Substitution of Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum — Oak Academic Publishing
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Substitution of Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum
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Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Bilbao, Spain
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2 Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Bilbao, Spain
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 Δ nirA :: 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.
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