Carbon Metabolism of Azospirillum brasilense

To understand the nature of bacteria-plant associations both plant and bacterial metabolism studies are important. Research into the biochemistry of photosynthesis has revealed that besides the reductive pentose phosphate cycle, many grasses also possess the C-4 dicarboxylic acid cycle. Grasses possessing this pathway utilize their available nitrogen more efficiently in producing dry matter and fixing atmospheric C02. The general importance of the C-4 dicarboxylic acid pathway in tropical grasses had previously been widely accepted. Azospirillum spp. are thought to be mainly responsible for the nitrogen fixation that occurs in grasses. The preference of these bacteria for malate and other organic acids as the carbon source of choice has been demonstrated in several studies. Malate is the primary product of photosynthesis in some of the C-4 grasses and it has been hypothesized that malate accumulation in the plant leaf leads to its transport into the roots, favoring nitrogen fixation. Child et al demonstrated the induction of nitrogenase by S. lipoferum and Rhizobium spp. grown in direct association with plant cell tissue culture. They showed that the plant tissue culture supplied TCA cycle intermediates needed to induce nitrogenase in both organisms. The positive chemotactic response of this bacterium to arabinose, galactose, fructose, malate, pyruvate, succinate, glutamate, leucine, and alanine was recently demonstrated. The above compounds are found in plant root exudates. Among other substances occurring in plant root exudates is lactate which is one of the best growth substrates for A. brasilense. Chemotaxis may play a role in the association of this bacterium with the roots of appropriate grasses. Kyung et al demonstrated a great increase in root exudation by sorghum seedlings inoculated with A. brasilense and Azotobacter vinelandii. This indicates a close association of these nitrogen fixers with certain plant roots. Little attention has been paid to metabolic pathways in A. brasilense not concerned with nitrogen fixation. Okon et al measured oxygen uptake by cell suspensions and crude cell-free extracts of S. lipoferum following the addition of Krebs cycle intermediates, sugar phosphates, and sugars. Their results suggested that the bacterium possesses an operative Krebs cycle, but that glycolytic and pentose phosphate pathways are only weakly functional. An oxidative pathway by which L-arabinose is converted to 2-ketoglutarate has been demonstrated in crude cell-free extracts of A. brasilense by Novick and Tyler. In the present study I examined carbon metabolic pathways in A. brasilense by measuring certain enzyme activities in crude cell free extracts obtained from cells grown singly on gluconate, glycerol, lactate, and malate as growth substrates. Two mutants CW-1 and CW-2, which grow very poorly on either malate, lactate, or glycerol, were also studied to determine the phenotypic site of mutation, to study regulation of carbon metabolism, and to ascertain the role of carbon metabolism enzymes in A. brasilense