Substitution of Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum
Affiliation(s)
. 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.
. 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.
ABSTRACT
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.
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.
Cite this paper
M. Buxens, J. Serra and M. Llama, "Substitution of Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum," Advances in Microbiology, Vol. 3 No. 6, 2013, pp. 69-79. doi: 10.4236/aim.2013.36A009.
M. Buxens, J. Serra and M. Llama, "Substitution of Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum," Advances in Microbiology, Vol. 3 No. 6, 2013, pp. 69-79. doi: 10.4236/aim.2013.36A009.
References
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http://dx.doi.org/10.1007/BF00021199 [12] S. Naithani, J. M. Hou and P. R. Chitnis, “Targeted Inactivation of the psaK1, psaK2 and psaK3 Genes Encoding Subunits of Photosystem I in the Cyanobacterium Synechocystis sp. PCC 6803,” Photosynthesis Research, Vol. 63, No. 3, 2000, pp. 225-236.
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