CHARACTERIZATION OF A GLUTAMINE SYNTHETASE II MUTANT IN Bradyrhizobium diazoefficiens USDA110

LAMELZA, F.; LÓPEZ, M.F.; HEGEL, V.; LÓPEZ GARCÍA, S.L.

Mar del Plata

Congreso; X CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL- SAMIGE 2014; 2014

Sociedad Argentina ed Microbiología General- SAMIGE

Bradyrhizobium diazoefficiens exists as a free-living organism, growing at the expense of soil nitrogen, or as a symbiont, reducing dinitrogen to ammonia for itself and its soybean host.Particularly, in free-living rhizobia, N assimilation proceeds through the high-affinity glutamine-synthetase (GS) - glutamate-synthase (GOGAT) cycle. Bacteria of the Rhizobiaceae, including Bradyrhizobium diazoefficiens USDA110, the subject of this study, are distinct from other bacteria in that they have at least two isoforms of GS: GSI y GSII. The most striking difference between GSI (encoded by glnA) and GSII (encoded by glnII) is how these enzymes are controlled. The activity of GSI is altered post-translationally by adenylylation but the level of the protein remains relatively constant. In contrast, GSII synthesis is under the control of the NtrBC system that responds to the nitrogen and carbon sources.Both control systems depend on the relative amounts of intracellular α-cetogutarate and glutamine. A high α-cetoglutarate/glutamine ratio, signal of N limitation, activates a Nitrogen Stress Response cascade promoting deadenylylation of GSI and the synthesis of GSII to increase NH4+ assimilation. These two control levels operating on different GS enzymes might help the rizobia become highly efficient for scavenging, in their natural environments, the low levels of N that are needed to establish a productive nitrogen-fixation symbiosis.In order to advance in this topic we generated a mutant in glnII, LP4169 strain, by a double homologous recombination strategy. The first step to characterize this mutant strain was analyzing the growth in Evans media with a low concentration of N source (0.1 µM of NH4Cl) and in Evans cero nitrogen (N0, 0.02 µM of NH4Cl). In both cases it was found that there was no significant difference in the OD500nm between LP4169 and the wild type (WT) strain. Nevertheless, when the colony forming unit per milliliter (cfu/ml) were analyzed, it was found that at stationary state (after 7 days of growth) LP4169 reduces drastically the cfu/ml and then (at the 11th day approximately) the number of cfu/ml increases again. This happens both in low N and N0 media so, taking this into account, it could be thought that the mutant is not able to survive in a proper way, as the WT strain does, during stationary state.To increase our results, the growth of LP4169 in different N sources like nitrate and glutamate (0.02%) was studied. Unfortunately, no differences could be noticed in the growth between the mutant and the WT strain.Finally, we studied GS activity through the g-glutamyl transferase assay. The results showed that total GS activity was the same for both WT and mutant strains but GSI activity was lower in LP4169 strain. These results could suggest that another enzyme is involved in this system but we need to carry on more studies to elucidate it.