Carbon and nitrogen metabolism in Rhizobium

We began our studies in the determination of the cycle of the glutamine synthetase in the fungus Neurospora crassa and the enzymes that participate in the glutamate-glutamine interconversion in N. crassa and in the new model organism Rhizobium etli .

R. etli does not present the glutamate dehydrogenase (GDH) gene. Together with the GS-GOGAT pathway, GDH constitutes the enter point of nitrogen (as ammonium) to all the cells. We expressed the GDH activity in R etli in nodule by means of specific promoters from the symbiotic stage, and it caused a strong inhibition of the nitrogen fixation, demonstrating that the ammonium assimilation and the fixation are incompatible processes.

We reported, for the first time for Rhizobium , the characterization of GOGAT enzyme. GOGAT synthesizes glutamate, and along with the Glutamine synthetase (GS), are responsible of the ammonium assimilation. The mutation of GOGAT in symbiosis simultaneously produced greater nitrogen contribution to the plant and nitrogen fixing capacity. The GS/GOGAT activity is fundamental in the nitrogen metabolism of the bacteria and it is closely related to the carbon metabolism.

We found that R. etli presents two GS´s that are specifically expressed according to the ammonium availability. Also, we reported that the GS-II sequence can serve as a phylogenetic marker in these organisms.

R. etli does not grow in subcultures because it enters into a "fermentative" metabolic state. Since the bacterium is an aerobic-strict organism, this finding had an extreme importance. The "fermentative" state represents profound metabolic changes, such as: alteration of some enzymatic activities of the Krebs (or Tricarboxilic Acid, TCA) cycle because two of their key enzymes have low activities;
also it accumulates a reserve polymer named PHB (poly-beta-hydroxybutyrate), it excretes a great amount of glutamate and also precipitates in cell conglomerates. This situation is very
similar to that within the nodules. The metabolic change is mainly due to the low synthesis and oxydation of two vitamins, thiamin and biotin, that are cofactors of the enzymes that disappear in the TCA cycle.

In free living and symbiotic conditions, R. etli produces the polymer PHB that represents a carbon and reducing power reserve. Apparently, the deviation of carbon and reducing power to the PHB synthesis allows that in microaerobiosis (as in the nodule) Rhizobium can maintain an active carbon metabolism. We obtained a mutant that does
not accumulate the polymer and we found that it excreted great amounts of organic acids and that the proportion of NADH/NAD+ was increased. It is, the mutation produced a diminished oxydative capacity in the cell and it was filled of reducing power. In symbiosis, the mutation
produced a better nitrogen fixation capacity possibly by deriving the excess of carbon to the energy-consuming process of the nitrogenase.

Functional Genomics of Prokaryotes Program