Rates Of Nitrogenase Activity Research Articles

Studies Using Mutant Strains of Azospirillum brasilense Reveal That Atmospheric Nitrogen Fixation and Auxin Production Are Light Dependent Processes.

As the use of microbial inoculants in agriculture rises, it becomes important to understand how the environment may influence microbial ability to promote plant growth. This work examines whether there are light dependencies in the biological functions of Azospirillum brasilense, a commercialized prolific grass-root colonizer. Though classically defined as non-phototrophic, A. brasilense possesses photoreceptors that could perceive light conducted through its host's roots. Here, we examined the light dependency of atmospheric biological nitrogen fixation (BNF) and auxin biosynthesis along with supporting processes including ATP biosynthesis, and iron and manganese uptake. Functional mutants of A. brasilense were studied in light and dark environments: HM053 (high BNF and auxin production), ipdC (capable of BNF, deficient in auxin production), and FP10 (capable of auxin production, deficient in BNF). HM053 exhibited the highest rate of nitrogenase activity with the greatest light dependency comparing iterations in light and dark environments. The ipdC mutant showed similar behavior with relatively lower nitrogenase activity observed, while FP10 did not show a light dependency. Auxin biosynthesis showed strong light dependencies in HM053 and FP10 strains, but not for ipdC. Ferrous iron is involved in BNF, and a light dependency was observed for microbial 59Fe2+ uptake in HM053 and ipdC, but not FP10. Surprisingly, a light dependency for 52Mn2+ uptake was only observed in ipdC. Finally, ATP biosynthesis was sensitive to light across all three mutants favoring blue light over red light compared to darkness with observed ATP levels in descending order for HM053 > ipdC > FP10.

Loss of nitrogen fixing capacity in a montane lichen is linked to increased nitrogen deposition

AbstractThe circumboreal/circumpolar N2‐fixing lichenStereocaulon vesuvianumis among the most widespread and abundant fruticose species in montane Britain but has lost the capacity to fix N2over large areas of the country.To investigate whether loss of N2‐fixation inS. vesuvianumis linked to increased N deposition, we examined thallus morphology, physiology and chemistry at twelve locations representing an N deposition gradient of 3–40 kg ha−1 year−1. Measurements were made in parallel on a non‐N2‐fixing reference species (Parmelia saxatilis). The presence or absence of cephalodia (N2‐fixing nodules containing the cyanobacteriumStigonemasp) was recorded in over 500 herbarium specimens ofS. vesuvianumdating back to 1820.Cephalodium abundance inS. vesuvianum, and15N concentration inS. vesuvianumandP. saxatilis, were strongly negatively correlated with N deposition and particularly with dry deposited N; cephalodia do not form at total N deposition rates ≥8–9 kg ha−1 year−1. Other morphological oddities inS. vesuvianumat N‐polluted sites include increased apothecium (fungal reproductive structure) production and green algal biofilm development. Biofilm covered thalli without cephalodia lacked nitrogenase activity and cephalodia at sites where they rarely develop had nitrogenase activities typical for this species. The presence or absence of cephalodia in herbarium specimens ofS. vesuvianumsuggest that the present‐day N‐deposition linked gradient in N2‐fixing capacity did not exist in the 19th century and largely developed between 1900–1940.Synthesis.We provide clear evidence that N2‐fixing capacity inS. vesuvianumhas been lost in regions subjected to many decades of enhanced atmospheric N deposition. This loss is consistent with established models of diazotrophy, which identify supply of combined N as an inhibitor of N2‐fixation. Progressive depletion of thallus15N with increasing N deposition is in line with available data indicating that much atmospheric N pollution is15N‐depleted. Rates of nitrogenase activity inS. vesuvianumare low compared to other symbiotic systems and perhaps more likely supplanted by elevated N deposition. We suggest that other ecosystem compartments with low rates of fixation (e.g. soils) might also be susceptible to N pollution and merit investigation.

Antenna Modification Leads to Enhanced Nitrogenase Activity in a High Light-Tolerant Cyanobacterium.

ABSTRACTBiological nitrogen fixation is an energy-intensive process that contributes significantly toward supporting life on this planet. Among nitrogen-fixing organisms, cyanobacteria remain unrivaled in their ability to fuel the energetically expensive nitrogenase reaction with photosynthetically harnessed solar energy. In heterocystous cyanobacteria, light-driven, photosystem I (PSI)-mediated ATP synthesis plays a key role in propelling the nitrogenase reaction. Efficient light transfer to the photosystems relies on phycobilisomes (PBS), the major antenna protein complexes. PBS undergo degradation as a natural response to nitrogen starvation. Upon nitrogen availability, these proteins are resynthesized back to normal levels in vegetative cells, but their occurrence and function in heterocysts remain inconclusive. Anabaena 33047 is a heterocystous cyanobacterium that thrives under high light, harbors larger amounts of PBS in its heterocysts, and fixes nitrogen at higher rates compared to other heterocystous cyanobacteria. To assess the relationship between PBS in heterocysts and nitrogenase function, we engineered a strain that retains large amounts of the antenna proteins in its heterocysts. Intriguingly, under high light intensities, the engineered strain exhibited unusually high rates of nitrogenase activity compared to the wild type. Spectroscopic analysis revealed altered PSI kinetics in the mutant with increased cyclic electron flow around PSI, a route that contributes to ATP generation and nitrogenase activity in heterocysts. Retaining higher levels of PBS in heterocysts appears to be an effective strategy to enhance nitrogenase function in cyanobacteria that are equipped with the machinery to operate under high light intensities.

Microbial Nitrogen Fixation in the Intestine of Tipulidae Tipula maxima Larvae

Nitrogen-fixing activity of microorganisms in the intestines of Crane fly (Tipulidae) larvae and their influence on the processes of microbial nitrogen fixation in soil have been studied. Two independent methods have shown high rates of nitrogenase activity in the digestive tract of larvae, which is determined mainly by transit nitrogen-fixing microorganisms. Nitrogen fixers actively developing in the intestines of larvae stimulate a sharp increase in nitrogenase activity in the soil: after 3 months of incubation, the activity increases eightfold; it can contribute to the accumulation of nitrogen in the habitats of the larvae.

ACTIVITY OF NITROGEN FIXATION IN LUCERNE AGROCENOSES AND PRODUCTIVITY OF CULTURE UNDER INFLUENCE OF FERTILIZERS AND MICROBIAL PREPARATION

In a long field experiment on common black soil, the influence of various fertilization systems and presowing bacterization of seeds on the activity of symbiotic nitrogen fixation in lucerne agrocenoses and crop productivity was studied. Stimulation of the process of fixation of atmospheric nitrogen was observed when mineral fertilizers were used in the doses not exceeding N60P60K60, when they were introduced in the year of sowing. High rates of nitrogenase activity were noted in conditions of aftereffect of manure, compost, organic mineral fertilizer (manure + N30P30K30), as well as systematic use of crop residues in the crop rotation. The plant mass in above variants was characterized by high protein content. At the same time, the greatest influence on protein synthesis was provided by the use of a microbial preparation. The combination of ecologically acceptable agricultural backgrounds with presowing bacterization of lucerne seeds ensured the obtaining of parameters of crop productivity.

The study of symbiotic traits – nodulation and activity of nitrogen fixation – in different cultivars of spring vetch (Vicia sativa L.)

Common vetch Vicia sativa L. is one of the major annual fodder legume crops in Russia. Like other legumes, it can enter into symbiosis with nodule bacteria of the genus Rhizobium and to fix molecular nitrogen from the air. This ability contributes to the enrichment of soils with nitrogen and the possibility of growing crops on poor soils. Vetch symbiotic genes have not yet been identified, so the breeding of this crop is confined to the assessment of breeding material with regard to nodulation and very rarely to the activity of nitrogen fixation measured by the acetylene method. This paper presents the results of studies of nodulation and nitrogen-fixing activity that we conducted with four spring vetch cultivars raised at the Siberian Research Institute for Plant Industry and Breeding. Two varieties of forage pea, namely Druzhnaya and traditional cv. Falenskaya 42, were taken as references. The activity of nitrogen fixation was assessed from nitrogenase activity, assayed by the acetylene method. The study revealed significant varietal differences in nodulation and nitrogen fixation, determined on a gas chromatograph Tsvet 500, Russia. The nitrogenase activity rates in vetch cultivars under study varied from 2811 to 6890 С2Н4 nmol/ plant/h. Two of them, Lena and Priobskaya 25, were prominent in high nodulation nitrogen fixation rates. Nodulation in the two reference pea cultivars was weaker than in vetch. However, their nitrogen fixation activities were higher.

Nitrogen fixation in shallow‐water sediments: Spatial distribution and controlling factors

Nitrogenase activity (NA) in shallow‐water (< 1 m) sediments was investigated at 60 randomly selected sites along a 150 km stretch on the brackish‐water Swedish west coast, without targeting any specific type of sediments, such as microbial mats. Benthic nitrogen (N) fixation and diazotrophs (nifH genes) were found at all sites, regardless of the presence of cyanobacterial or microbial mats. The majority of sites showed N fixation rates between 0.03 and 1 mmol N m−2 d−1. These rates were similar to those of benthic denitrification previously measured in the area. Maximum rates up to 3.4 mmol N m−2 d−1 were measured. A structural equation model was used to investigate direct and indirect effects of biogeochemical and physical factors on NA. Number of nifH genes had the largest direct positive influence on NA, whereas increasing wave exposure had an indirect negative effect on NA through its influence on the diazotrophic abundance. Increased salinity, previously been shown to suppress NA in coastal waters, was found to directly stimulate benthic N fixation, likely by generating favorable conditions for diazotrophic sulfate‐reducing bacteria. Our field data confirmed previously observed negative effects of dissolved inorganic nitrogen on NA, which have so far mainly been experimentally studied. Both NA rates and the number of nifH genes correlated positively with pore‐water dissolved inorganic phosphorus concentrations. These findings show that the potential for N fixation in illuminated sediments can be considerable, stretching beyond cyanobacterial mats, being controlled by complex interactions between biotic and abiotic factors.

Biochar application reduces nodulation but increases nitrogenase activity in clover

Biochar is produced from the pyrolysis of organic materials, and when buried in soil can act as a long term soil carbon (C) store. Evidence suggests that biochar can also increase crop yields, reduce nutrient leaching and increase biological nitrogen fixation in leguminous plants. However, the potential for increasing biological N2 fixation in agroecosystems is poorly understood, with inconsistent reports of root nodulation following biochar application. Therefore, the aim of this study was to determine the effect of biochar application rate and time since application on nodulation and nitrogenase activity in nodules of clover grown in a temperate agricultural soil. We used replicated field plots with three biochar application rates (0, 25 and 50 t ha−1). Three years after biochar amendment, the plots were further split and fresh biochar added at two different rates (25 and 50 t ha−1) resulting in double-loaded reapplications of 25 + 25 and 50 + 50 t ha−1. Three years after biochar application, there was no significant difference in the total number of root nodules between biochar-amended and unamended soil, regardless of the application rate. However, despite clover root nodules being of a similar number and size the level of nitrogenase activity of individual nodules in biochar-amended soil was significantly higher than in unamended soil. Reapplication of biochar resulted in decreased nodulation, although the rate of nitrogenase activity per nodule remained unaffected. In the short term, biochar influences root nodule number and localised N2 fixation per nodule; however, total nitrogenase activity for the whole root system remained unaffected by the application rate of biochar or time since its application. These results emphasise the importance of long-term field studies, with a variety of applications rates for determining the influence of biochar applications on N2-fixing organisms and in providing data that can meaningfully inform agronomic management decisions and climate change mitigation strategies.

N<sub>2 fixation rates and associated diversity (nifH) of microbialite and mat-forming consortia from different aquatic environments in Mexico

The present study describes patterns and rates of N2 fixation and associated diversity (based on the nifH gene) of microbialite and mat-forming bacterial consortia from different aquatic environments in Mexico. All of the communities shared a diurnal pattern of N2 fixation with peak activity during the day while showing a unique genetic composition. Our study sug- gests the importance of heterocystous cyanobacteria as the main diazotrophic organisms, although nifH sequences were also affiliated to Oscillatoriales, Chroococcales, and several Pro- teobacteria. Genetic composition did not relate to the environmental variables analysed, although rates of nitrogenase activity associated to microbial C and N explained 11% of the variation among consortia.

Nitrogen fixation by a marine non‐heterocystous cyanobacterium requires a heterotrophic bacterial consort

Cultures of the non-heterocystous cyanobacterium, Leptolyngbya nodulosa, could be grown indefinitely in media devoid of combined nitrogen. Acetylene reduction assays showed that these cultures fixed nitrogen in the dark period of a diurnal cycle under micro-oxygenic or anaerobic conditions. Addition of DCMU to cultures induced much higher rates of nitrogenase activity, most of which occurred in the light. Measurements of activity in the presence of chloramphenicol indicated that nitrogenase is synthesized in darkness and probably destroyed in the subsequent light period. Neither the dark-mediated nitrogenase in the absence of DCMU nor light-mediated activity in the presence of DCMU could be sustained for more than 3 days without a photoperiodic light/dark cycle. Axenic cultures could not be grown in the absence of combined nitrogen and did not demonstrate any acetylene reduction activity. An identical nifH gene sequence was found in axenic and non-axenic cultures of L. nodulosa. RT-PCR demonstrated that this gene was expressed only in non-axenic cultures. Western blotting showed that the Fe-protein of nitrogenase is absent in cultures that are incapable of acetylene reduction, indicating that the lack of nitrogenase activity is likely due to the absence of the enzyme. These observations strongly indicate that L. nodulosa contains a functional nitrogenase which is not expressed in the absence of heterotrophic bacteria.

Comparison of Nitrogen Fixation for North- and South-facing Robinia pseudoacacia Stands in Central Korea

The nitrogenase activity, root nodule biomass, and rates of nitrogen (N) fixation were measured in 25-year-old pure north- and south-facing Robinia pseudoacacia stands in an urban forest of Seoul (Kkachisan Mountain) in central Korea. The nitrogenase activity was estimated using an acetylene reduction (AR) assay, which showed an increasing trend during the early growing season, with sustained high rates from June through to September with a decrease thereafter. July had the highest nitrogenase activity rate (micromoles C2H4 per gram dry nodule per hour), averaging 95.8 and 115.1 for the north- and south-facing stands, respectively. The maximum root nodule biomass (kilograms per hectare) was 45.7 and 9.1 for the north- and south-facing stands in July, respectively. The AR rate appeared to be strongly correlated to the soil temperature (r 2 = 0.68, P < 0.001) and soil pH (r 2 = 0.59, P < 0.001) while root nodule biomass was correlated to the soil temperature (r 2 = 0.36, P < 0.01) and water content (r 2 = 0.35, P < 0.05). The soil temperature showed clear differences between seasons, while there was a significant difference in soil pH, organic matter, total N concentrations, and available phosphorus between the north- and south-facing stands. The N2 fixation rates during the growing season varied from 0.1 to 37.5 kg N ha−1 month−1 depending on the sampling location and time. The annual N2 fixation rate (kg N per hectare per year) was 112.3 and 23.2 for the north- and south-facing stands, respectively. The differences in N2 fixation rate between the two stands were due mainly to the differences in total nodule biomass.

Biological Nitrogen Fixation in Two Tropical Forests: Ecosystem-Level Patterns and Effects of Nitrogen Fertilization

Humid tropical forests are often characterized by large nitrogen (N) pools, and are known to have large potential N losses. Although rarely measured, tropical forests likely maintain considerable biological N fixation (BNF) to balance N losses. We estimated inputs of N via BNF by free-living microbes for two tropical forests in Puerto Rico, and assessed the response to increased N availability using an on-going N fertilization experiment. Nitrogenase activity was measured across forest strata, including the soil, forest floor, mosses, canopy epiphylls, and lichens using acetylene (C2H2) reduction assays. BNF varied significantly among ecosystem compartments in both forests. Mosses had the highest rates of nitrogenase activity per gram of sample, with 11 ± 6 nmol C2H2 reduced/g dry weight/h (mean ± SE) in a lower elevation forest, and 6 ± 1 nmol C2H2/g/h in an upper elevation forest. We calculated potential N fluxes via BNF to each forest compartment using surveys of standing stocks. Soils and mosses provided the largest potential inputs of N via BNF to these ecosystems. Summing all components, total background BNF inputs were 120 ± 29 μg N/m2/h in the lower elevation forest, and 95 ± 15 μg N/m2/h in the upper elevation forest, with added N significantly suppressing BNF in soils and forest floor. Moisture content was significantly positively correlated with BNF rates for soils and the forest floor. We conclude that BNF is an active biological process across forest strata for these tropical forests, and is likely to be sensitive to increases in N deposition in tropical regions.

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

Endophytic colonization ofTypha australisby a plant growth-promoting bacteriumKlebsiella oxytocastrain GR-3

To isolate and characterize endophytic diazotrophic bacteria from a semi-aquatic grass (Typha australis) which grows luxuriantly with no addition of any nitrogen source. Ten endophytic diazotrophic bacteria from surface-sterilized roots and culm of T. australis were isolated and screened for plant growth-promoting activities employing standard methods. Based on the rate of nitrogenase activity, indole acetic acid (IAA) production and phosphate (P) solubilization, one root isolate namely GR-3 was found to be the most efficient one. This isolate was identified as Klebsiella oxytoca on the basis of 16S rDNA sequence analysis. Amplification of nifH by polymerase chain reaction (PCR) and detection of dinitrogenase reductase by western blot confirmed the diazotrophic nature of GR-3. It was tagged with gusA fused to a constitutive promoter and the resulting transconjugant was inoculated onto endophyte-free rice variety Malviya dhan-36 seedlings to express cross-infection ability which resulted in a significant increase in root/shoot length and chlorophyll a content. Roots and culm of T. australis harbour several endophytic diazotrophic bacteria. One root isolate, identified as K. oxytoca GR-3, seems to be an efficient plant growth-promoting bacterium. Plant growth-promoting properties of GR-3 suggest that this promising isolate merits further investigations for potential application in agriculture.

Pesticides reduce symbiotic efficiency of nitrogen-fixing rhizobia and host plants.

Unprecedented agricultural intensification and increased crop yield will be necessary to feed the burgeoning world population, whose global food demand is projected to double in the next 50 years. Although grain production has doubled in the past four decades, largely because of the widespread use of synthetic nitrogenous fertilizers, pesticides, and irrigation promoted by the "Green Revolution," this rate of increased agricultural output is unsustainable because of declining crop yields and environmental impacts of modern agricultural practices. The last 20 years have seen diminishing returns in crop yield in response to increased application of fertilizers, which cannot be completely explained by current ecological models. A common strategy to reduce dependence on nitrogenous fertilizers is the production of leguminous crops, which fix atmospheric nitrogen via symbiosis with nitrogen-fixing rhizobia bacteria, in rotation with nonleguminous crops. Here we show previously undescribed in vivo evidence that a subset of organochlorine pesticides, agrichemicals, and environmental contaminants induces a symbiotic phenotype of inhibited or delayed recruitment of rhizobia bacteria to host plant roots, fewer root nodules produced, lower rates of nitrogenase activity, and a reduction in overall plant yield at time of harvest. The environmental consequences of synthetic chemicals compromising symbiotic nitrogen fixation are increased dependence on synthetic nitrogenous fertilizer, reduced soil fertility, and unsustainable long-term crop yields.

Seasonal changes in periphyton nitrogen fixation in a protected tropical wetland

Cyanobacteria are important for global nitrogen cycle and often form complex associations referred to as cyanobacterial mats or periphyton that are common in tropical, limestone-based wetlands. The objective of this study was to monitor the nitrogen fixation rate using the acetylene reduction assay of these cyanobacterial mats in a tropical, unfertilized, and protected wetland. To account for temporal and spatial variation of nitrogenase activity, we were interested in seasons in a hydrological cycle (dry, rains, and end of rains), sites with different vascular vegetation, and rates of nitrogenase activity in a 24-h cycle. The annual average of nitrogenase activity was 22 nmol C2H4 cm−2 h−1, with a range of <6 to 35 nmol C2H4 cm−2 h−1, and the annual nitrogen fixation rate of our study site (9.0 g N m−2 year−1) is higher than similar estimates from other freshwater wetlands. There was a clear temporal pattern in nitrogenase activity with a maximum rate occurring during the rainy season (August) and a maximum nitrogenase activity occurring between 0600 and 1200 hours. We found spatial differences in nitrogenase activity among the four sites that could be attributed to variations in species composition within the periphyton.

Modeling the dynamic regulation of nitrogen fixation in the cyanobacterium Trichodesmium sp.

A physiological, unbalanced model is presented that explicitly describes growth of the marine cyanobacterium Trichodesmium sp. at the expense of N(2) (diazotrophy). The model involves the dynamics of intracellular reserves of carbon and nitrogen and allows the uncoupling of the metabolism of these elements. The results show the transient dynamics of N(2) fixation when combined nitrogen (NO(3)(-), NH(4)(+)) is available and the increased rate of N(2) fixation when combined nitrogen is insufficient to cover the demand. The daily N(2) fixation pattern that emerges from the model agrees with measurements of rates of nitrogenase activity in laboratory cultures of Trichodesmium sp. Model simulations explored the influence of irradiance levels and the length of the light period on fixation activity and cellular carbon and nitrogen stoichiometry. Changes in the cellular C/N ratio resulted from allocations of carbon to different cell compartments as demanded by the growth of the organism. The model shows that carbon availability is a simple and efficient mechanism to regulate the balance of carbon and nitrogen fixed (C/N ratio) in filaments of cells. The lowest C/N ratios were obtained when the light regime closely matched nitrogenase dynamics.

Nitrogen fixation in coarse woody debris of Thuja plicata and Tsuga heterophylla forests on northern Vancouver Island

Asymbiotic nitrogenase activity in coarse woody debris was measured using the acetylene reduction assay under ambient conditions in three different stand ages (5, 53, and 88 years old) of an unmanaged second-growth Tsuga heterophylla (Raf.) Sarg. – Abies amabilis (Dougl. ex Loud.) Dougl. ex J. Forbes forest type and a Thuja plicata Donn. ex D. Don – Tsuga heterophylla old-growth forest on northern Vancouver Island, British Columbia, Canada. Four different decay classes of coarse woody debris, different species in the early decay stages, and sapwood and heart wood were sampled separately. Mean nitrogenase activity ranged between 1.3 and 19.5 nmol C2H4·d–1·(g dry mass)–1, with an overall mean of 5.7. High variability of the activity rates between logs and within logs was observed in all four stands. Mean activity rates were, in most cases, significantly different between decay classes, with generally increasing nitrogenase activity with the progress of decay. Moisture content of the samples was a good predictor of nitrogenase activity and could explain differences between decay classes. Only minor differences in nitrogenase activity were found between the different stands. Estimates of nitrogen fixation ranged from 1.0 to 2.1 kg N·ha–1·year–1, the magnitude of these values depending more on the mass of coarse woody debris substrate available for asymbiotic nitrogen-fixing bacteria (103–158 t·ha–1 in this study) than on differences in nitrogenase activity rates. The measured nitrogenase activity and the resultant estimates of nitrogen fixation are among the highest values reported in the literature.

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N2-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO3− or NH4+, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO3− and NH4+. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.

Effects of O2 on N2 fixation in heterocystous cyanobacteria from the Baltic Sea

The effect of O2 on nitrogenase activity in natural samples of heterocystous cyanobacte- ria from the Baltic Sea was studied using on-line laser photo-acoustic trace-gas detection. This technique records nitrogenase activity in near real-time and allows measurements in continuously changing O2 concentrations. Our results showed that under non-steady state conditions the optimum concentration of O2 for N2 fixation differed from that at steady-state O2 levels. The optimum O2 concentration depended upon whether the O2 concentration was increasing or decreasing, with decreasing concentrations yielding higher O2 optima for dark nitrogenase activity than increasing O2 concentrations. The cyanobacteria rapidly adapt to changes in O2, and therefore measurements also reflect the history of O2 concentrations to which organisms have been exposed. Steady-state and non- steady-state O2 concentrations both decreased their optimum O2 concentration for nitrogenase activity rates with increasing irradiance. However, the optimum O2 concentration was always higher than zero, even at saturating irradiances. Hence, it appears that low levels of O2 are an obligatory requirement for maximum nitrogenase activity in the field. Low levels of respiration served as a source of additional energy, suggesting that even at light saturation, photosynthetic energy genera- tion in the heterocyst can not saturate the demand of nitrogenase for ATP. The large changes in nitro- genase activity due to the combined effect of variations in O2 concentration and light emphasize the necessity of including these effects in models that calculate the daily integral of N2 fixation.