IDENTIFICATION OF NITRATE TRANSPORTER GENES IN CHLAMYDOMONAS-REINHARDTII

The Chlamydomonas reinhardtii nar-2, nar-3, and nar-4 genes, which are within a nitrate regulated gene cluster containing the nitrate reductase structural gene nit-1, have been related to nitrate transport. Mutant strains defective in nitrate transport and having an active nitrate reductase have been genetically constructed. Their nitrate non-utilizing phenotype has been directly complemented by transformation using the pCO-5 plasmid which carries the nar-2, nar-3, and nar-4 clustered genes. Integration of pCO-5 DNA in the genome of nitrate transport mutants resulted in the expression of these nar transcripts and the recovery of a high affinity nitrate transport activity. Complementation of the nitrate non-utilizing phenotype of the constructed strains was also achieved by co-transformation with plasmids containing nar-2 and nar-3 genes or nar-2 and nar-4, but not with single plasmids containing each individual gene. In addition, DNA sequences of a practically complete cDNA of nar-3 and a partial one of nar-4 have been generated and the deduced amino acid sequences showed a very significant identity with that of the nitrate transporter gene (crnA) from Aspergillus nidulans. These data strongly support the hypothesis that the nitrate transport system in C. reinhardtii contains at least two protein components encoded by the nar-2 and nar-3 genes. The nar-4 gene would produce a protein with a high identity to that of nar-3.