in nodules, while serine also occurs in relatively large amounts. In xylem tissue citrulline and glutamic acid are prominent.<br/>The activities of N <sub>2</sub> -ase, GS, GDH and OCT in root nodule homogenates are reported. From the K <sub>m</sub> values of GDH for NH <sub>4</sub><sup>+</sup> (16 mM) and glutamate (0.9 mM) and the concentrations in the nodule of NH <sub>4</sub><sup>+</sup> (1.5 mM) and glutamate (0.5 mM) it is concluded that GDH in alder nodules probably is responsible for the deamination of glutamate and not for the synthesis of this key amino acid. The important function of GDH in the nitrogen metabolism of alder nodules is confirmed by the much higher activity of this enzyme in homogenates of nodules as compared to that in homogenates of root-tips and leaves.<br/>The vesicle clusters, which contain the N <sub>2</sub> -ase activity, did not show activity of GS, GDH and OCT. The cytoplasm of the host cells was shown to possess the GS activity, while GDH and OCT are localized in the organelles of the host cells. No activity of NADH-dependent GOGAT was observed.<br/>In Chapter 111.2 it is shown that the activity of NADH-dependent GOGAT from root nodules of lupins is inhibited by some compound in the homogenate of alder nodules.<br/>Simultaneously with and independently of the present research, Schubert <em>et al.</em> (1981) analyzed the composition of the pool of free amino acids in nodules and the xylem tissue of <em>Alnus glutinosa</em> grown in the field in East Lansing, Michigan. The only difference with respect to these amino acid pools between the American alders and the European alders studied in the present research, is the relatively high amount of serine in nodules of the latter, whereas in nodules of the former this amino acid is not found. The results of both Schubert <em>et al.</em> (1981) and the present research confirm the hypothesis that in alder,citrulline is the main transport vehicle of fixed nitrogen (Miettienen and Virtanen, 1952; Leaf, Gardner and Bond, 1958; Wheeler and Bond, 1970).<br/>From the results shown in Chapter 111.2 it can be concluded that our failure to find any GOGAT activity may be ascribed to the presence of an inhibiting compound in the homogenate of alder nodules, so that it is not excluded that GOGAT is active in alder nodules <em>in vivo</em> .<br/>The data reported in Chapter III are in accordance with the following model: The nitrogen fixed as ammonia in the vesicle clusters of the endophyte, is assimilated in the cytoplasm of the host cell into glutamate by the action of GS and presumably GOGAT. Glutamate is in part deaminated in the plant organelles by the action of GDH to supply the NH <sub>4</sub><sup>+</sup> required for the synthesis of carbamyl phosphate. Another part of the glutamate is converted to ornithine, which in the organelles of the host cell<br/>reacts with carbamyl phosphate to form citrulline according to the OCT reaction. Citrulline is excreted from the nodule and serves as nitrogen source for the plant.<p/&gt