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

Abstract

~Crude extracts of Rhizobium trifolii strain 7000 contained enzymes of the Entner-Doudoroff and pentose phosphate pathways. No phosphofructokinase (EC 2.7.1 . 1 1) 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 characterized 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. trgolii 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 carbon 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 nitrogen 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.

Properties of Organic Acid Utilization Mutants of Rhizobium leguminosarum Strain 300

“…The latter study of R. leguminosarum mutants which were defective in sugar utilization suggested that the capacity to utilize C6 or C1 sugars, which are the major components of the plant photosynthate, was not essential for the establishment of an effective symbiosis. This finding was in accord with the data of Ronson & Primrose (1979); however, Duncan (1981) reported that a fructokinase mutant of R. meliloti was ineffective in symbiotic N2 fixation.…”

“…The nutritional and symbiotic properties of bacterial mutants with specific defects in their capacities to utilize carbon compounds have been examined in Rhizobium trifolii (Ronson & Primrose, 1979;Ronson et al, 1981), R . meliloti (Duncan, 1981) and R .…”

“…The nutritional requirements and preferences of Rhizobium, both as free living cells and as bacteroids, have been studied by genetic and biochemical analyses. Biochemical studies include examination of catabolic enzyme activities (Ronson & Primrose, 1979;Glenn et al, 1984b), solute transport (de Vries, 1980;Finan et al, 1981 ;Ronson et al, 1981 ;Saroso et al, 1984) and substrate oxidation (Glenn & Dilworth, 1981).…”

An exoB mutant of Rhizobium sp. is effective in indeterminate nodules of Hedysarum coronarium

“…Like other Rhixobitlm exoB mutants, CallO is able to grow on galactose as sole carbon source, in contrast to E. coli galE mutants, where galactose as the unique carbon source is lethal (Adhya, 1987 (Ronson & Primrose, 1979) and R . meliloti (Arias & Cervenans ky, 1986).…”

Biochemical Genetics of Nitrogen Fixation

“…Regulation of N2 fixation in legumes by fixed N does not seem to be at the level of nitrogenase synthesis (23; D. Noel and W. J. Brill, manuscript in preparation), but rather is at the level of nodulation. In the case of R. trifolii and clover, fixed N seems to prevent the lectin from being available to the bacteria (54 (186). A mutant of R. meliloti with a defective a-ketoglutarate dehydrogenase forms ineffective nodules (69).…”