Isolation and characterization of the VnfEN genes of the cyanobacterium Anabaena variabilis

Abstract

The filamentous cyanobacterium Anabaena variabilis fixes nitrogen in the presence of vanadium (V) and in the absence of molybdenum (Mo), using a V-dependent nitrogenase (V-nitrogenase) encoded by the vnfDGK genes. Downstream from these genes are two genes that are similar to the vnfEN genes of Azotobacter vinelandii. Like the vnfDGK genes, the vnfEN genes were transcribed in the absence of Mo, whether or not V was present. A mutant with an insertion in the vnfN gene lacked V-nitrogenase activity; thus, the vnfEN genes were essential for the V-nitrogenase system in A. variabilis. Growth and acetylene reduction assays with wild-type and mutant strains suggested that the V-nitrogenase reduced dinitrogen better than acetylene. The similarity of the vnfEN genes of A. variabilis and A. vinelandii was not strong. The vnfEN genes of A. variabilis showed greater similarity to the vnfDK genes just upstream than to the A. vinelandii vnfEN genes. Sequence comparisons provide support for the idea that if the vnf genes were transferred laterally among bacterial strains, the vnf cluster was not transferred intact. It appears likely that the structural genes were transferred before a duplication event led to the evolution of the vnfEN genes independently in the two strains. The divergence of the vnfEN genes from the vnfDK genes suggests that this duplication, and hence the transfer of vnf genes, was an ancient event.Anabaena variabilis ATCC 29413 is a filamentous cyanobacterium that fixes dinitrogen under a variety of environmental conditions. Under aerobic conditions, nitrogen fixation occurs exclusively in cells called heterocysts (18,27). Heterocysts are terminally differentiated cells that develop in a pattern from certain vegetative cells in the filament in response to limitation in fixed nitrogen (reviewed in reference 44). The primary Modependent nitrogenase (Mo-nitrogenase) in this organism is encoded by a contiguous cluster of nif1 genes, including nifB1, fdxN, nifS1, nifU1, nifD1, nifK1, nifE1, and nifN1, that are transcribed only in heterocysts (8,22,39). These genes are very similar to the nif cluster of Anabaena sp. strain PCC 7120 (reviewed in reference 21), which is also transcribed exclusively in heterocysts (16). We have recently characterized an alternative Mo-nitrogenase gene cluster in this strain that is absent in Anabaena sp. strain PCC 7120; this second cluster contains genes nifB2, nifS2, nifU2, nifH2, nifD2, nifK2, nifEN2, nifX2, and nifW2, which are transcribed in vegetative cells and in heterocysts only under strictly anaerobic conditions (39). These nif2 genes are homologs of the nif1 genes; however, this cluster differs from the nif1 cluster: there is no fdxN gene, nor is there the 11-kb excision element that is present in nif1 and in the nif cluster of Anabaena sp. strain PCC 7120. In addition, the nifE1 and nifN1 homologs are fused into a single open reading frame, nifEN2. Certain fdx genes in this organism that are expressed only under anaerobic conditions may also be part of this second system (...

Tansley Review No. 107. Heterocyst and akinete differentiation in cyanobacteria

“…However, the nif2 cluster, which lacks both the 11-kb and 55-kb elements, is expressed in vegetative cells, but only under anaerobic conditions, and in heterocysts (Thiel et al, , 1997. The vnf cluster codes for an alternative nitrogenase system containing a vanadium cofactor, instead of the usual molybdenum cofactor, and is expressed in the absence of molybdenum, but only in heterocysts (Thiel, 1993(Thiel, , 1996.…”

Lichen-symbiotic cyanobacteria associated withPeltigerahave an alternative vanadium-dependent nitrogen fixation system

“…Azotobacter spp.) (Thiel, 1996). A third system encoded by the anfHDGK operon (which is dependent upon Fe only and expressed under both Mo-and V-limited conditions) is known from lineages outside of the phylum Cyanobacteria .…”

High-Affinity Vanadate Transport System in the CyanobacteriumAnabaena variabilisATCC 29413

“…Nitrogen fixation is mediated by a family of highly conserved nitrogenases that have a unique metal cofactor containing homocitrate (12,22,34). Most of these enzymes have a molybdenum cofactor; however, a few bacteria have an alternative V-nitrogenase that has vanadium in place of molybdenum in the cofactor (5,11,45,47). Most cyanobacteria have the molybdoenzyme nitrate reductase, and many cyanobacteria fix nitrogen with a Mo-nitrogenase; hence, molybdate is important for their growth.…”

“…Similar putative tungstate transport genes have been identified in the genomes of many other bacteria, although tungstoenzymes have not been characterized in most of these strains. Vanadium cofactors have been identified for three types of enzymes, V-dependent haloperoxidases, found in a variety of algae and fungi (25, 26); V-dependent nitrate reductases, found in two strains of bacteria (2-4); and V-nitrogenases, found in a several strains of nitrogen-fixing bacteria (5,11,45,47). In a completely different role, vanadium functions in anaerobic respiration as an electron acceptor in several strains of bacteria (8,29,36,37).…”