INVESTIGADORES
MASSA Gabriela Alejandra
congresos y reuniones científicas
Título:
Heterotrophic nitrification coupled with denitrification in Azospirillum brasilense Sp245
Autor/es:
MOLINA FAVERO CELESTE; ARRUABARRENA DI PALMA ANDRÉS; MASSA GABRIELA ALEJANDRA; AMENTA MELINA; LAMATTINA LORENZO; CREUS CECILIA
Lugar:
La Falda
Reunión:
Workshop; II Taller Latinoamericano Sobre Rizobacterias Promotoras de Crecimiento Vegetal; 2014
Resumen:
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.