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Amino acid metabolism in Rhizobium

Poole, Philip (1986) Amino acid metabolism in Rhizobium. PhD thesis, Murdoch University.

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Addition of a broad range of L-amino acids and several D-amino acids to washed cells of Rhizobium leguminosarum strains WU235 and MNF3841 grown on glucose/NH4Cl elicited a low rate of O2 consumption. L-Glutamate, L-glutamine, L-aspartate, L-asparagine, L-alanine or L-histidine served as the sole source of nitrogen and carbon for growth of strain WU235 and each caused a several-fold increase in the amino acid dependent O2 consumption. In all these cultures excess ammonia was liberated, with the quantity depending on the number of nitrogen atoms per amino acid molecule. A very high dicarboxylic acid dependent O2 consumption in cells of WU235 grown on aspartate was found to be due to the presence of aspartase (EC

R. leguminosarum WU235 only expressed aspartase when grown on L-aspartate or L-asparagine as the sole carbon source. Cells grown on glucose plus L-aspartate, or fumarate plus L-aspartate, did not express aspartase. Although these results suggested catabolite control of an inducible enzyme, induction of aspartase could not be demonstrated. Aspartase-producing cells continued to synthesize the enzyme after repeated subculture on glucose plus NH4Cl. Cells grown in glucose plus NH4Cl and plated onto aspartate produced different colony sizes; the larger (0.1% of the total) expressed aspartase, while the smaller did not. At dilutions sufficient to exclude the large aspartase-producing colonies, all initial colonies were the same size. They later developed papillae or became cluster colonies and produced aspartase. The data suggest that strain WU235 is unable to produce aspartase unless a mutation occurs which leads to constitutive enzyme synthesis.

Rhizobium leguminosarum MNF3841 grown on glucose/NH4Cl constitutively transported several L-amino acids. Transport rates were elevated 1.5-4 fold after growth in the absence of anmonia. Uptake of L-glutamate, L-glutamine, L-asparagine and L-leucine was inhibited to varying extents by a broad range of L-amino acids. The use of structural analogues of Lglutanvate and metabolic inhibitors suggested that L-glutamate transport was an active process requiring the L-isaner to have a free alpha hydrogen and a free amino group. Cells loaded with either L-(14C) leucine or L-(14C) glutamate exhibited exchange with a wide range of amino acids. The apparent Km for L-glutamate transport was 81 nM and both Laspartate and L-alanine were competitive inhibitors of Lglutamate uptake. Thus there appears to be an extremely high affinity carrier for L-glutamate that is not only very sensitive to inhibition by L-aspartate but also capable of being inhibited by a broad range of amino acids at an order of magnitude higher concentration.

Batch cultures of R. leguminosarum MNF3841, R. leguminosarum WU235, R. phaseoli WU15, R. trifolii TA1 and R. meliloti WU38 used amnonia faster than glutamate when presented with an equimolar mixture of the two. Only the cowpea strain NGR234 used both nitrogen sources at the same rate. R. leguminosarum MNF3841 grew faster on ammonia than on glutamate as the nitrogen source. In chemostat culture grown under phosphate limitation strain MNF3841 did not release excess ammonia when grown on either mannitol/L-glutamate or fumarate/L-glutamate, showing that L-glutamate catabolism was tightly regulated to meet the cells nitrogen requirement. Furthermore the rate of consumption of ammonia was similar to that for L-glutamate when either was supplied as the sole nitrogen source. However with L-histidine or L-alanine as the nitrogen source large quantities of excess ammonia were released. When chemostat cultures of R. leguminosarum MNF3841 were supplied with an equimolar mixture of ammonia and Lglutamate, 81-100% of the nitrogen consumed was ammonia. Similarly with mixtures of L-glutamate/L-histidine or Lglutamate/ L-alanine almost no L-glutamate was consumed, a result attributable to the release of excess ammonia from either L-histidine or L-alanine. The use of 14C labelled fructose or L-glutamate suggested that the intra and extracellular L-glutamate pools were isolated. This indicated that the ammonia preference must be exerted by a restriction in Lglutamate transport. L-Glutamate transport rates were low in L-glutamate/NH4Cl containing chemostats, which suggests ammonia restricts L-glutamate transport both by repression and perhaps by inhibition by seme metabolic intermediate.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Environmental and Life Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Dilworth, Michael and Glenn, Andrew
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