Resumo

Gluconacetobacter diazotrophicus is endophytic bacteria of sugarcane know by your benefit on growth and nitrogen accumulation in this plant, attributed principally to biological nitrogen fixation. Screening of a Tn5 mutant library of Gluconacetobacter diazotrophicus strain PAL5 led to the identification of a mutant (Gdiaa31) with a single Tn5 insertion in the predicted promoter region of a tonB homologue. In Gram-negative bacteria, tonB genes are required for formation of the TonB-ExbB-ExbD protein complex, which is responsible for energization of membrane receptors involved in uptake of siderophore-iron complexes and other cationic molecules such as vitamin B12. The expression of genes involved in iron acquisition is strictly regulated by the Ferric-uptake regulator protein (Fur). To investigate whether a mutation in the promoter region of gene tonB influenced iron uptake, real-time PCR quantification was applied to measure transcript levels of GdtonB, GdexbB, GdexbD1, GdexbD2 and Gdfur. G. diazotrophicus PAL5 wild-type and Gdiaa31 strains were grown in liquid medium with or without addition of 37 µM of FeCl3. Total RNA was isolated with Trizol and used for cDNA synthesis with the Superscript III First-strand synthesis kit. The qPCR analysis revealed that in Gdiaa31 transcript levels of GdtonB, GdexbB, GdexbD1 and GdexbD2 were always higher than in the same treatments with the wild-type. This suggests that these genes are regulated in a common manner. In contrast, transcript levels of the Gdfur gene in Gdiaa31 were not affected when compared to the wild-type in the absence of iron, suggesting a distinct regulation mechanism. This observation corroborates with our hypothesis that the Fur protein could be a regulator of the GdtonB, GdexbB, GdexbD1 and GdexbD2 genes. Also, the Fur protein bound to iron ions binds to the fur promoter and acts as an auto-inhibitor of fur transcription. Therefore, it could be expected that in Gdiaa31 after addition of extracellular iron, this autorepression is alleviated. This hypothesis is in accordance with the observation that Gdfur transcript levels are diminished in the wild-type in the presence of iron, which was not the case in Gdiaa31.