In many filamentous cyanobacteria nitrogen fixation occurs in differentiated cells called heterocysts. Filamentous strains that do not form heterocysts may ri nitrogen in vegetative cells, primarily under anaerobic conditions. We describe here two functional Mo-dependent nitrogenases in a single organism, the cyanobacterium Anabaena variabilis. Filamentous cyanobacteria of the genus Anabaena serve as a simple prokaryotic model for developmental control of gene expression. When deprived of a source of fixed nitrogen, about every 10th photosynthetic vegetative cell in the cyanobacterial filament differentiates into a morphologically and physiologically distinct cell called a heterocyst (1, 2). The primary function of heterocysts is nitrogen fixation, the reduction of atmospheric dinitrogen to ammonia mediated by the enzyme nitrogenase. Nitrogenase is very oxygen labile; hence, nitrogen fixation is restricted to anaerobic environments. Heterocysts provide the requisite anaerobic environment because their cell envelope limits oxygen entry and they lack oxygen-evolving photosystem II, which is characteristic of vegetative cells (2). Within a filament heterocysts differentiate in a semiregular pattern, thus providing spatial separation of nitrogen fixation from oxygenic photosynthesis in what is functionally a onedimensional multicellular organism (3).Among nitrogen-fixing cyanobacteria that do not differentiate heterocysts there does not appear to be a single mechanism for protection of nitrogenase from oxygen and different strains show a range in oxygen tolerance (4,5). In many nonheterocystous cyanobacteria, photosynthesis is temporally separated from nitrogen fixation, which occurs only at night (6-8). For other nonheterocystous cyanobacteria that fix nitrogen aerobically in the light without apparently differentiated cells, little is known of the mechanisms for protecting nitrogenase from oxygen (9, 10); however, nitrogenase activity in laboratory-grown cultures is significantly enhanced by lower oxygen tensions (4). Thus, low oxygen tensions are probably necessary for optimal nitrogenase activity.The heterocystous cyanobacterium, Anabaena sp. strain PCC 7120 (hereafter, Anabaena PCC 7120), has a large cluster of nif genes (including nifBSUHDKEN) that encode a Modependent nitrogenase system (11). The nifB-fdxN-nifS-nifU operon is interrupted by a 55-kb insertion infdxN and the nifD gene has an 11-kb insertion, both of which are excised during heterocyst differentiation (12-14). The 11-kb element is prevalent in heterocystous cyanobacteria (15) but is missing in all nonheterocystous cyanobacteria examined to date (4). The nif genes of Anabaena variabilis ATCC 29413 homologous to those of Anabaena PCC 7120 have been cloned and partially mapped (16); they contain the 11-kb excision element, but not the 55-kb excision element (17). In addition to that nifHDK cluster, a different putative nifHD segment, transcribed within hours after the onset of nitrogen starvation under anaerobic conditions, was cloned from ...

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.

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium containing both a Mo-dependent nitrogenase encoded by the nif genes and V-dependent nitrogenase encoded by the vnf genes. The nifB, nifS, and nifU genes of A. variabilis were cloned, mapped, and partially sequenced. The fdxN gene was between nifB and nifS. Growth and acetylene reduction assays using wild-type and mutant strains indicated that the nifB product (NifB) was required for nitrogen fixation not only by the enzyme encoded by the nif genes but also by the enzyme encoded by the vnf genes. Neither NifS nor NifU was essential for nitrogen fixation in A. variabilis.Anabaena spp. are filamentous cyanobacteria capable of fixing dinitrogen in specialized morphologically distinct cells called heterocysts (18,41). Under diazotrophic growth conditions, vegetative cells compose 90 to 95% of the cells in a filament and contain both photosystems I and II. The other 5 to 10% of cells are heterocysts that have only photosystem I and therefore evolve no oxygen (40). Additional envelope layers in heterocysts may protect nitrogenase from oxygen (38, 39).Many nif genes have been identified in Anabaena sp. strain PCC 7120: one large cluster contains nifB, fdxN, nifS, nifU, nifH, nifD, nifK, nifN, nifX, open reading frame 3, nifW, open reading frame 1, open reading frame 2, and fdxH (8,23,30,33,36,37,42). nifJ is not part of the cluster (4). The nifB-fdxNnifS-nifU operon is interrupted by a 55-kb insertion in fdxN, and the nifD gene has an 11-kb insertion, both of which are excised during heterocyst differentiation (19,20). The heterocyst ferredoxin gene of Anabaena sp. strain PCC 7120, fdxH, is downstream of the other characterized nif genes in the cluster (8). The nif genes of A. variabilis ATCC 29413 that are the homologs of the nif genes of Anabaena sp. strain PCC 7120 have been cloned and mapped (24), and the 11-kb excision element, but not the 55-kb excision element, has been found in that nif cluster (9).Besides the nif genes that encode nitrogenase 1, which requires a Mo cofactor, Anabaena variabilis also contains vnf genes that encode nitrogenase 2, which requires a V cofactor (53). Nitrogenases that do not require Mo were first described in Azotobacter vinelandii, which has nitrogenase 1, nitrogenase 2, and nitrogenase 3. Nitrogenase 3, which requires only Fe in the cofactor, is encoded by the anf genes (6, 10, 27). The vnf and anf systems of A. vinelandii do not have homologs of all the genes of the nif system. Known vnf and anf genes include vnfDGK, homologous to nifDK; vnfEN, homologous to nifEN; vnfA, homologous to nifA; and anfHDGK, homologous to nif HDK (6). The nifB, nifS, nifU, nifM, and nifV genes are required for the activity of all three nitrogenases of A. vinelandii (28,29). The vnfEN genes may also function in the anf system (6). The vnf-and anf-encoded nitrogenases are repressed by Mo and have the ability to reduce acetylene not only to ethylene but also to ethane, which is one criterion for the presence of these altern...

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