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.
Highlights
Nitrate assimilation in cyanobacteria involves three main steps
Thermosynechococcus elongatus BP-1 was selected as a surrogate host and its nirA gene was substituted by the homologous gene of P. laminosum
We have constructed transgenic cells of T. elongatus BP-1 in which nirA was substituted by the homologous gene of P. laminosum
Summary
Nitrate assimilation in cyanobacteria involves three main steps. Firstly, the anion entry inside the cell mediated by Nrt, the active ABC (ATP-Binding-Cassette) transporter [1,2,3]. The intracellular nitrate is reduced to ammonium by two consecutive reactions catalyzed by nitrate reductase (NarB) and nitrite reductase (NirA). We reported the purification and characterization of the proteins responsible of the uptake and reduction of nitrate and nitrite in the thermophilic cyanobacterium P. laminosum [7,8,9,10]. In P. laminosum these proteins (NrtA, NrtB, NrtC, NrtD, NarB and NirA) are encoded by genes that are clustered together into an operon that presents the sequence nirA-nrtA-nrtB-nrtC-nrtD-narB [11]. Even though nitrogen assimilation has been studied in a large number of cyanobacteria, the structure and function of the proteins codified by the genes of this operon have been scarcely studied and poorly understood. The phylogenetic relationship among NirA from some cyanobacteria is shown in supplementary Figure S1
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