SummaryAnabaena variabilis ATCC 29413 fixes nitrogen in specialized cells called heterocysts using either a Mo-nitrogenase or a V-nitrogenase. V-nitrogenase structural genes, vnfDGK, as well as vnfEN form an operon with ava4025, located upstream of vnfDG that is repressed by fixed nitrogen and by Mo. The ava4025-vnfDGKEN operon is under the control of a Morepressible promoter located nearly 600 bp upstream of ava4025. Levels of vnfDG transcript were about 500-fold higher than ava4025, the first gene of the operon. This may be the result of RNA processing at a site 87 bp upstream of vnfDG that was initially identified as the transcription start site. A strain with a deletion in the coding region of ava4025 grew diazotrophically with Mo or with V. Two similar proteins, VnfR1 and VnfR2, whose genes are located some distance from the ava4025-vnfDGKEN operon, each repressed transcription from the ava4025-vnfDGKEN promoter and a mutant lacking both VnfR1 and VnfR2 made the V-nitrogenase in the presence of Mo. Overexpression of the V-nitrogenase in the double vnfR1 vnfR2 mutant resulted in decreased activity of the Mo-nitrogenase. VnfR1 bound specifically, in vitro, to a region upstream of the ava4025 promoter.