Open reading frame all0601 from Anabaena sp. strain PCC 7120 represents a novel gene, cnaT, required for expression of the nitrate assimilation nir operon

Abstract

Expression of the nitrate assimilation nir operon in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 requires the action of both the global nitrogen control transcription factor NtcA and the pathway-specific transcriptional regulator NtcB. In the genome of this cyanobacterium, the ntcB gene is found in a cluster of genes located in the complementary strand, upstream from the nir operon. Just downstream of ntcB, there is an open reading frame, all0601 (previously designated orf356 and now designated the cnaT gene), that putatively encodes a protein similar to proteins with glycosyl transferase activity and that is also present clustered together with ntcB homologues or nitrate assimilation structural genes in other cyanobacterial genomes. An insertional mutant of cnaT was generated and found to be unable to assimilate nitrate, although it could use ammonium or dinitrogen as a source of nitrogen for growth. In the mutant, under derepression conditions, nir operon mRNA (as determined by RNA-DNA hybridization and primer extension analysis) and enzymes of the nitrate reduction system (i.e., nitrate reductase and nitrite reductase) were expressed at low or undetectable levels. Inactivation of cnaT did not impair expression of ntcB, and expression of cnaT itself was constitutive and regulated by neither NtcA nor NtcB. Regulation of expression of the nir operon in Anabaena sp. strain PCC 7120 by CnaT and the previously described regulatory elements, NtcA and NtcB, is discussed.