CHARACTERIZATION OF NtrY, A SENSOR PROTEIN OF A TWO COMPONENT SYSTEMS IN Bradyrhizobium diazoefficiens USDA 110.

HEGEL, VALERIA A.; LÓPEZ, MARÍA F.; LAMELZA, FLORENCIA; LÓPEZ GARCÍA, SILVINA L.

Córdoba

Congreso; XI Congreso Argentino de Microbiología General- SAMIGE 2015; 2015

Sociedad Argentina de Microbiología General (SAMIGE)

Rhizobia are soil bacteria able to establish a nitrogen-fixing symbiosis within the root nodule cells of legume host plants.The transition from the free-living to the symbiotic state is accompanied by drastic changes in bacterial metabolism, eventually leading to the formation of bacteroids specialized fornitrogen (N) fixation.Our research focuses on the symbiotic relation between soybean legume and Bradyrhizobiumdiazoefficiens USDA 110. We have already advanced in the study of nutritional effects of N in B.diazoefficiens cultures in free-living bacteria as well as in different stages of the symbiotic interaction.Moreover, we have demonstrated that N-limited cultures stimulate early events of symbiosis and N2 fixation.The major aim of our work is the comprehension of the role of the regulators involved in N metabolism.One of these regulators is NtrBC, a two-component system (TCS) that senses the intracellular variations of C/N ratio and controls the expression of some genes implicated in ammonium assimilation. When the C/N ratio is high (under nitrogen starvation) NtrB (sensor protein)phosphorylates NtrC (response regulator) that in turn, activates transcription of genes required for nitrogen uptake and metabolism.B. diazoefficiens, as other alphaproteobacteria, has a second two-component system named NtrY-NtrX located immediately downstream of the nifR3-ntrB-ntrC operon and whose function remains unknown. NtrY exhibits a high degree of homology with sensor histidine kinase proteins while NtrX with response regulator proteins. This system has been reported in Azorhizobium caulinodans,Azospirillum brasilense, Rhodobacter capsulatus and Brucella spp. to be involved in nitrogen metabolism, symbiotic nodulation and low-oxygen response.Taking into account that nifR3-ntrB-ntrC is located next to ntrY-ntrX, we studied if these genes belong to the same operon analysing by RT-PCR the intergenic region. These results demonstrated that ntrY-ntrX are located in a different operon than ntrB-ntrC.In order to study the role of NtrY/X in N metabolism, we generated ntrY null mutant strain (namedLP4489) in B. diazoefficiens USDA 110. This mutant was generated by crossover-PCR so as to avoid polar effect in ntrX. We compared the growth of this mutant with the wild type strain in Evans media with high concentration of N (20 mM of NH4Cl) and in Evans media with low concentration of N source (0.1 mM of NH4Cl). In both cases we found that there was no significant difference in the OD 500nmneither in the colony forming unit per mililiter (cfu/ml). However, it is necessary to analyze the growth of this mutant in other N sources.Finally, swimming assays were performed in AG media with 0.3% w/v of agar. Unexpectedly, LP4489 strain was less motile than the parent USDA 110. These results could suggest that NtrY has an effect on bacteria motility. Indeed, more studies are required to investigate the role of this sensor protein in B. diazoefficiens.