Rhizobium sullae, a nitrogen fixing symbiotic bacterium, induces nodule formation on Hedysarum coronarium (Casella et al., 1984; Squartini et al., 2002). R. sullae strain HCNT1 is known to possess a truncated denitrification chain, harbouring only a gene (nirK) that encodes for a dissimilatory nitrite reductase (Toffanin et al., 1996). Since this strain does not obtain any obvious benefits from it, the conservation of this gene is currently unexplained. R. sullae HCNT1 was recently found able to grow on high concentrations of selenite and to reduce this oxyanion to the less toxic elemental selenium. Since its nirK-minus mutant grows weakly in the presence of even low selenite and it is not able to grow at higher concentrations, a defence mechanism for detoxification of selenite was proposed to explain one of the potential roles of nirK (Basaglia et al., 2007). Nitrite reductase activity of R. sullae was then evaluated in different environmental conditions, in the presence of nitrite, selenite or both. In this approach it was possible to clarify if nitrite reductase and selenite reductase are, in this strain, the same protein that works in different way, according to the substrate and to the atmospheric conditions, or they are two distinct proteins. Firstly, it was observed that selenite reduction activity occurred either under aerobic or anaerobic atmosphere, while the reduction of nitrite to NO can be attained only after a microaerobic preincubation, and that the presence of nitrite in the cultural medium together with selenite did not influence selenite reduction to elemental red selenium. On the contrary, the addition of selenite to cultures containing nitrite inhibits the production of nitrogen oxides. The presence of selenite has no effect on the expression level of nirK; mRNA analysis confirmed that nirK is always expressed, as the related transcript is present either during aerobic or under microaerophilic conditions. Moreover, the use in the culture of a specific chelator for a copper-containing nitrite reductase such as NirK, together with selenite, inhibited also the reduction of this oxyanion to elemental red form. This latest result indicates that the putative selenite reductase in R. sullae contains copper, like nitrite reductase. The above evidences seem to suggest that nitrite and selenite reductase could be the same protein; the enzyme studied could be a selenite reductase, rather than a nitrite reductase, which reduce selenite under aerobic condition and that becomes able to reduce also nitrite but only after a treatment under microaerobic condition. Therefore, protein purification was essential to better understand its properties. A recombinant-proteins approach (His-tag) allowed to purify a subunit of about 40 kDa encoded by nirK gene, that is a subunit which form the overall architecture of the homotrimeric enzyme (120 kDa). Preliminary in vitro experiments with the purified protein, for testing its ability to reduce nitrite and selenite separately, were undertaken. Although NirK showed the ability to reduce nitrite, medium-long term experiments are required to obtain the optimal conditions under which the protein could also reduce selenite.

A potential new role of nirK gene in Rhizobium sullae

BOTTEGAL, MARIANGELA;BASAGLIA, MARINA;POVOLO, SILVANA;CASELLA S.
2009

Abstract

Rhizobium sullae, a nitrogen fixing symbiotic bacterium, induces nodule formation on Hedysarum coronarium (Casella et al., 1984; Squartini et al., 2002). R. sullae strain HCNT1 is known to possess a truncated denitrification chain, harbouring only a gene (nirK) that encodes for a dissimilatory nitrite reductase (Toffanin et al., 1996). Since this strain does not obtain any obvious benefits from it, the conservation of this gene is currently unexplained. R. sullae HCNT1 was recently found able to grow on high concentrations of selenite and to reduce this oxyanion to the less toxic elemental selenium. Since its nirK-minus mutant grows weakly in the presence of even low selenite and it is not able to grow at higher concentrations, a defence mechanism for detoxification of selenite was proposed to explain one of the potential roles of nirK (Basaglia et al., 2007). Nitrite reductase activity of R. sullae was then evaluated in different environmental conditions, in the presence of nitrite, selenite or both. In this approach it was possible to clarify if nitrite reductase and selenite reductase are, in this strain, the same protein that works in different way, according to the substrate and to the atmospheric conditions, or they are two distinct proteins. Firstly, it was observed that selenite reduction activity occurred either under aerobic or anaerobic atmosphere, while the reduction of nitrite to NO can be attained only after a microaerobic preincubation, and that the presence of nitrite in the cultural medium together with selenite did not influence selenite reduction to elemental red selenium. On the contrary, the addition of selenite to cultures containing nitrite inhibits the production of nitrogen oxides. The presence of selenite has no effect on the expression level of nirK; mRNA analysis confirmed that nirK is always expressed, as the related transcript is present either during aerobic or under microaerophilic conditions. Moreover, the use in the culture of a specific chelator for a copper-containing nitrite reductase such as NirK, together with selenite, inhibited also the reduction of this oxyanion to elemental red form. This latest result indicates that the putative selenite reductase in R. sullae contains copper, like nitrite reductase. The above evidences seem to suggest that nitrite and selenite reductase could be the same protein; the enzyme studied could be a selenite reductase, rather than a nitrite reductase, which reduce selenite under aerobic condition and that becomes able to reduce also nitrite but only after a treatment under microaerobic condition. Therefore, protein purification was essential to better understand its properties. A recombinant-proteins approach (His-tag) allowed to purify a subunit of about 40 kDa encoded by nirK gene, that is a subunit which form the overall architecture of the homotrimeric enzyme (120 kDa). Preliminary in vitro experiments with the purified protein, for testing its ability to reduce nitrite and selenite separately, were undertaken. Although NirK showed the ability to reduce nitrite, medium-long term experiments are required to obtain the optimal conditions under which the protein could also reduce selenite.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2458516
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