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ABSTRACT In Anabaena variabilis , the nif1 genes, which are activated by CnfR1, produce a Mo-nitrogenase that is expressed only in heterocysts. Similarly, the nif2 genes, which are activated by CnfR2, make a Mo-nitrogenase that is expressed only in anaerobic vegetative cells. However, CnfR1, when it was expressed in anaerobic vegetative cells under the control of the cnfR2 promoter or from the Co 2+ -inducible coaT promoter, activated the expression of both nifB1 and nifB2 . Activation of nifB2 , but not nifB1 , by CnfR1 required NtcA. Thus, expression of the nif1 system requires no heterocyst-specific factor other than CnfR1. In contrast, CnfR2, when it was expressed in heterocysts under the control of the cnfR1 promoter or from the coaT promoter, did not activate the expression of nifB1 or nifB2 . Thus, activation of the nif2 system in anaerobic vegetative cells by CnfR2 requires additional factors absent in heterocysts. CnfR2 made from the coaT promoter activated nifB2 expression in anaerobic vegetative cells grown with fixed nitrogen; however, oxygen inhibited CnfR2 activation of nifB2 expression. In contrast, activation of nifB1 and nifB2 by CnfR1 was unaffected by oxygen. CnfR1, which does not activate the nifB2 promoter in heterocysts, activated the expression of the entire nif2 gene cluster from a nifB2 :: nifB1 :: nifB2 hybrid promoter in heterocysts, producing functional Nif2 nitrogenase in heterocysts. However, activity was poor compared to the normal Nif1 nitrogenase. Expression of the nif2 cluster in anaerobic vegetative cells of Nostoc sp. PCC 7120, a strain lacking the nif2 nitrogenase, resulted in expression of the nif2 genes but weak nitrogenase activity. IMPORTANCE Cyanobacterial nitrogen fixation is important in the global nitrogen cycle, in oceanic productivity, and in many plant and fungal symbioses. While the proteins that mediate nitrogen fixation have been well characterized, the regulation of this complex and expensive process is poorly understood in cyanobacteria. Using a genetic approach, we have characterized unique and overlapping functions for two homologous transcriptional activators CnfR1 and CnfR2 that activate two distinct nitrogenases in a single organism. We found that CnfR1 is promiscuous in its ability to activate both nitrogenase systems, whereas CnfR2 depends on additional cellular factors; thus, it activates only one nitrogenase system. 

Species of the floating, freshwater fern Azolla form a well-characterized symbiotic association with the non-culturable cyanobacterium Nostoc azollae , which fixes nitrogen for the plant. However, several cyanobacterial strains have over the years been isolated and cultured from Azolla from all over the world. The genomes of 10 of these strains were sequenced and compared with each other, with other symbiotic cyanobacterial strains, and with similar strains that were not isolated from a symbiotic association. The 10 strains fell into three distinct groups: six strains were nearly identical to the non-symbiotic strain, Nostoc ( Anabaena ) variabilis ATCC 29413; three were similar to the symbiotic strain, Nostoc punctiforme , and one, Nostoc sp. 2RC, was most similar to non-symbiotic strains of Nostoc linckia . However, Nostoc sp. 2RC was unusual because it has three sets of nitrogenase genes; it has complete gene clusters for two distinct Mo-nitrogenases and an alternative V-nitrogenase. Genes for Mo-nitrogenase, sugar transport, chemotaxis and pili characterized all the symbiotic strains. Several of the strains infected the liverwort Blasia , including N. variabilis ATCC 29413, which did not originate from Azolla but rather from a sewage pond. However, only Nostoc sp. 2RC, which produced highly motile hormogonia, was capable of high-frequency infection of Blasia . Thus, some of these strains, which grow readily in the laboratory, may be useful in establishing novel symbiotic associations with other plants.