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The filamentous cyanobacterium Anabaena variabilis ATCC 29413 fixes nitrogen under aerobic conditions in specialized cells called heterocysts that form in response to an environmental deficiency in combined nitrogen. Nitrogen fixation is mediated by the enzyme nitrogenase, which is very sensitive to oxygen. Heterocysts are microxic cells that allow...
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... measuring RNA by RT-qPCR at various times after the addition of rifampin, which inhibits initiation of transcription by RNA polymerase, we determined the half-lives of most of the genes in the nif1 gene cluster [69]. The half-life of nifH1 is much longer than the genes upstream of the processing site and the half-lives of the transcripts downstream of nifH1 decline with increasing distance from the processing site [69] (Table 1), suggesting that transcript stability plays a major role in controlling the relative amount of transcript. A mutant strain in which the stem-loop structure located at the processing site of nifH1 is abolished shows a shorter half-life for the nifH1 transcript than the wild-type strain and nitrogen fixation is strongly inhibited in this mutant (Thiel; unpublished data). ...
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Increase in desiccation is one of the adverse impacts of global climate change that limits the growth of microorganisms including cyanobacteria. Reduction in growth and nitrogen fixation by cyanobacteria due to desiccation is expected to severely restrict their application of cyanobacteria as biofertilizer. Therefore, in the present study the desic...
Anabaena variabilis ATCC 29413 has two distinct nitrogenases that function in heterocysts, a conventional Mo-nitrogenase and an alternative V-nitrogenase. Synthesis of these two enzymes was repressed in cells growing with a source of fixed nitrogen, such as ammonium; however, the V-nitrogenase was also repressed by Mo. Expression of the V-nitrogena...
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 contr...
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In the filamentous cyanobacterium Anabaena variabilis two Mo-nitrogenase gene clusters, nif1 and nif2, function under different environmental conditions in different cell types. Little is known about the regulation of transcription from the promoter upstream of the first gene of the cluster, which drives transcription of each of these...
Citations
... NifW is not directly active in the FeMo-protein assembly, but correlated with it under aerobic conditions, being part of an O2 protection system (Kim and Burgess, 1996). NifZ is important for P-cluster maturation , and the function of NifT remains unknown (Thiel and Pratte, 2014). It has been suggested that electron transfer from the Fe protein to the MoFe protein within the proteinprotein complex normally alterations conformational changes which enhance the affinity of the Fe protein for the MoFe protein (Lanzilotta et al., 1997). ...
Nitrogenase is a complicated enzyme that actives the ATP-dependent reduction of dinitrogen (N2) to ammonia (NH3). The aim of this manuscript is to review the nitrogenase evolution with considering nitrogenase, structure of nitrogenase, action mechanism of nitrogenase and oxygen sensitive mechanism of nitrogenase. The searches focused on publications from 1980 to February 2023, using PubMed, Google Scholar, Science Direct, and Scopus databases. In the term of evolution, the nitrogen cycle has experienced highly changes; at the beginning of life and suggested the exact anoxic scenario, the comparatively sufficient ammonium was possibly used in an assimilation/mineralization cycle by protocellular organisms. The main nif gene products which are active in nitrogen fixation are nifH, nifD, nifK, nifT, nifY/nafY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifL, nifA, nifB, fdxN, nifQ, and nifJ. The main vnf gene products which are active in nitrogen fixation are vnfA, vnfE, vnfN, vnfX, vnfH, vnfFd, vnfD, vnfG, vnfK, and vnfY. Oxygen can be either detrimental or beneficial for diazotrophs in organisms suitable for an aerobic catabolism, and it supports the production of a substrate for nitrogenase (ATP), but it can also impede the activity and suppress the synthesis of this enzyme.
... Furthermore, it is usual to find more than one operon in some strains [97]. In the P. brasiliensis CCIBt3594 genome, the genes nifK, nifH, nifD, nifB, nifS, and nifJ were identified, as well as iscU and fdx within the nif gene cluster, in the same order previously reported for Anabaena variabilis ATCC29413 [98] and other strains ( Figure 1). The gene fdx plays a crucial role in heterocytous-forming cyanobacteria. ...
The freshwater Pannus genus comprises cyanobacterial unicellular species with a particular morphology, forming free-floating rounded colonies with thin, homogenous, and colorless colonial mucilage. There is little literature on the taxonomy of the Pannus and none on its metabolism. This study presents the first genomic characterization of a Pannus strain isolated from Pantanal Biome, Brazil. The genome was assembled into 117 contigs with a total size of 5.1 Mb and 99.12% completeness. It contained 4988 protein-encoding genes, including some involved in secondary metabolite biosynthesis, such as cyanobactin and terpenes. Interestingly, P. brasiliensis CCIBt3594 has a complete set of nitrogen fixation genes and is a non-heterocytou unicellular cyanobacterium. Finally, the phylogenomic analyses revealed the lack of information on closely related strains and anchored the genus Pannus within the order Chroococcales, Microcystaceae family, closest to Microcystis spp. representatives. This work presents novel evidence concerning a sparsely characterized genus of the Cyanobacteria phylum and contributes to elucidating taxonomic and systematic issues within the group of unicellular cyanobacteria.
... Calcium (Ca) has been found to be integral for metabolism and intercellular communication [14]. Remarkably, environmental factors, such as trace metal concentrations, influence the expression of genes involved in enzymatic activity [13,14]. ...
... Nevertheless, the taxa within phylum can have different nitrogenases that can function in a different way depending on the environmental conditions and physiological features [58]. For example, it was demonstrated that Anabaena variabilis possesses two types of nitrogenase that function under different environmental conditions and express in different types of cells [59]. In our study, we show that two types of nitrogenase operons are present in the Rhodovulum species that probably results in different nitrogen fixation rates, so it is necessary to further investigate this phenomenon. ...
The genome of the moderately haloalkaliphilic diazotrophic anoxygenic phototrophic bacterium Rhodovulum tesquicola A-36sT isolated from an alkaline lake was analyzed and compared to the genomes of the closest species Rhodovulum steppense A-20sT and Rhodovulum strictum DSM 11289T. The genomic features of three organisms are quite similar, reflecting their ecological and physiological role of facultative photoheterotrophs. Nevertheless, the nitrogenase activity of the pure cultures of the studied bacteria differed significantly: the highest rate (4066 nmoles C2H2/mg of dry weight per hour) was demonstrated by Rhodovulum strictum while the rates in Rhodovulum tesquicola and Rhodovulum steppense were an order of magnitude lower (278 and 523 nmoles C2H2/mg of dry weight per hour, respectively). This difference can be attributed to the presence of an additional nitrogenase operon found exclusively in R. strictum and to the structural variation in nitrogenase operon in R. tesquicola.
... The cyanobacterial [V]-nitrogenase was first discovered and described in detail in the strain A. variabilis [49,50]. Cells of cyanobacteria in a Mo-poor and V-rich medium reduced acetylene to ethylene, producing high yield of H 2 [51]. ...
Due to the side effects of greenhouse gases, interest in alternative energy sources is growing, and research into hydrogen (Н2) production from cyanobacteria has become a promising direction for the industry. The article provides an overview of cyanobacterial hydrogen production strategies and their current economic efficiency. It also describes metabolic, genetic and technical methods for obtaining H2 from cyanobacteria. Cyanobacteria are considered potential producers of hydrogen energy that will be economically viable shortly, as they only need cheap salts, water and solar energy to grow. However, producing hydrogen from cyanobacteria still requires extensive work, and the main problem is the small amount of hydrogen energy obtained. To produce large amounts of cyanobacterial hydrogen, the most active wild-type strains must be selected and technological, modular and genetic research must be carried out simultaneously. The low energy efficiency of hydrogen from cyanobacteria also shows the need for comprehensive research through international programs.
... Recently, genes encoding for a vanadium nitrogenase (vnf) were reported in four plants Nostoc cyanobionts (Nelson et al., 2019). A notable exception is Anabaena variabilis, which expresses three different nitrogenases: a heterocyst-specific Mo-nitrogenase, a vegetative cell-specific Mo-nitrogenase, and a heterocyst-specific V-nitrogenase (Thiel and Pratte, 2014). No other wellcharacterized cyanobacterial strain expresses two or three nitrogenases. ...
... Some of the best characterized cyanobacterial nif gene clusters (Thiel, 2019) include A. variabilis ATCC 29413 (Thiel and Pratte, 2014), Cyanothece sp. ATCC 51142 (Stockel et al., 2011;Liu et al., 2018), Leptolyngbya sp. ...
... Additional players in this intricate biosynthetic pathway include NifX (metallochaperone of FeMo-co precursors), NifW (involved in apo-NifDK binding prior to P-cluster maturation), NifZ (involved in P-cluster maturation), NifT (unknown role), FdxN (involved in the formation of NifB-co), NifJ (pyruvate-flavodoxin (Fld) oxidoreductase required for nitrogenase activity in iron deficiency in Anabaena (Bauer et al., 1993), and NifP (serine acetyltransferase involved in the expression of nitrogenase activity). The genes for NifQ (involved in the processing of molybdenum for incorporation into FeMo-co), NifM (involved in the maturation of NifH), and NafY (involved in stabilization of apo-NifDK and binding of FeMo-co) are not reported in cyanobacterial genomes (Thiel and Pratte, 2014). ...
Sustainable production of N-fertilizers is essential to hold the increasing demand for food expected over the next decades. While reduced access to N-fertilizers causes food shortages in some regions of the world, incorrect management in other areas causes detrimental effects on the environment. An increase in the current agricultural exploitation of biological N2-fixation promises a great benefit on both the economic and environmental aspects. To that end, a variety of novel strategies have been proposed and were actively investigated and improved over the last decade. One such strategy consists in the production of biomass of N2-fixing cyanobacteria and its application as a fertilizer and soil conditioner. Separating biological fertilizer production under optimized conditions for that sole purpose from soil fertilization and crop cultivation might bypass the probabilities of the microorganisms’ adaptation to the agronomic conditions and facilitate the management of the fertilizer. Last decade’s advances in different fields of science and technology might lead to mature technology for this strategy to contribute an alternative source of N-fertilizer for sustainable agriculture. This review will compile the main recent advances in (1) massive cultivation of cyanobacteria, including the use and recycling of nutrients in domestic or industrial wastewater; (2) biomass mineralization in the soil; (3) cyanobacterial production of accessory bioactive compounds for stimulating plant growth and/or improving soil characteristics; (4) empirical evidence for efficient plant fertilization and soil conditioning; (5) genetic engineering tools for the optimization of selected traits in cyanobacteria for their use as organic fertilizer; and (6) multi-institutional projects, as well as the main present and future challenges toward the industrial application of this technology.
... The microoxic environment of the heterocysts results from their cell wall structure, their lack of oxygen-evolving photosystem II, and their high rate of respiration (13)(14)(15)(16)(17). All heterocyst-forming cyanobacteria express a Mo-nitrogenase in heterocysts, but some strains also have a heterocyst-specific V-nitrogenase that is made only under conditions of Mo deficiency (18,19). ...
... The genes for nitrogen fixation in cyanobacteria are well conserved and typically include a few clusters of nif genes, some of them comprising over a dozen genes (20). These large clusters of genes have been best characterized in Anabaena variabilis ATCC 29413 (19), Leptolyngbya boryana (21,22), and Cyanothece sp. (7,23). ...
... A. variabilis is unusual because it has two nif-encoded Mo-nitrogenases. The nif1 genes are expressed only in heterocysts, while the nif2 genes are expressed only in anaerobic vegetative cells (19,20,24). The expression of the nif1 genes depends on the differentiation of heterocysts, a complex developmental process that requires a carefully regulated cascade of regulatory factors, including NtcA, HetR, NrrA, DevH, and other proteins (9,11,12,25). ...
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 Co2+-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.
... However, we realize that there are examples of 2-3 nifH copies in heterocyst-forming cyanobacteria such as Anabaena variabilis and Fischerella sp. 88,89 , or in the firmicutes Clostridium pasteurianum 90 , and that we are not taking into account the polyploidy effect. ...
Nitrogen fixation has a critical role in marine primary production, yet our understanding of marine nitrogen-fixers (diazotrophs) is hindered by limited observations. Here, we report a quantitative image analysis pipeline combined with mapping of molecular markers for mining >2,000,000 images and >1300 metagenomes from surface, deep chlorophyll maximum and mesopelagic seawater samples across 6 size fractions (<0.2–2000 μm). We use this approach to characterise the diversity, abundance, biovolume and distribution of symbiotic, colony-forming and particle-associated diazotrophs at a global scale. We show that imaging and PCR-free molecular data are congruent. Sequence reads indicate diazotrophs are detected from the ultrasmall bacterioplankton (<0.2 μm) to mesoplankton (180–2000 μm) communities, while images predict numerous symbiotic and colony-forming diazotrophs (>20 µm). Using imaging and molecular data, we estimate that polyploidy can substantially affect gene abundances of symbiotic versus colony-forming diazotrophs. Our results support the canonical view that larger diazotrophs (>10 μm) dominate the tropical belts, while unicellular cyanobacterial and non-cyanobacterial diazotrophs are globally distributed in surface and mesopelagic layers. We describe co-occurring diazotrophic lineages of different lifestyles and identify high-density regions of diazotrophs in the global ocean. Overall, we provide an update of marine diazotroph biogeographical diversity and present a new bioimaging-bioinformatic workflow.
... Many cyanobacteria, even those with only one nif system, have additional nifH copies (Fig. 6) that have not been studied. N. variabilis has five copies of nifH genes (nifH5 is mentioned above) of which only three, nifH1, nifH2 and vnfH, function as part of a complete nitrogenase [83]. A phylogeny of all the nifH genes in the strains described in this study shows that the nifH copies that are most closely related to each other also share similar genes surrounding them, even (in the case of nifH5) when these nearby genes have no similarity to known nitrogenase genes. ...
... However, Nostoc 2RC differs significantly from these other strains because it has two additional sets of nitrogenase genes, also present in all of the N. variabilis-like strains and in Nostoc Moss5 and Moss6. These are the nif2 genes, encoding an Mo-nitrogenase that functions in vegetative cells under anoxic conditions [66,83] and the vnf genes that make the alternative V-nitrogenase [78,79], which are absent in all the Nostoc strains closely related to Nostoc 2RC, except for the vnfH-like and vnfR-like genes. The presence of these two conserved vnf-like genes suggests that these Nostoc strains may have once had the full cluster of vnf genes. ...
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.
... The former cluster contains five of the six genes (missing nifB), whereas the latter cluster contains three genes (missing nifENK); regardless, all six required genes are represented among the two clusters. The expression of the genes in the two clusters may differ under distinct growth conditions [53]. ...
Cyanobacteria are known as rich repositories of natural products. One cyanobacterial-microbial consortium (isolate HT-58-2) is known to produce two fundamentally new classes of natural products: the tetrapyrrole pigments tolyporphins A–R, and the diterpenoid compounds tolypodiol, 6-deoxytolypodiol, and 11-hydroxytolypodiol. The genome (7.85 Mbp) of the Nostocales cyanobacterium HT-58-2 was annotated previously for tetrapyrrole biosynthesis genes, which led to the identification of a putative biosynthetic gene cluster (BGC) for tolyporphins. Here, bioinformatics tools have been employed to annotate the genome more broadly in an effort to identify pathways for the biosynthesis of tolypodiols as well as other natural products. A putative BGC (15 genes) for tolypodiols has been identified. Four BGCs have been identified for the biosynthesis of other natural products. Two BGCs related to nitrogen fixation may be relevant, given the association of nitrogen stress with production of tolyporphins. The results point to the rich biosynthetic capacity of the HT-58-2 cyanobacterium beyond the production of tolyporphins and tolypodiols.
