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Anabaena variabilis ATCC 29413 is a filamentous heterocystous cyanobacterium that fixes nitrogen under a variety of environmental conditions. Under aerobic growth conditions, nitrogen fixation depends upon differentiation of heterocysts and expression of either a Mo-dependent nitrogenase or a V-dependent nitrogenase in those specialized cells. Under anaerobic conditions, a second Mo-dependent nitrogenase gene cluster, nifII, was expressed in vegetative cells long before heterocysts formed. A strain carrying a mutant gene in the nifII cluster did not fix nitrogen under anaerobic conditions until after heterocysts differentiated. The nifII cluster was similar in organization to the nifI cluster that is expressed in heterocysts and that includes nifBSUHDKENXW as well as three open reading frames that are conserved in both cyanobacterial nif clusters.

In many filamentous cyanobacteria nitrogen fixation occurs in differentiated cells called heterocysts. Filamentous strains that do not form heterocysts may fix nitrogen in vegetative cells, primarily under anaerobic conditions. We describe here two functional Mo-dependent nitrogenases in a single organism, the cyanobacterium Anabaena variabilis. Using a lacZ reporter with a fluorescent beta-galactoside substrate for in situ localization of gene expression, we have shown that the two clusters of nif genes are expressed independently. One nitrogenase functions only in heterocysts under either aerobic or anaerobic growth conditions, whereas the second nitrogenase functions only under anaerobic conditions in vegetative cells and heterocysts. Differences between the two nif clusters suggest that the nitrogenase that is expressed in heterocysts is developmentally regulated while the other is regulated by environmental factors.