Simultaneous bioprecipitation of cadmium to cadmium sulfide nanoparticles and nitrogen fixation by Rhodopseudomonas palustris TN110.

This study investigated the abilities of a purple non-sulfur bacterium, Rhodopseudomonas palustris TN110 to bioremediate cadmium through the biosynthesis of CdS nanoparticles and to fix nitrogen simultaneously. Under microaerobic-light conditions, R. palustris TN110 synthesized CdS nanoparticles. The produced CdS nanoparticles had a spherical shape and an average size of 4.85 nm. The Fourier transform infrared spectrum of the nanoparticles reveals the carbonyl groups, bending vibrations of the amide I and II bands of proteins, and CN stretching vibrations of aromatic and aliphatic amines. These bands and groups suggest protein capping/binding on the surface of the nanoparticles. R. palustris TN110 converted 25.61% of 0.2 mM CdCl2 to CdS nanoparticles under optimal conditions (pH 7.5, 30 °C and 3000 lux). The half maximal inhibitory concentration (IC50) value of the produced CdS nanoparticles was 1.76 mM. The produced CdS nanoparticles at IC50 up-regulated two genes associated with nitrogen fixation: Mo-Fe nitrogenase gene (nifH) and V-Fe nitrogenase gene (vnfG) at 2.83 and 2.27 fold changes, respectively. On the contrary, the produced CdS nanoparticles slightly down-regulated Fe-Fe nitrogenase gene (anfG). The amounts of ammonia released by the strain support the gene expression results. R. palustris TN110 has great potential to serve concurrently as a cadmium bioremediation agent and a nitrogen fixer. The strain could be beneficial to paddy fields that are contaminated with Cd through run off from mining and chemical fertilizer applications.

Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness.

Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness, the so-called switch-off effect. This is due to reversible modification of the Fe-protein, one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to P(II) protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.

Effects of monosulfuron on growth, photosynthesis, and nitrogenase activity of three nitrogen-fixing cyanobacteria.

Application of monosulfuron, a new sulfonylurea herbicide, produced a simulative effect on heterocyst formation and nitrogenase activity but an inhibitory effect on photosynthesis, i.e., a lower net photosynthetic rate, fewer photosynthetic pigments, and a smaller Fv/Fm ratio at increasingly higher monosulfuron concentrations (0.001-10 mg/l) for three nonspecific filamentous nitrogen-fixing cyanobacteria: Anabaena azollae, A. flos-aquae, and A. azotica. The decrease in biliprotein of algal cells was less than that of carotenoid and chlorophyll-a. Monosulfuron was more readily degraded and less accumulated in A. azotica compared with A. azollae and A. flos-aquae. The three algae exhibited varying degrees of sensitivity to monosulfuron: Calculated 50% inhibition concentrations (IC(50)s) of algal growth and no observed-effect concentration (NOEC) values after 4 days of treatment were 0.014 and 0.005, 0.029 and 0.019, and 0.22 and 0.075 mg/l for A. flos-aquae, A. azollae, and A. azotica, respectively. Normal agricultural use of monosulfuron at postemergence rates of 0.3-0.8 mg/l in rice fields will likely be toxic to these three ubiquitous nitrogen-fixing cyanobacteria. Low-dose monosulfuron application (<0.1 mg/l) enables growth of the more tolerant A. azotica as biofertilizer, and the use of photosynthetic efficiency and growth rates as sensitive-indicator indexes of toxicity to nitrogen-fixing cyanobacteria are recommended.

Interactions between CCM and N2 fixation in Trichodesmium.

In view of the current increase in atmospheric pCO(2) and concomitant changes in the marine environment, it is crucial to assess, understand, and predict future responses of ecologically relevant phytoplankton species. The diazotrophic cyanobacterium Trichodesmium erythraeum was found to respond strongly to elevated pCO(2) by increasing growth, production rates, and N(2) fixation. The magnitude of these CO(2) effects exceeds those previously seen in other phytoplankton, raising the question about the underlying mechanisms. Here, we review recent publications on metabolic pathways of Trichodesmium from a gene transcription level to the protein activities and energy fluxes. Diurnal patterns of nitrogenase activity change markedly with CO(2) availability, causing higher diel N(2) fixation rates under elevated pCO(2). The observed responses to elevated pCO(2) could not be attributed to enhanced energy generation via gross photosynthesis, although there are indications for CO(2)-dependent changes in ATP/NADPH + H(+) production. The CO(2) concentrating mechanism (CCM) in Trichodesmium is primarily based on HCO(3)(-) uptake. Although only little CO(2) uptake was detected, the NDH complex seems to play a crucial role in internal cycling of inorganic carbon, especially under elevated pCO(2). Affinities for inorganic carbon change over the day, closely following the pattern in N(2) fixation, and generally decrease with increasing pCO(2). This down-regulation of CCM activity and the simultaneously enhanced N(2) fixation point to a shift in energy allocation from carbon acquisition to N(2) fixation under elevated pCO(2) levels. A strong light modulation of CO(2) effects further corroborates the role of energy fluxes as a key to understand the responses of Trichodesmium.

Sewage sludge application can induce changes in antioxidant status of nodulated alfalfa plants.

A greenhouse experiment was conducted to investigate the oxidative stress produced by sewage sludge addition on nodulated alfalfa (Medicago sativa L. cv. Aragón) plants. Two types of sludge were incorporated into substrate: anaerobic mesophilic digested (AM) and autothermal thermophilic aerobic digested (ATAD) sludge. Pots without sludge but with inoculated plants were used as control treatment for comparison. Results showed that sludge amended plants had increased tissue accumulation of heavy metals that induced oxidative stress. This is characterized by induction of the antioxidant enzymatic activities and alterations in the redox state of ascorbate. ATAD sludge application produced a reduction in nodulation, increased nodule antioxidant enzyme activities and decreased ascorbate/dehydroascorbate ratio. As a consequence, nodules of ATAD treatment suffered from oxidative damages as evidenced by high malondialdehyde levels. By contrast, AM application enhanced plant growth and no deleterious effects on nodulation were found. Nodules developed in AM sludge had increased antioxidant enzyme activities, ascorbate/dehydroascorbate ratio and improved capacity for thiol synthesis. Results clearly showed that nodulated alfalfa performed better in AM than in ATAD sludge and suggest that differential response appears to be mediated by plant ability to thiol synthesis and to maintenance of a more equilibrated antioxidant status.

Effects of herbicide butachlor on soil microorganisms and on nitrogen-fixing abilities in paddy soil.

The composition of culture-independent microbial communities and the change of nitrogenase activities under the application of butachlor in paddy soil were investigated. Nitrogen-fixation ability was expressed by the amount of acetylene reduction, and changes of microbial communities were studied by using denaturing gradient gel electrophoresis (DGGE) technique; afterward, minimum distance (MD, in brief) statistics was applied to determine the cluster numbers in UPGMA dendrograms. The results showed that the reduction of acetylene was suppressed shortly after butachlor application but was augmented after 37 days in both upper and lower layer soils. From UPGMA dendrograms, the diazotrophic divergences ranged from 33% to 64% throughout rice growth stages. For general bacterial communities, the diversities ranged from 28% to 52%. The divergences became higher with the cultivation period, and the application of butachlor imposed a significant variation on microbial community shift, which may be a reason for the boosting nitrogen-fixation ability in paddy soils.

RbrA, a cyanobacterial rubrerythrin, functions as a FNR-dependent peroxidase in heterocysts in protection of nitrogenase from damage by hydrogen peroxide in Anabaena sp. PCC 7120.

The heterocyst is a specialized cell for nitrogen fixation in some filamentous cyanobacteria. Here we report that a rubrerythrin (RbrA) from Anabaena sp. PCC 7120 functions as a peroxidase in heterocysts and plays an important role in protection of nitrogenase. The electron donor for RbrA in H(2)O(2) reduction is NADPH and the electron transfer from NADPH to RbrA depends on ferredoxin:NADP(+) oxidoreductase. A rbrA mutant (r27) grew much more slowly than the wild type under diazotrophic conditions. Its nitrogenase activity measured in air was only 8% of that measured under anoxic conditions. Staining r27 filaments with 2',7'-dichlorodihydrofluorescein diacetate indicated that heterocysts had a higher H(2)O(2) concentration than the vegetative cells. The expression of rbrA was controlled by two promoters and the promoter for the smaller transcript was regulated by HetR. Spatial expression of rbrA was studied and the results showed that the transcription is localized predominantly in heterocysts. In a mutant lacking nifH and rbrA, the H(2)O(2) concentration in heterocysts was lower than that in the vegetative cells, suggesting that NifH is involved in H(2)O(2) generation. Our results demonstrate that RbrA is a critical enzyme for H(2)O(2) decomposition and provide evidence that nitrogenase autoprotection is important in heterocysts.

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase.

Posttranslational regulation of nitrogenase, or switch-off, in the methanogenic archaeon Methanococcus maripaludis requires both nifI(1) and nifI(2), which encode members of the PII family of nitrogen-regulatory proteins. Previous work demonstrated that nitrogenase activity in cell extracts was inhibited in the presence of NifI(1) and NifI(2), and that 2-oxoglutarate (2OG), a potential signal of nitrogen limitation, relieved this inhibition. To further explore the role of the NifI proteins in switch-off, we found proteins that interact with NifI(1) and NifI(2) and determined whether 2OG affected these interactions. Anaerobic purification of His-tagged NifI(2) resulted in copurification of NifI(1) and the dinitrogenase subunits NifD and NifK, and 2OG or a deletion mutation affecting the T-loop of NifI(2) prevented copurification of dinitrogenase but did not affect copurification of NifI(1). Similar results were obtained with His-tagged NifI(1). Gel-filtration chromatography demonstrated an interaction between purified NifI(1,2) and dinitrogenase that was inhibited by 2OG. The NifI proteins themselves formed a complex of approximately 85 kDa, which appeared to further oligomerize in the presence of 2OG. NifI(1,2) inhibited activity of purified nitrogenase when present in a 1:1 molar ratio to dinitrogenase, and 2OG fully relieved this inhibition. These results suggest a model for switch-off of nitrogenase activity, where direct interaction of a NifI(1,2) complex with dinitrogenase causes inhibition, which is relieved by 2OG. The presence of nifI(1) and nifI(2) in the nif operons of all nitrogen-fixing Archaea and some anaerobic Bacteria suggests that this mode of nitrogenase regulation may operate in a wide variety of diazotrophs.

[Calcium as a control factor of nitrogenase activity in Vicia faba L. root nodules: calcium release from symbiosomes and the accompanying decrease in their nitrogenase activity is blocked by verapamil]. / Kal'tsii v kontrole nitrogenasnoi aktivnosti kornevykh kluben'kov Vicia faba L.: vykhod kal'tsiia iz simbiosom i soputstvuiushchee emu snizhenie ikh nitrogenaznoi aktivnosti blokiruiutsia verapamilom.

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron microscopy demonstrates a considerable calcium depletion in symbiosomes of the infected nodule cells by EGTA and A23187. Ca2+ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca2+ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached the baseline in symbiosomes. The experiments on isolated symbiosomes and arsenazo III demonstrated that verapamil inhibited Ca2+ transport induced by valinomycin at the background of K+ ions. These data suggest the presence of a verapamil-sensitive transporter on the peribacteroid membrane responsible for Ca2+ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of the root nodules is discussed.

Effect of some abiotic factors on the biological activity of Gluconacetobacter diazotrophicus.

AIMS: The effect of some abiotic factors, dryness, heat and salinity on the growth and biological activity of Gluconacetobacter diazotrophicus, and the influence of a salt stress on some enzymes involved in carbon metabolism of these bacteria is studied under laboratory conditions. METHODS AND RESULTS: Strain PAL-5 of G. diazotrophicus was incubated under different conditions of drying, heat and salinity. Cells showed tolerance to heat treatments and salt concentrations, and sensitivity to drying conditions. Higher NaCl dosage of 150 and 200 mmol l -1 limited its growth and drastically affected the nitrogenase activity and the enzymes glucose dehydrogenase, alcohol dehydrogenase, fumarase, isocitrate dehydrogenase and malate dehydrogenase. CONCLUSIONS: Gluconacetobacter diazotrophicus, despite its endophytic nature, tolerated heat treatments and salinity stress, but its nitrogenase activity and carbon metabolism enzymes were affected by high NaCl dosage. SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation of the biological activity of G. diazotrophicus in response to different abiotic factors led to more knowledge of this endophyte and may help to clarify pathways involved in its transmission into the host plant.

Leghaemoglobin oxygenation gradients in alfalfa and yellow sweetclover nodules.

Respiration in support of N(2) fixation by rhizobia in legume root nodules depends on an adequate supply of O(2), but excessive O(2) can damage nitrogenase, the key enzyme. The movement of O(2) into and within the nodule is driven by gradients in the concentration of O(2) or in the oxygenation of the O(2)-carrier, leghaemoglobin. Steeper gradients may increase flux to the sites of respiration, but gradients also raise the possibility of inadequate O(2) in some nodule zones and excessive O(2) in others. No detailed study of O(2) gradients in the interior of nodules has been published previously. Spectral changes in leghaemoglobin with oxygenation, previously used to measure the average O(2) status of the nodule interior, were used to map longitudinal gradients in O(2) and in respiratory capacity in the elongated nodules of alfalfa (Medicago sativa L.) and sweetclover (Melilotus officinalis L.). Variability among nodules under air in the magnitude and direction of internal O(2) gradients was seen in both species. Despite consistently higher respiratory capacity near the meristematic tip, a majority of nodules had higher O(2) towards the tip than towards the base. These results contrast with a previous report, apparently based on limited data, but they are consistent with anatomical and tracer studies showing higher gas permeability near the tip.

Leaf-atmosphere NH(3) exchange of white clover (Trifolium repens L.) in relation to mineral N nutrition and symbiotic N(2) fixation.

Plant-atmosphere NH(3) exchange was studied in white clover (Trifolium repens L. cv. Seminole) growing in nutrient solution containing 0 (N(2) based), 0.5 (low N) or 4.5 (high N) mM NO(3)(-). The aim was to show whether the NH(3) exchange potential is influenced by the proportion of N(2) fixation relative to NO(3)(-) supply. During the treatment, inhibition of N(2) fixation by NO(3)(-) was followed by in situ determination of total nitrogenase activity (TNA), and stomatal NH(3) compensation points (chi(NH(3))) were calculated on the basis of apoplastic NH4(+) concentration ([NH4(+)]) and pH. Whole-plant NH(3) exchange, transpiration and net CO(2) exchange were continuously recorded with a controlled cuvette system. Although shoot total N concentration increased with the level of mineral N application, tissue and apoplastic [NH4(+)] as well as chi(NH(3)) were equal in the three treatments. In NH(3)-free air, net NH(3) emission rates of <1 nmol m(-2) s(-1) were observed in both high-N and N(2)-based plants. When plants were supplied with air containing 40 nmol mol(-1) NH(3), the resulting net NH(3) uptake was higher in plants which acquired N exclusively from symbiotic N(2) fixation, compared to NO(3)(-) grown plants. The results indicate that symbiotic N(2) fixation and mineral N acquisition in white clover are balanced with respect to the NH4(+) pool leading to equal chi(NH(3)) in plants growing with or without NO(3)(-). At atmospheric NH(3) concentrations exceeding chi(NH(3)), the NH(3) uptake rate is controlled by the N demand of the plants.

Cyanobacteria in Uruguayan rice fields: diversity, nitrogen fixing ability and tolerance to herbicides and combined nitrogen.

It has been established that cyanobacteria play a vital role in the maintenance of flooded rice field fertility. To evaluate the potential use of nitrogen-fixing cyanobacteria as a natural biofertilizer for rice in Uruguay, the diversity, abundance and nitrogen fixing ability of these microorganisms were studied in the field and in the laboratory. The effect of urea fertilization on population density and diversity of heterocystous cyanobacteria was determined on a 3-year assay. The highest number of cyanobacteria, 1.6x10(4) CFU x m(-2), was found at the control 8 weeks after flooding. About 90% of the heterocystous cyanobacteria found in both treatments belong to the genera Nostoc and Anabaena. Maximal nitrogenase activity was reached after 12 weeks of flooding in both treatments, with an average of about 20 micromol C2H4 x m(-2) x h(-1). To improve the understanding of the environmental factors that can limit nitrogenase activity in rice fields, two of the most abundant cyanobacteria isolates were tested for tolerance to combined nitrogen and two herbicides. In both isolates 0.2 mM ammonium inhibited nitrogenase activity after 24 h of culture. The addition of field-recommended doses of quinclorac and propanil affected oxygen photoevolution but nitrogenase activity was only inhibited by propanil.

Metalaxyl effect on nitrogenase activity (acetylene reduction) and yield of mungbean (Vigna radiata (L.) wilzek).

In an experiment, application of different levels of metalaxyl to a sandy loam soil significantly affected the nodulation and nitrogenase activity of mungbean. In both the compost amended and unamended soils, 0.5 mg kg(-1) of metalaxyl enhanced acetylene reduction activity and yield of mungbean, where as higher concentrations (1 mg and 2.5 mg kg(-1) of fungicide) inhibited the nodulation traits as well as economic traits of mungbean.

Analysis of genes encoding an alternative nitrogenase in the archaeon Methanosarcina barkeri 227.

Methanosarcina barkeri 227 possesses two clusters of genes potentially encoding nitrogenases. We have previously demonstrated that one cluster, called nif2, is expressed under molybdenum (Mo)-sufficient conditions, and the deduced amino acid sequences for nitrogenase structural genes in that cluster most closely resemble those for the Mo nitrogenase of the gram-positive eubacterium Clostridium pasteurianum. The previously cloned nifH1 from M. barkeri shows phylogenetic relationships with genes encoding components of eubacterial Mo-independent eubacterial alternative nitrogenases and other methanogen nitrogenases. In this study, we cloned and sequenced nifD1 and part of nifK1 from M. barkeri 227. The deduced amino acid sequence encoded by nifD1 from M. barkeri showed great similarity with vnfD gene products from vanadium (V) nitrogenases, with an 80% identity at the amino acid level with the vnfD gene product from Anabaena variabilis. Moreover, there was a small open reading frame located between nifD1 and nifK1 with clear homology to vnfG, a hallmark of eubacterial alternative nitrogenases. Stimulation of diazotrophic growth of M. barkeri 227 by V in the absence of Mo was demonstrated. The unusual complement of nif genes in M. barkeri 227, with one cluster resembling that from a gram-positive eubacterium and the other resembling a eubacterial V nitrogenase gene cluster, suggests horizontal genetic transfer of those genes.

Dinitrogenase reductase-activating glycohydrolase can be released from chromatophores of Rhodospirillum rubrum by treatment with MgGDP.

Dinitrogenase reductase-activating glycohydrolase (DRAG), involved in the regulation of nitrogenase activity in Rhodospirillum rubrum, is associated with chromatophore membranes in cell extracts. We show that DRAG can be specifically released by treatment with MgGDP; other nucleotides studied had no effect. The DRAG activity released corresponds to the release of DRAG protein.

Circular dichroism and x-ray spectroscopies of Azotobacter vinelandii nitrogenase iron protein. MgATP and MgADP induced protein conformational changes affecting the [4Fe-4S] cluster and characterization of a [2Fe-2S] form.

Nucleotide interactions with nitrogenase are a central part of the mechanism of nitrogen reduction. Previous studies have suggested that MgATP or MgADP binding to the nitrogenase iron protein (Fe protein) induce protein conformational changes that control component protein docking, interprotein electron transfer, and substrate reduction. In the present study, we have investigated the effects of MgATP or MgADP binding to the Azotobacter vinelandii nitrogenase Fe protein on the properties of the [4Fe-4S] cluster using circular dichroism (CD) and x-ray absorption spectroscopies. Previous CD and magnetic CD studies on nitrogenase Fe protein suggested that binding of either MgATP or MgADP to the Fe protein resulted in identical changes in the CD spectrum arising from transitions of the [4Fe-4S]2+ cluster. We present evidence that MgADP or MgATP binding to the oxidized nitrogenase Fe protein results in distinctly different CD spectra, suggesting distinct changes in the environment of the [4Fc-4S] cluster. The present results are consistent with previous studies such as chelation assays, electron paramagnetic resonance, and NMR, which suggested that MgADP or MgATP binding to the nitrogenase Fe protein induced different conformational changes. The CD spectrum of a [2Fe-2S]2+ form of the nitrogenase Fe protein was also investigated to address the possibility that the MgATP- or MgADP-induced changes in the CD spectrum of the native enzyme were the result of a partial conversion from a [4Fe-4S] cluster to a [2Fe-2S] cluster. No evidence was found for a contribution of a [2Fe-2S]2+ cluster to the CD spectrum of oxidized Fe protein in the absence or presence of nucleotides. A novel two-electron reduction of the [2Fe-2S]2+ cluster in Fe protein was apparent from absorption, CD, and electron paramagnetic resonance data. Fe K-edge x-ray absorption spectra of the oxidized Fe protein revealed no changes in the structure of the [4Fe-4S] cluster upon MgATP binding to the Fe protein. The present results reveal that MgATP or MgADP binding to the oxidized state of the Fe protein result in different conformational changes in the environment around the [4Fe-4S] cluster.

Effect of herbicides on nitrogen fixation (C2H2 reduction) associated with rice rhizosphere.

In a field study nitrogenase activity associated with rice rhizosphere was differently influenced by the applied herbicides. Pretilachlor at two application levels had no effect on nitrogenase activity while butachlor and benthiocarb exerted marginal stimulation. Cinmethylin consistently stimulated nitrogenase activity throughout the plant growth period. Anilofos when applied singly had no substantial effect on nitrogenase activity but in combination with 2,4-D the activity was enhanced. Populations of anaerobic nitrogen-fixing bacteria and Azospirillum sp. and Azotobacter sp. were stimulated in such a combination.

MgATP-induced conformational changes in the iron protein from Azotobacter vinelandii, as studied by small-angle x-ray scattering.

Small angle x-ray scattering experiments have been carried out on the purified iron proteins of nitrogenase from wild-type Azotobacter vinelandii and from a Nif- mutant strain, A. vinelandii UW91 (which has an A157S mutation). This study was designed to investigate the influence of MgATP and MgADP binding on the protein structure in solution. For the wild-type protein, the binding of MgATP induces a significant conformational change that is observed as a decrease of about 2.0 A in the radius of gyration. In contrast, the binding of MgADP to the wild-type iron protein does not detectably affect the radius of gyration. In the absence of nucleotides, the radius of gyration for the UW91 mutant is indistinguishable from that of the wild-type. However, unlike for the wild-type protein, the radius of gyration of the UW91 iron protein is unaffected by the addition of MgATP. We have previously shown that the UW91 iron protein has a normal [4Fe-4S] cluster and MgATP binding ability but that it is completely blocked for electron transfer and MgATP hydrolysis (Gavini, N., and Burgess, B. K. (1992) J. Biol. Chem. 267, 21179-21186). These x-ray scattering measurements suggest that a conformation different from that of the native state is therefore required for the iron protein to perform electron transfer to the MoFe protein. These results also support the hypothesis that Ala-157 is crucial for the iron protein to establish the electron-transfer-favored conformation induced by MgATP binding.

Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism.

Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.