Nitrite reductase (NirA, EC 1.7.7.1) from the thermophilic, unicellular, non-N2-fixing cyanobacte-rium Thermosynechococcus elongatus BP-1 has been cloned and expressed in Escherichia coli. Analysis by SDS-PAGE of the ...Nitrite reductase (NirA, EC 1.7.7.1) from the thermophilic, unicellular, non-N2-fixing cyanobacte-rium Thermosynechococcus elongatus BP-1 has been cloned and expressed in Escherichia coli. Analysis by SDS-PAGE of the pure recombinant protein (His6NirA) showed two protein bands, one of 58 kDa (corresponding to the theoretical His6NirA molecular mass) and another of 44 kDa. Western blotting and mass spectrometry analyses confirmed that the 44 kDa protein resulted from proteolysis of the intact His6NirA, and suggested the existence, at the C-terminal domain of the 58 kDa form, of a region particularly sensible to proteolysis or accessible to proteases. A sample of both forms of His6NirA was used to obtain anti-NirA polyclonal antibodies. These antibodies were used to assess, by SDS-PAGE followed by Western blotting, the in vivo expression of NirA in wild-type cells of T. elongatus BP-1 growing in cultures with nitrate, nitrite or ammonium which were inoculated with cells grown with different nitrogen sources. These analyses revealed that protein bands corresponding to the complete (58 kDa) and truncated (44 kDa) forms of NirA can also be detected in solubilized cells. Moreover, the presence of each of these forms depended on the nitrogen source used to grow cells. Thus, expression of the complete NirA generally predominates in cells growing in medium with nitrate or nitrite. However, the truncated form prevails in cells grown in nitrate or nitrite and then transferred to medium with ammonium. The fact that the patterns of in vivo expression of NirA are different depending on the nitrogen source used possibly relies on a post-translational regulatory mechanism by proteolysis.展开更多
文摘Nitrite reductase (NirA, EC 1.7.7.1) from the thermophilic, unicellular, non-N2-fixing cyanobacte-rium Thermosynechococcus elongatus BP-1 has been cloned and expressed in Escherichia coli. Analysis by SDS-PAGE of the pure recombinant protein (His6NirA) showed two protein bands, one of 58 kDa (corresponding to the theoretical His6NirA molecular mass) and another of 44 kDa. Western blotting and mass spectrometry analyses confirmed that the 44 kDa protein resulted from proteolysis of the intact His6NirA, and suggested the existence, at the C-terminal domain of the 58 kDa form, of a region particularly sensible to proteolysis or accessible to proteases. A sample of both forms of His6NirA was used to obtain anti-NirA polyclonal antibodies. These antibodies were used to assess, by SDS-PAGE followed by Western blotting, the in vivo expression of NirA in wild-type cells of T. elongatus BP-1 growing in cultures with nitrate, nitrite or ammonium which were inoculated with cells grown with different nitrogen sources. These analyses revealed that protein bands corresponding to the complete (58 kDa) and truncated (44 kDa) forms of NirA can also be detected in solubilized cells. Moreover, the presence of each of these forms depended on the nitrogen source used to grow cells. Thus, expression of the complete NirA generally predominates in cells growing in medium with nitrate or nitrite. However, the truncated form prevails in cells grown in nitrate or nitrite and then transferred to medium with ammonium. The fact that the patterns of in vivo expression of NirA are different depending on the nitrogen source used possibly relies on a post-translational regulatory mechanism by proteolysis.
