Diazotrophic Organisms Research Articles

Is the distribution of nitrogen‐fixing cyanobacteria in the oceans related to temperature?

Approximately 50% of the global natural fixation of nitrogen occurs in the oceans supporting a considerable part of the new primary production. Virtually all nitrogen fixation in the ocean occurs in the tropics and subtropics where the surface water temperature is 25°C or higher. It is attributed almost exclusively to cyanobacteria. This is remarkable firstly because diazotrophic cyanobacteria are found in other environments irrespective of temperature and secondly because primary production in temperate and cold oceans is generally limited by nitrogen. Cyanobacteria are oxygenic phototrophic organisms that evolved a variety of strategies protecting nitrogenase from oxygen inactivation. Free-living diazotrophic cyanobacteria in the ocean are of the non-heterocystous type, namely the filamentous Trichodesmium and the unicellular groups A-C. I will argue that warm water is a prerequisite for these diazotrophic organisms because of the low-oxygen solubility and high rates of respiration allowing the organism to maintain anoxic conditions in the nitrogen-fixing cell. Heterocystous cyanobacteria are abundant in freshwater and brackish environments in all climatic zones. The heterocyst cell envelope is a tuneable gas diffusion barrier that optimizes the influx of both oxygen and nitrogen, while maintaining anoxic conditions inside the cell. It is not known why heterocystous cyanobacteria are absent from the temperate and cold oceans and seas.

Nitrogen fixation in the western English Channel (NE Atlantic Ocean)

In temperate Atlantic waters (18.8 to 20.1°C), biological nitrogen fixation has been demonstrated by 2 independent measurements: 15 N-N2 incorporation and nifH identification in the DNA and expressed messenger RNA (mRNA). At 2 stations in the western English Channel, bulk waters were incubated with 15 N-N2. At the high levels of particulate nitrogen (≤11.5 µmol N l -1 ), absolute fixation rates of 18.9 ± 0.01 and 20.0 nmol N l -1 d -1 were determined. While a caveat must accompany the magnitude of the rates presented due to the limited number of data, the presence and activity of diazotrophic organisms in these waters is of ecological significance and may affect current attitudes to nitrogen and carbon budgets. In particular, our estimate of the rate of N fixation (0.35 mmol N m -2 d -1 ) is comparable to that of denitrification rates in UK shelf seas. Molecular analy- sis identified a diversity of expressed nifH genes, and 21 different prokaryotic nifH transcripts were identified.

Heme‐Copper Oxidases and Their Electron Donors in Cyanobacterial Respiratory Electron Transport

Cyanobacteria are the paradigmatic organisms of oxygenic (plant-type) photosynthesis and aerobic respiration. Since there is still an amazing lack of knowledge on the role and mechanism of their respiratory electron transport, we have critically analyzed all fully or partially sequenced genomes for heme-copper oxidases and their (putative) electron donors cytochrome c(6), plastocyanin, and cytochrome c(M). Well-known structure-function relationships of the two branches of heme-copper oxidases, namely cytochrome c (aa(3)-type) oxidase (COX) and quinol (bo-type) oxidase (QOX), formed the base for a critical inspection of genes and ORFs found in cyanobacterial genomes. It is demonstrated that at least one operon encoding subunits I-III of COX is found in all cyanobacteria, whereas many non-N(2)-fixing species lack QOX. Sequence analysis suggests that both cyanobacterial terminal oxidases should be capable of both the four-electron reduction of dioxygen and proton pumping. All diazotrophic organisms have at least one operon that encodes QOX. In addition, the highly refined specialization in heterocyst forming Nostocales is reflected by the presence of two paralogs encoding COX. The majority of cyanobacterial genomes contain one gene or ORF for plastocyanin and cytochrome c(M), whereas 1-4 paralogs for cytochrome c(6) were found. These findings are discussed with respect to published data about the role of respiration in wild-type and mutated cyanobacterial strains in normal metabolism, stress adaptation, and nitrogen fixation. A model of the branched electron-transport pathways downstream of plastoquinol in cyanobacteria is presented.

Nitrogenase Activity in Intertidal Sediment Along the Tanzanian Coast, Western Indian Ocean

Nitrogenase activity was determined using the acetylene reduction assay in the littoral areas near Tanga, Dar es Salaam and Mafia with the aim of investigating its spatial and temporal distribution along the Tanzanian coastal line. Ethylene (C 2 H 4 ) production rates ranged from 0.07 - 13.5 nmol C 2 H 4 cm- 2 h- 1 in Tanga area, 0.30 - 5.43 nmol C 2 H 4 cm- 2 h- 1 in Dar es Salaam and from 0.10 - 6.25 nmol C 2 H 4 cm- 2 h- 1 in Mafia. Significantly higher rates of nitrogenase activity were measured during night-incubated samples compared to daytime incubated samples suggesting that the sediments were mostly composed of non-heterocystous diazotrophic organisms. However, there were no significant differences in rates of nitrogenase activity between the rainy and dry season and between the sandy and muddy beaches. Also, there were no significant differences in nitrogenase activity between the upper, mid and lower littoral zones, and between the three sampling locations, i.e. Tanga, Dar es Salaam and Mafia. A nitrogen fixation rate of 38 mmol N m- 2 y- 1 was estimated. We conclude that diazotrophs contribute a substantial amount of nitrogen input to the Tanzania coastal ecosystems. Keywords : Nitrogen fixation, Acetylene Reduction Assay, diazotrophs, intertidal sediment, Tanzania West Indian Ocean Journal of Marine Science Vol. 5 (2) 2006: pp. 133-140

Nitrogen Fixation in Streams in Jackson Hole, WY

Nitrogen fixation, the conversion of unreactive N2 gas from the atmosphere into a biologically usable form NH3, is the least studied process of the N cycle in stream ecology (Grimm and Petrone 1997). Tapping into the atmospheric N pool can only be accomplished naturally by N-fixers. These diazotrophic organisms can contribute significantly to the autochthonous inputs of nitrogen into streams (Grimm and Petrone 1997). Without further investigations into N-fixation we would ignore a potentially major N flux and transfonnation. Not only do we need to quantify this process, but also examine what factors control N-fixation in streams.

NITROGEN CONTRIBUTIONS TO AGRICULTURE

For the last three decades, nitrogen fertilizers have contributed in a significant way to increase food production. The increase attained in the production of many crops has surpassed the population Nitrogen (N) provided by these fertilizers is the main mineral element that plants absorb, and it intervenes in essential physiological processes for their growth and development. Some plants can use atmospheric nitrogen, by associating themselves to prokaryotic or diazotrophic organisms, but most crops rely on the external supplementation of N from organic matter mineralization and the addition of fertilizers, so they can complete their growth cycle. The primary source of N is atmospheric nitrogen submitted to a biological or industrial fixation process. Both processes have a high energetic cost. The application of these fertilizers has been taking place in a plethoric way under the premise: supplement increase, equal to production increase. This assumption has had considerable environmental and economic costs, so, currently, strategies are being developed to allow for a reasonable supplementation of nitrogen to crops; to help crops reach their maximum production potential and, at the same time, preserving the environment.

Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR.

A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-microm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the alpha- and gamma-subdivisions of the division Proteobacteria (alpha- and gamma-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, alpha-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

Genetic Analysis of nif Regulatory Genes by Utilizing the Yeast Two-Hybrid System Detected Formation of a NifL-NifA Complex That Is Implicated in Regulated Expression of nif Genes

In diazotrophic organisms, nitrogenase synthesis and activity are tightly regulated. Two genes, nifL and nifA, are implicated as playing a major role in this regulation. NifA is a transcriptional activator, and its activity is inhibited by NifL in response to availability of excess fixed nitrogen and high O(2) tension. It was postulated that NifL binds to NifA to inhibit NifA-mediated transcriptional activation of nif genes. Mutational analysis combined with transcriptional activation studies clearly is in agreement with the proposal that NifL interacts with NifA. However, several attempts to identify NifA-NifL interactions by using methods such as coimmunoprecipitations and chemical cross-linking experiments failed to detect direct interactions between these proteins. Here we have taken a genetic approach, the use of a yeast two-hybrid protein-protein interaction assay system, to investigate NifL interaction with NifA. A DNA fragment corresponding to the kinase-like domain of nifL was PCR amplified and was used to generate translation fusions with the DNA binding domain and the DNA activation domain of the yeast transcriptional activator GAL4 in yeast two-hybrid vectors. Similarly, a DNA fragment corresponding to the catalytic domain of nifA was PCR amplified and used to generate translation fusions with the DNA-binding domain and the DNA-activation domain of GAL4 in yeast two-hybrid vectors. After introducing appropriate plasmid combinations in yeast cells, the existance of direct interaction between NifA and NifL was analyzed with the MATCHMAKER yeast two-hybrid system by testing for the expression of lacZ and his3 genes. These analyses showed that the kinase-like domain of NifL directly interacts with the catalytic domain of NifA.

Metalloclusters of the nitrogenases.

Biological nitrogen fixation, the reduction of dinitrogen to ammonia, is catalysed by nitrogenases. These enzymes are found in relatively few groups of bacteria and until recently it appeared that N2 fixation occurred by a single route involving a molybdenum-containing enzyme. However, it is now clear that Mo is not an essential metal for N2 fixation. The soil bacterium Azotobacter chroococcum has an Mo-containing nitrogenase with properties very similar to those found in many other diazotrophic organisms, but it also has a vanadium-containing nitrogenase which functions in the presence of V if Mo is not available. The closely related species A. vinelandii has, in addition to Mo and V nitrogenases, a third nitrogenase which functions only under conditions where Mo and V levels are very low [1].

Nitrogen fixation associated with grasses and cereals: Recent results and perspectives for future research

Over the last few years research in the area of biological nitrogen fixation (BNF) associated with cereals and grasses has become divided into two areas. On the one hand there have been a large number of reports of responses of field-grown plants to inoculation with N2-fixing bacteria, principallyAzospirillum spp. On the other hand there have been several reports of significant contributions of associated BNF to the nutrition of several crops, including wetland rice, sugar cane and some forage grasses. However, where BNF contributions have definitely been established no certain information is available as to the diazotrophic organisms responsible. Furthermore, certain recent reports indicate that, at least in some cases, responses of plants to inoculation withAzospirillum spp. have been shown not to be due to BNF contributions.