Effect of the Inorganic Nitrogen Source in the Expression of Nitrite Reductase (NirA) in <i>Thermosynechococcus elongatus</i>BP-1

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.

Substitution of Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum

Even though the nitrate assimilation operon has been extensively studied in Phormidium laminosum, some aspects still remain unclear. The genetic manipulation of this cyanobacterium is problematic that hinders the elucidation of further aspects of nitrogen metabolism. To circumvent this, Thermosynechococcus elongatus BP-1 was selected as a surrogate host and its nirA gene was substituted by the homologous gene of P. laminosum. This process, based on Long Flanking Homology Polymerase Chain Reaction and the natural competence of T. elongatus BP-1, required an intermediate T. elongatus BP-1 ΔnirA::kat mutant, which carries a gene encoding a thermostable kanamycin nucleotidyl transferase in place of nirA_Te. In the presence of nirA_Pl, nirA defective mutants of T. elongatus BP-1 recovered the ability to grow with nitrate as the sole nitrogen source, and showed a phenotype similar to that observed in wild-type cells. The procedure could be useful to substitute other genes from T. elongatus BP-1 with the homologues from P. laminosum in order to study this particular operon. Furthermore, it may be used as a general tool to explore phenotypic changes due to the exchange of a single gene between cyanobacteria.