CHARACTERIZATION OF A GLUTAMINE SYNTHETASE II MUTANT IN Bradyrhizobium diazoefficiens USDA110

Mar del Plata

Congreso; X Congreso Argentino de Microbiología General SAMIGE; 2014

Sociedad Argentina de Microbiología General (SAMIGE)

CHARACTERIZATION OF A GLUTAMINE SYNTHETASE II MUTANT IN Bradyrhizobium diazoefficiens USDA110 Rhizobia are Gram-negative soil bacteria which fix nitrogen during symbiosis with legume plants. This Biological Nitrogen Fixation is carried on within specific structures, called nodules, in legumes roots. Our research group is focused on the symbiosis of the soybean legume and its symbiont Bradyrhizobium diazoefficiens USDA110. Up to now, we have advanced in the study of nutritional effects of nitrogen in B. diazoefficiens cultures in free-living bacteria as well as in different stages of the symbiotic interaction. Particularly, is known that in free-living rhizobia N assimilation proceeds throughout the high-affinity glutamine-synthetase (GS) -glutamate-synthase (GOGAT) cycle. B. diazoefficiens, as many other rhizobia, has at least two isoforms of GS: GSI y GSII. The most striking difference between GSI and GSII is how the enzymes are controlled in response to N availability. The activity of GSI is altered post-translationally, but the level of the protein remains relatively constant. In contrast, GSII (encoded by the glnII gene) is induced when N is limited. Both GS activities depend on the relation between a cetogutarate/glutamine (intracellular relation C/N). When this relation is high, because the N is low, a Nitrogen Stress Response regularity cascade is triggered, the expression of GSII is induced and both GSs are activated. As we are interested in the comprehension of the role of these two enzymes in N metabolism we generated a mutant strain in glnII, named LP4169, by a double homologous recombination in which we deleted the most part of the gene of interest. The first step to characterize the mutant strain was to analyze the growth in Evans media with a low concentration of N source (0.1 μM of NH4Cl) and in a media Evans cero nitrogen (N0 ) with 0.02 μM of NH4Cl. In both cases we found that there was no significant difference in the OD500nm between the mutant LP4169 and the wild type (WT) strain. But, when we analyzed the colony forming unit per milliliter (cfu/ml) we found that at a stationary state (after 7 days of growth) LP4169 reduce drastically the cfu/ml and then (at the 11th day approximately) increase again the number of cfu/ml. This happens both in low N and N0 media and makes us think that the mutant is not able to survive in a proper way as the WT strain does during a stationary state. To increase our results, we also studied the growth of LP4169 in other N sources like nitrate or glutamate using a low concentration of them (0.02%). Unfortunately, we could not find any difference in the growth between the mutant and the WT strain. Finally, it was also studied GS activity using the g-glutamyl transferase assay. The results showed us the total GS activity was the same for both WT and mutant strains, but the values of the heat sample (that measure only GSI activity) was much lower in LP4169. This result was surprising for us and we need to carry on more studies to elucidate how these enzymes are acting in this mutant.