A possible role of nirK gene in Rhizobium sullae

Background: Rhizobium sullae HCNT1 does not obtain any obvious benefits from its truncated denitrification chain, so that the conservation of the unique gene of denitrification that encodes for a dissimilatory nitrite reductase is apparently inexplicable. Recently, a defence mechanism for detoxification of selenite was proposed. Objectives: Investigation on the possible meaning of nirK gene in Rhizobium sullae and in depth examination on the nature of the encoded enzyme. Methods: Physiological responses to the presence of selenite was determined by growth in liquid and solid medium, by gaschromatograph analyses and scanning electron microscopy. Molecular investigation were carried out by RT-PCR and SDSPAGE analyses. Results: 1. Selenite reduction activity occurred either under aerobic or anaerobic atmosphere, while the reduction of nitrite to NO can be attained only after a preincubation under microaerobic conditions. 2. 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. 3. The presence of selenite has no effect on the expression level of nirK: cDNA analysis confirmed that nirK is always expressed, as the transcript is present either during aerobic or under microaerophilic conditions. 4. 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). Conclusions: Previous findings suggested that nitrite and selenite reductases could be the same protein. The enzyme here studied seems to be more likely a selenite reductase, able to reduce selenite under any condition tested, and that becomes able to also reduce nitrite, but only after a microaerobic incubation.