Bacterial transport and transformation of nitrate and nitrite in the nitrogen cycle

Atmospheric nitrogen gas is very stable but it can be combined with hydrogen, either in an industrial chemical process or biologically to give ammonia. The latter can be used by plants and bacteria to provide the nitrogen content of proteins and DNA. Some soil bacteria can effectively react ammonia with oxygen to give nitrate and nitrite. Other bacteria can reduce nitrite and nitrate back to nitrogen gas, thus completing a nitrogen cycle. A newly discovered variant of this process is catalysed by novel bacteria which can react to ammonia with nitrite to give nitrogen gas. There is no chemical precedent for this and we wish to discover how this works. Nitrate and nitrite can also be used by plants and bacteria for protein and DNA production. Nitrate and nitrite are negatively charged and water-loving chemicals. These properties raise fundamental questions as to how these chemical species pass into and out of biological cells. Such cells have a surrounding barrier that has properties that normally oppose the passage of nitrate and nitrite. We intend to increase considerably understanding as to how such passage occurs.

The first step in dentrification in most species of bacteria is movement of nitrate from the external medium to its site of reduction by a membrane-bound enzyme whose active site faces the cytoplasm. The reaction product nitrite then has to be exported from the cell to the periplasm where it is reduced. The movement of the hydrophilic nitrate and nitrite across the membrane is little understood, in part because there is no suitable radioisotope with which to trace nitrate movement and also because separating putative transport events from metabolism is not easy. Entry of nitrate has to be understood in the context of a membrane potential, approximately 180mV negative on the cytoplasmic side. Nitrate and nitrite also has to enter bacteria for purposes of assimilation ¿ again there is the problem of entry against the membrane potential. In previous work with Paracoccus provided evidence for a dual function protein with separate nitrate/proton symporter and nitrate/nitrite antiporter activities. Recent genome analysis for this organism shows the presence of very probably nitrate/proton and nitrite/proton symporters associated with nitrogen assimilation. The main aim of this proposal is to perform a series of judicious switch of function experiments in which dentrification will be supplied by the assimilatory transporters and vice versa. Through such experiments the energetics of each transporter will be established. In a parallel programme we will conduct tests of our recent hypothesis as to the biochemistry of the novel Anammox organism in which ammonia is oxidised anaerobically by nitrite to give nitrogen gas.