Carbohydrate Metabolism in Rhizobium trifolii: Identification and Symbiotic Properties of Mutants

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Abstract
Crude extracts of R. trifolii strain 7000 contained enzymes of the Entner-Doudoroff [ED] and pentose phosphate pathways. No phosphofructokinase (EC 2.7.1.11) activity and only a low activity of fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) were found, suggesting that the Embden-Meyerhof-Parnas pathway was not physiologically important in this strain. Independent carbohydrate-negative mutants of R. trifolii were isolated and charqcterized as deficient in glucokinase (glk; EC 2.7.1.2), fructose uptake (fup), the Entner-Doudoroff pathway (edp) and pyruvate carboxylase (pyc; EC 6.4.1.1). Glucokinase was essential for glucose phosphorylation in R. trifolii and was also required for growth on sucrose. The edp mutant was impaired in growth on all hexoses tested except galactose, suggesting that the ED pathway was the major pathway used by R. trifolii for the catabolism of these sugars. Galactose may be catabolized via a different pathway, possibly involving an NADP+-linked galactose dehydrogenase. Pyruvate carboxylase was an important anaplerotic enzyme in R. trifolii required for growth on all C sources tested, except succinate. All the mutants, including a glk fup double mutant, formed an effective symbiosis on red clover, suggesting that neither glucose, fructose nor sucrose are used by the bacteroids to provide ATP and reductant for N2 fixation. The bacteroids probably receive a supply of tricarboxylic acid cycle intermediates from the plant cytosol, and these may be their major source of energy.