CarbonMetabolism EnzymesofRhizobium meliloti Cultures and Bacteroids andTheirDistribution within Alfalfa Nodulest

Bacteria ofthegenusRhizobium canformsymbioses with certain members ofthefamily Leguminosae, resulting inthe reduction ofatmospheric dinitrogen toammonium.The Rhizobium bacteria invade theroots viainfection threads formed bytheplant cells. Theplant rootcells undergo rapid cortical division toformatumorlike growth referred toasa nodule. During nodule development, thebacteria undergo morphological andphysiological changes that leadtoaform, called abacteroid, capable ofsymbiotic dinitrogen fixation. Theenergy tosupport symbiotic dinitrogen fixation isprovided bycarbon metabolites fromtheplant rootnodule cells. Thecarbon metabolism ofRhizobium meliloti bacteroids is notaswellcharacterized asthatofotherstrains ofRhizobiumsymbionts. Herewe report acomparative metabolic study ofalfalfa rootnodule bacteroids, free-living cultures of R.meliloti grownonseveral carbon substrates, andtheplant nodule cytosol. R.meliloti 102F51 (Nitragin Co.,Milwaukee, Wis.) was grownonaminimal single-carbon-source mediumthat consisted ofthefollowing ingredients (perliter): 0.36g of KH2PO4, 1.4gofK2HPO4, 0.2gofNaCl,2.5mgofH3B03, 1mgofCUSO4, 2mgofNa2MnO4, 0.27gofNH4Cl,0.25g ofMgSO4,15mgofdisodium EDTA,10mgofMnSO4,1mg ofCoCI2, 6.5mgofFeCl3, 1mgofNiCl2, 1mgofZnSO4,1 mgofthiamine, 2mgofcalcium pantothenate, 0.01mgof biotin, and5mM carbon source (mannitol, glucose, sucrose, succinate, malate, oracetate). Themediumwasadjusted to pH7.0before autoclaving. R.meliloti cultures inthemid-tolate exponential phase of growth werecentrifuged at8,000 x gfor10min,washed twice in50mM potassium phosphate buffer (pH7.8), and suspended in20mlof50mM potassium phosphate buffer (pH7.8) containing 0.2%(vol/vol) 2-mercaptoethanol