Carbon metabolism in free-living and bacteroid forms of cowpea Rhizobium strain NGR234

Growth of NGR234, a fast-growing cowpea Rhizobium. was investigated on a variety of carbon sources. The results indicate that this organism has the capacity to grow on a wide range of sugars. polyols, organic acids, amino acids, and aromatic acids as sole carbon source. Glucose, C4-dicarboxylic acids, and glutamate supported the fastest growth, while 4-hydroxybenzoate resulted in the slowest growth. Oxidation studies indicated that NGR234 possessed constitutive oxidation systems for glucose, glutamate, or aspartate and inducible systems for the other substrates investigated. Cells grown on mannitol or sucrose had the capacity to oxidize fructose, and cells grown on fumarate or malate or succinate were capable of oxidizing the other two of these C4-dicarboxylic acids. Bacteroids isolated from snake bean nodules were able to oxidize C4-dicarboxylie acids, with succinate supporting the highest O2 consumption, but they failed to oxidize the other substrates studied. The capacity of these bacteroids to oxidize C4-dicarboxylic acids was about 50% of those found in cultured cells. Looking at the ability of NGR234 cells to transport substrates revealed that arabinose was the only substrate transported into the cell via a constitutive transport system, the transport of all other substrates examined being inducible. These transport systems were inhibited by azide (2 mM) , carbonyl cyanide m-chlorophenylhydrazone (CCCP, 0.025 mM), and 2,4-dinitrophenol (DNP, 1 mM). Arsenate (5 mM) was slightly or moderately inhibitory to the transport systems. Isolated bacteroids had the capacity to take up arabinose or succinate, but failed to take up fructose or glucose or ribose or sucrose, suggesting the possibility that such sugars were not available to the bacteroids in sufficient concentrations to induce their transport systems. Further studies on enzyme systems indicated that cell-free extracts derived from sugar-grown cells (except arabinose-grown) contained enzymes of the Entner-Doudoroff (ED) pathway, the pentose phosphate (P?) pathway and the TCA cycle, but lacked phosphofructokinase (PFK), the key enzyme of the Embden-Meyerhof-Parnas (EMP) pathway. Cells grown on arabinose or succinate possessed low activities of 6-phosphogluconate dehydrogenase, the key enzyme for entry to the pentose phosphate pathway. Such cells lack significant activities of invertase, fructokinase, glucose-6-phosphate dehydrogenase or the Entner-Doudoroff enzymes. The regulation of these catabolic enzymes suggested a way to use their activities in bacteroids as a probe of the environment of the bacteroid in the nodules. Extracts prepared from bacteroids of snake bean nodules separated on Percoll gradients contained only low or zero activities of invertase, fructokinase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes, and 6-phosphogluconate dehydrogenase. All of the TCA cycle enzymes were present at significant levels. These results suggested that sugars were not entering these bacteroids in quantities sufficient to induce these enzymes. Control experiments showed that dicarboxylic acids did not prevent induction by sugars of the ED enzymes or glucose-6-phosphate dehydrogenase. Leakage of such enzymes from the bacteroids was excluded because bacteroid marker enzymes were not detectable outside the cells. Taken together, these experiments suggest that sugars do not enter the NGR234 bacteroid in any significant quantities, and that the peribacteroid membrane may be an important regulatory device by which the plant may control the type and quantity of carbon compounds reaching the bacteroid