Role of the GlnK protein (PII) in the nitrogen stress response circuit in Bradyrhizobium diazoefficiens USDA 110

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

Córdoba

Congreso; Congreso Argentino de Microbiología General SAMIGE; 2015

Symbiosis between rhizobia and legume plants is a model of nutritional complementation. Plants reduce atmospheric CO2 and provide carbon compounds to the bacteria inside the nodule while rhizobia use these compounds and fix atmospheric N2 to ammonia, a nitrogen (N) source that plants can metabolize. Bradyrhizobium diazoefficiens USDA 110, our bacteria of study, is an alphaproteobacterium that is able to exist as a free-living organism growing at the expense of soil nutrient, or in symbiotic association with soybean plants. This symbiotic interaction requires limited amounts of soil N sources to take place. However, agricultural soils are N fluctuating environments and free-living bacteria have developed specific mechanism to acquire and metabolize N in an efficient way in order to survive. The Nitrogen Stress Response (NSR) circuit is an example of these mechanisms and it has been studied in rhizobia such us Ensifer meliloti and other bacteria like Escherichia coli and Rhodopseudomonas palustris. Nevertheless, we don´t know anything about this response in B. diazoefficiens USDA 110. In most microorganisms, the core elements of the NSR regulation cascade include a bifunctional uridylyltransferase/uridylyl-cleavage enzyme GlnD (glnD) and two PII proteins: GlnB (glnB) and GlnK (glnK). The uridylylation state of these PII proteins is regulated by the a- cetoglutarate/glutamine intracellular ratio. In nitrogen-starved cells, when theconcentration of a-ketoglutarate is high, GlnD uridylylates PII proteins which, in turn, activate the bacterial NSR, leading to more efficient ammonia assimilation by increasing glutamine synthetase (GS) activity. Using bioinformatics tools we found homologous genes in B. diazoefficiens USDA 110 that codify two forms of PII proteins: one copy of glnB and two copies of glnK(glnK1 and glnK2). Generally, rhizobia have only one copy of glnK, so we proposed to find out whether both copies are functional as well as to understand the role of these PII proteins in the complex nitrogen metabolism. To achieve our objective, null mutants of glnK1 (DglnK1 strain) and glnK2 (DglnK2 strain) were generated. Although in other rhizobia the deletion of glnK don´t produce striking phenotypes, it seems to be different in B.diazoefficiens. The results obtained when GS activity was assayed led us to think that both copies of GlnK are functional in our bacteria. Moreover, preliminary growth studies of DglnK1strain showed a significant difference when a nitrate source was added to the culture after ammonium starvation. The DglnK1 strain was not able to grow as well as the wild type strain with this N source and the final OD500nm was lower. This result suggests that GlnK proteins could be involved, not only in ammonium assimilation but also in nitrate metabolism or uptake; so it is necessary to continue studying the performance of both mutant strains in media with nitrate as N source.