CONICET | Buscador de Institutos y Recursos Humanos

Introduction The well-known PGPR A. brasilense produces phytohormones like auxin and other signalling molecules like nitric oxide (NO), both involved in plant root development and growth. A. brasilense produces NO mainly by denitrification of nitrate. Nevertheless, in media with ammonium as N source, it potentially could produce NO through a heterotrophic nitrification pathway coupled with denitrification. Objectives Our aims were to determine heterotrophic nitrification activity in A. brasilense Sp245 and NO production by this pathway coupled with denitrification. Materials and Methods A. brasilense Sp245 wild type (wt) and its periplasmic nitrate reductase mutant (Faj164) were grown in OAB liquid media with NH4+ as N source. At the end of log phase it was determined: i) the production of intermediaries of heterotrophic nitrification, hydroxylamine and nitrite (neutral red method), and nitrate (nitration of salicylic acid); ii) the production of nitrite induced by incubation for additional 2 h with hydroxylamine; iii) the NO production by electronic paramagnetic resonance (determined also at the middle of log phase); iv) the NO production induced by incubation for additional 2 h with different concentrations of hydroxylamine or nitrite with the NO specific fluorescent probe DAF-2DA. The presence of putatives enzymes of the heterotrophic nitrification pathway (ammonium monooxigenase AMO, hydroxylamine oxidoreductase HAO and nitrite oxidoreductase NitOR) in A. brasilense Sp245 genome was analyzed in silico and by PCR with specific primers (amoA). Finally, the expression of amoA was determined at middle and end of log phase by semiquantitative RT-PCR Results In cultures growing with NH4+, both wt and Faj164 produced 3 to 4 μM hydroxylamine and 2.8 to 3 μM nitrite at the end of log phase. The wt strain also produced 36.4 μM nitrate. The production of ca. 3 μM nitrite was also induced by incubation with hydroxylamine. At the same time, both strain produced NO, showing the Faj164 mutant a higher production (ca. 9 nmol NO.g-1 bacteria) than the wt (ca. 4 nmol NO.g-1 bacteria). On the contrary, at the middle of log phase only Faj164 produced NO. Incubation with hydroxylamine or nitrite induced NO production by wt and Faj164 in a dose-dependent manner. In the genome of A. brasilense Sp245 there are two sequences annotated as putatives AMO (accession number: CCC99091.1 and NC_016617.1), which show homology with several putatives AMO of other heterotrophic microorganisms and have domains associated with AMO in the families pfam05145, TIGR03082 and COG3180. We could not identify HAO or NitOR sequence genes. The amplified amo gene from A. brasilense wt showed 71% nucleotide identity with amo the gene of Rhodopirillum centenum SW. The expression of the gene amoA was observed in cultures of wt and Faj164 at the middle and the end of log phase. The transcript levels were similar for wt and Faj164 at the end of log phase, but 75% higher for Faj164 than wt at the middle of the log phase. Conclusions The results strongly suggest that A. brasilense Sp245 is capable of performing the heterotrophic nitrification pathway coupled with denitrification. In cultures with ammonium the microorganism produces the intermediaries of heterotrophic nitrification, hydroxylamine, nitrite and nitrate, and the intermediary of denitrification NO. Besides, hydroxylamine induces the synthesis of nitrite, and hydroxylamine and nitrite induce the production of NO. In the genome of A. brasilense Sp245 there are two putatives AMO, one of them, at least, is a functional gene and is expressed in cultures with ammonium. The absence of sequences for HAO and NitOR suggests that these steps are accomplished by other enzymes as it was reported for other heterotrophic nitrifiers. The activity of these pathways producing NO can be relevant for the promoting effect of A. brasilense on root growth and development.

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