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Carbon metabolism in Rhizobium leguminosarum MNF 3841

McKay, Ian (1988) Carbon metabolism in Rhizobium leguminosarum MNF 3841. PhD thesis, Murdoch University.

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So much depends upon a red wheel barrow glazed with rain water beside the white chickens - William Carlos Williams 1923.

Carbon catabolism of Rhizobium lequminosarum MNF3841 was studied in free-living cells grown in chemostat and in bacteroids.

Enzymes of the Entner-Doudoroff (ED) pathway, the pentose phosphate (PP) pathway and the TCA cycle were present, though the absence of phosphofruetokinase prec1uded the operation of the complete Embden- Meyerhof-Parnas (EMP) pathway. The low activity of fructose-bisphosphate aldolase in sugar-grown cells indicated that recycling of glyceraldehyde 3-phosphate (produced by the ED pathway) to fructose 6-phosphate is unlikely. Further catabolism of glyceraldehyde 3-phosphate is probably achieved via the enzymes of the latter part of the EMP pathway which were shown to be present in this organism.

In phosphate-limited chemostat culture the activities of invertase, glucose-6-phosphate de hydro- genase, the ED enzymes and 6-phosphogluconate dehydrogenase were two- to three-fold lower in cells grown on fumarate compared to the activities in cells grown on sucrose. Glucose-6-phosphate dehydrogenase also showed modulation of activity due to the nature of the growth limitation with oxygen-limited cells possessing only 50% of the activity of phosphate-limited cells when fumarate was the carbon source. None of the other sugar catabolic enzymes, nor any of those of the TCA cycle showed any modulation in response to the growth substrate or the nature of the growth limitation.

Since modulation of some sugar catabolic enzymes was demonstrated in free-living cells in response to growth substrate, the preferences of free-living cells for C4-dicarboxylates, or sugars, were further investigated. In chemostat culture under phosphate-limitation MNF3841 co-utilised fumarate in combination with glucose, or sucrose, or glucose plus fructose. A slight preference for Ca-dicarboxylates was indicated, since the inhibition of sugar utilisation by fumarate was greater than the inhibition of fumarate utilisation by equivalent concentrations of glucose, or sucrose, or glucose plus fructose.

Though the obvious importance of Ca-dicarboxylates as carbon sources for both free-living rhizobia and bacteroids is recognised, the ancillary enzymes required for their catabolism have not yet been identified. R. lequminosarum MNF3841 catabolised Ca-dicarboxylates and L-arabinose vi a the TCA cycle with the requirement for acetyl CoA being met by the action of malic enzyme and pyruvate dehydrogenase. Malic enzyme was present in sugar-grown free-living cells though higher levels were observed when fumarate or L-arabinose was the growth substrate. Manganese-dependent malic enzyme activity was evident with either NADP* or NAD* as the cofactor and the activity was stimulated by the presence of KC1. The activity of pyruvate dehydrogenase, which is also required for the catabolism of sugars via the TCA cycle, was higher in sucrose-grown cells than those grown on fumarate.

In addition to the TCA cycle and the ancillary enzymes (malic enzyme and pyruvate dehydrogenase) the growth of rhizobia on C4-dicarboxylates (and other substrates which feed into the TCA cycle such as L-glutamate, L-aspartate, L-histidine and L-arabinose) also requires a system of gluconeogenesis. This is accomplished in MHF3841 vi a phosphoenolpyruvate carboxykinase (PEPCK), fruetose-bisphosphate aldolase and fructose-bisphosphatase in conjunction with enzymes of the EMP pathway. In addition R. lequminosarum MNF3085, a PEPCK-deficient mutant, failed to grow on succinate, pyruvate, L-arabinose or L-glutamate, yet grew as well as MNF3841 on glucose, sucrose and glycerol showing that PEPCK is essential for gluconeogenesis.

PEPCK and fruetose-bisphosphate aldolase were rapidly derepressed following transfer of cells from a medium with sucrose as the carbon source to one with fumarate as the carbon source. In chemostat culture, the addition of 0.1 mM sucrose caused an 80% inhibition of PEPCK and fructose-bisphosphate aldolase synthesis and 0.4 mM sucrose caused complete inhibition of PEPCK synthesis.

Although Ca-dicarboxylate transport was rapidly inducible in free-living cells, bacteroids of MNF3841 isolated from pea nodules could immediately transport 1 4 C-succinate. Furthermore, blocking pyruvate dehydrogenase with arsenite resulted in bacteroids immediately accumulating pyruvate and malate from fumarate indicating that bacteroids in the nodules are in receipt of C*- dicarboxylates.

Bacteroids isolated on a Percoll gradient had activities of TCA cycle enzymes, pyruvate dehydrogenase and malic enzyme up to six-fold higher than those in free-living cells, whereas the activities of sugar catabolic enzymes in bacteroids were 2- to 14- fold lower than those in free-living cells grown on sucrose. These activities are a further indication that Ca-dicarboxylates (and not sugars) are the principal form of carbon catabolised by bacteroids.

Additionally bacteroids of MNF3841 contained low levels of PEPCK and fruetose-bisphosphate aldolase. The bacteroid-associated PEPCK activity was clearly of bacterial and not plant origin because of its nucleotide requirement and the fact that bacteroids of MNF3085 (PEPCK deficient in the free-living form) contained no PEPCK activity. MNF3085 nodulated and fixed nitrogen as effectively as t he parent which demonstrates that the capacity to synthesise sugars via gluconeogenesis is not required for an effective symbiosis. Thus these data suggest that although bacteroids of MNF 3085 receive sufficient sugar to compensate for their gluconeogenic defect, there is insufficient sugar available to bacteroids of the wild type to completely repress the synthesis of PEPCK. A low amount of sugar available to the bacteroid suggested by these data would be in keeping with the very low activities of sugar catabolic enzymes in the bacteroid. These data in conjunction with the transport of C4-dicarboxylates by bacteroids immediately after their isolation and the elevated activities of the enzymes of C4-dicarboxylate catabolism in bacteroids indicate that C4-dicarboxylates are indeed the major carbon substrate used by them for N2 fixation.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Biological and Environmental 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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