Characterization of the gene encoding nitrite reductase and the physiological consequences of its expression in the non-denitrifying Rhizobium 'hedysari' strain HCNT1

Rhizobium ''hedysari'' HCNT1 is an unclassified rhizobium which contains a nitric oxide-producing nitrite reductase but is apparently incapable of coupling the reduction of nitrite to energy conservation. The gene encoding the nitrite reductase, nirK, has been cloned and sequenced and was found to encode a protein closely related to the copper-containing family of nitrite reductases. Unlike other members of this family, nirK expression in HCNT1 is not dependent on the presence of nitrogen oxides, being dependent only on oxygen concentration. Oxygen respiration of microaerobically grown Nir-deficient cells is not affected by concentrations of nitrite that completely inhibit oxygen respiration in wild-type cells. This loss of sensitivity suggests that the product of nitrite reductase, nitric oxide, is responsible for inhibition of oxygen respiration. By using a newly developed chemically modified electrode to detect nitric oxide, it was found that nitrite reduction by HCNT1 produces significantly higher nitric oxide concentrations than are observed in true denitrifiers. This indicates that nitrite reductase is the only nitrogen oxide reductase active in HCNT1. The capacity to generate such large concentrations of freely diffusible nitric oxide as a consequence of nitrite respiration makes HCNT1 unique among bacteria.