Characterization of nitrogenase genes nifP and nifVZT1 in the cyanobacterium Anabaena (Trichormus) variabilis ATCC 29413

Abstract

Anabaena (Trichormus) variabilis ATCC 29413 is a heterocyst-forming cyanobacterium with two Mo-nitrogenases encoded by the large nif1 and nif2 gene clusters. The nif1-encoded nitrogenase is expressed in heterocysts under oxic growth conditions, whereas the nif2-encoded nitrogenase is expressed only in anoxic vegetative cells. The nifP and nifVZT1 genes are contiguous and distinct from the major nif1 and nif2 clusters, suggesting that nifP and nifVZT1 may be cotranscribed. However, we identified primary transcription start sites for nifP and nifVZT1, indicating that each has its own promoter. The promoter region of nifP shares multiple conserved motifs with the nifB1 and nifB2 promoters, which are regulated by homologous transcriptional activators CnfR1 and CnfR2, respectively. These conserved motifs are not present in the promoter region of nifVZT1. Using nifP promoter fragments that included or excluded the conserved motifs fused to reporter gene lacZ, we determined which motifs contributed to nifP expression; however, these identified motifs differed from the conserved motifs shown previously to regulate nifB1 or nifB2 transcription. Further, the regulation of transcription requires a region inside the coding region of nifP. Mutant strains lacking nifP or nifVZT1 grew well in the absence of fixed nitrogen and showed no reduction in nitrogenase activity compared to the wild-type strain grown under oxic or anoxic conditions. Further, nifP was expressed constitutively in vegetative cells grown -N or +N, but its expression increased greatly in heterocysts by CnfR1 activation of the nifP promoter. In contrast, nifVZT1 was expressed only in heterocysts but, unlike most other nif1 genes, was not under the control of CnfR1.