Improved analysis of metabolite uptake by Bradyrhizobium japonicum bacteroids

Abstract

SALMINEN, S. O., and STREETER, J. G. 1991. Improved analysis of metabolite uptake by Bradyrhizobium japonicum bacteroids.Can. J. Microbiol. 37: 238-243. Uptake of a variety of metabolites by Bradyrhizobium japonicum bacteroids under aerobic conditions was studied with the goal of identifying factors that would influence the accuracy of kinetic parameters. Saturable and nonsaturable components of absorption were identified, allowing separate analysis of only the active component. A significant proportion of labeled substrate was released as CO,. The CO, release was linear with time and occurred without a noticeable lag period. The kinetic parameters of saturable, active uptake indicated that if the label converted to C 0 2 was ignored, this would result in underestimation of the total uptake of malate by more than 50%. The proportion of the labeled substrate converted to CO, depended on the compound but was high in all cases. Glucose showed the smallest difference, with 13% of the label converted to CO, in a 5-min incubation. The uptake of glutamate was inhibited by aspartate, whereas no effect by equimolar dicarboxylic acids was found. Succinate or fumarate inhibited malate uptake, whereas amino acids showed no effect. Thus, organic acids appeared to have a common carrier distinct from that for amino acids. Techniques for separation of active and passive uptake components and for collection and analysis of CO, respired are described; these techniques should be used for accurate analysis of metabolite uptake.Key words: Bradyrhizobium japonicum, bacteroids, metabolite uptake, CO,. SALMINEN, S. O., et STREETER, J. G. 199 1. Improved analysis of metabolite uptake by Bradyrhizobium japonicum bacteroids. Can. J. Microbiol. 37 : 238-243.L'absorption dans des conditions aerobiques d'une variete de metabolites par les bactero'ides Bradyrhizobium japonicum a ete etudiee en vue d'identifier les facteurs qui pourraient influencer la precision des parametres cynetiques. Des composCs saturables et non saturables de l'absorption ont ete identifies, mais seuls les composes actifs ont pu faire l'objet d'analyses separees. Une proportion significative des substrats marques a Ct C libCree sous forme de CO,. La liberation du CO, a kt6 linkaire avec le temps, mais sans Nriode de latence appreciable. Les parametres cynetiques des composCs actifs et saturables absorb& ont indique que si les substrats marquCs transformes en CO, Ctaient ignorCs, cette omission se traduirait par une sous-estimation de plus de 50% de l'absorption totale du malate. La proportion des substrats marques convertis en CO, s'est avCrCe dependante des composes, mais elle a kt6 ClevCe dans tous les cas. La difference la plus faible a Cte obtenue avec le glucose, soit 13% du marquage converti en CO, au cours d'une incubation de 5 min. L'absorption du glutamate a ete inhibee par l'aspartate, tandis que les acides dicarboxyliques Cquimolaires n'ont eu aucun effet. L'absorption du malate a etC inhibee par le succinate et le fumarate, alors que les acides aminks n'o...

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with 14CO2

“…1, Table II). This agrees with the data obtained with isolated bacteroids provided with 14C-labeled substrates (24,26). Although glutamate in R leguminosarum bacteroids was not initially labeled as rapidly as malate or alanine, by 6 min the radioactivity (dpm/300 mg) in glutamate was 2214, whereas the corresponding amounts in malate and alanine were 2853 and 2118, respectively.…”

“…1). Because the radioactivity in glutamate in the cytosol was low, the labeling of glutamate in the bacteroids would seem indicative of malate metabolism rather than uptake of labeled glutamate, especially since the uptake of malate by isolated bacteroids is 3.4 times faster than that of glutamate (26).…”

Aspartate transport in Rhizobium meliloti

“…However, Salminen & Streeter (1991) showed that uptake measurements using 14C-labelled substrates may underestimate the true rate due to evolution of 14C02 during metabolism. To provide an estimate of the magnitude of this effect in our experiments, we measured 14C02 production as the decrease of total radioactivity from R. meliloti cultures taking up ['4C]succinate, as described in Methods.…”

Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules

“…The labelling of the glutamate pool in soybeans turned over slowly, suggesting a low rate of metabolism [44]. The high rate of glutamate synthesis has been attributed to inhibition of the 2-ketoglutarate complex of the tricarboxylic acid (TCA) cycle under microaerobic conditions due to a lowered NADH/NAD + ratio [46]. Consistent with this, mutation of the 2-ketoglutarate complex results in the synthesis and secretion of large quantities of glutamate by Rhizobium leguminosarum b.v. 6iciae [47].…”

“…More recent results suggest that amino acids must also be considered. Exposure of Bradyrhizobium japonicum bacteroids to 14 C-succinate, or whole pea and soybean nodules to 14 CO 2 , resulted in substantial synthesis of 14 C-glutamate, 14 C-aspartate and 14 C-alanine [43][44][45][46]. The labelling of the glutamate pool in soybeans turned over slowly, suggesting a low rate of metabolism [44].…”