Variations in the Rhythms of Respiration and Nitrogen Fixation in Members of the Unicellular Diazotrophic Cyanobacterial Genus Cyanothece

In order to accommodate the physiologically incompatible processes of photosynthesis and nitrogen fixation within the same cell, unicellular nitrogen-fixing cyanobacteria have to maintain a dynamic metabolic profile in the light as well as the dark phase of a diel cycle. The transition from the photosynthetic to the nitrogen-fixing phase is marked by the onset of various biochemical and regulatory responses, which prime the intracellular environment for nitrogenase activity. Cellular respiration plays an important role during this transition, quenching the oxygen generated by photosynthesis and by providing energy necessary for the process. Although the underlying principles of nitrogen fixation predict unicellular nitrogen-fixing cyanobacteria to function in a certain way, significant variations are observed in the diazotrophic behavior of these microbes. In an effort to elucidate the underlying differences and similarities that govern the nitrogen-fixing ability of unicellular diazotrophic cyanobacteria, we analyzed six members of the genus Cyanothece. Cyanothece sp. ATCC 51142, a member of this genus, has been shown to perform efficient aerobic nitrogen fixation and hydrogen production. Our study revealed significant differences in the patterns of respiration and nitrogen fixation among the Cyanothece spp. strains that were grown under identical culture conditions, suggesting that these processes are not solely controlled by cues from the diurnal cycle but that strain-specific intracellular metabolic signals play a major role. Despite these inherent differences, the ability to perform high rates of aerobic nitrogen fixation and hydrogen production appears to be a characteristic of this genus.Nitrogen fixation is an important global phenomenon by which molecular nitrogen, one of the most abundant components of the earth’s atmosphere, is converted into a more reduced form suitable for incorporation into living systems. The majority of this nitrogen fixation is achieved by biological means through the activity of microorganisms (Burris and Roberts, 1993; Raymond et al., 2004; Rubio and Ludden, 2008). This process is energy intensive, and nitrogenase, the enzyme complex involved in the biological nitrogen fixation reaction, is generally known to be extremely sensitive to oxygen (Robson and Postgate, 1980; Hill et al., 1981; Berman-Frank et al., 2005). Thus, most microbes participating in this process fix nitrogen only when suitable anaerobic or microaerobic conditions are established in an otherwise oxygen-rich environment. However, some nitrogen-fixing (diazotrophic) microbes have the advantage of being able to fix nitrogen in aerobic environments. Outstanding among these are the photosynthetic prokaryotes called cyanobacteria, an extremely successful group of microbes with plant-like traits. These microbes are considered to be the progenitors of plant chloroplasts. Cyanobacteria perform both oxygen-evolving photosynthesis and oxygen-sensitive nitrogen fixation, thereby providing a platform to power the most metabolically expensive biological process (Simpson and Burris, 1984) with solar energy.Among the nitrogen-fixing cyanobacteria, filamentous strains have been extensively studied for their contribution to the nitrogen cycle in marine and terrestrial ecosystems (Mulligan and Haselkorn, 1989; Kaneko et al., 2001; Meeks et al., 2001; Sañudo-Wilhelmy et al., 2001; Wong and Meeks, 2001; Gomez et al., 2005). Some of these filamentous strains develop specialized cells called heterocysts that allow the spatial segregation of photosynthesis and nitrogen fixation. These heterocysts also have higher rates of respiratory oxygen consumption, which results in a virtually anoxic environment conducive for the nitrogenase enzyme (Bergman et al., 1997). All heterocystous strains are known to fix nitrogen aerobically. In contrast, nonheterocystous cyanobacteria lack any specialized oxygen-free compartments and often require incubation under microoxic or anaerobic conditions for nitrogen fixation (Rippka and Waterbury, 1977; Rippka et al., 1979; Brass et al., 1992). However, some nonheterocystous cyanobacterial strains can fix nitrogen under aerobic conditions. These include some filamentous genera like Trichodesmium spp., Lyngbya spp., and Oscillatoria spp. (Jones, 1990; Janson et al., 1994; Finzi-Hart et al., 2009) as well as unicellular genera like Gloeothece spp. and Cyanothece spp. (Wyatt and Silvey, 1969; Rippka and Waterbury, 1977; Huang and Chow, 1988; Van Ni et al., 1988; Schütz et al., 2004).In comparison with filamentous cyanobacteria, which have long been recognized for their nitrogen-fixing ability, the importance of unicellular cyanobacteria as key components of the environmental nitrogen cycle has only been recently uncovered. Studies over the last decade have established unicellular strains like Crocosphaera spp., Cyanothece spp., and UCYN-A as important players in the marine nitrogen cycle (Zehr et al., 2001; Montoya et al., 2004; Zehr, 2011). Since unicellular diazotrophic cyanobacteria utilize the same cellular platform for photosynthesis and nitrogen fixation, they are required to adjust their cellular metabolism to accommodate these two antagonistic processes. Systems-level studies in the unicellular genus Cyanothece have revealed a temporal separation of the two processes, photosynthesis occurring during the day and nitrogen fixation occurring at night (Stöckel et al., 2008; Toepel et al., 2008; Welsh et al., 2008). Cellular respiration plays a critical role during the transition from one phase to the next, rapidly freeing the intracellular environment of the photosynthetically generated oxygen and rendering it conducive for the induction of nitrogenase activity. In addition, respiration also sustains the process of nitrogen fixation, not only by maintaining a low-oxygen environment required for the functioning of the nitrogenase enzyme but also by mobilizing the stored solar energy to fuel this energy-intensive process.Unicellular diazotrophs exhibit great diversity in the efficiency of nitrogen fixation as well as in the physiological regulation of the process. For instance, members of the genus Gloeothece fix nitrogen aerobically during the day, but at 0% dissolved oxygen concentration, nitrogen fixation is shifted entirely to the dark period (Ortega-Calvo and Stal, 1991; Taniuchi et al., 2008). In contrast, some Synechococcus spp. strains can fix nitrogen only when incubated under anoxic conditions (Steunou et al., 2006). Members of the genus Cyanothece have been reported to engage in both aerobic and anaerobic nitrogen fixation, with nitrogenase activity peaking during the night (Reddy et al., 1993; Bergman et al., 1997; Turner et al., 2001). This suggests that, in addition to the regulations imposed by the diurnal cycle, strain-specific intracellular cues govern the process of nitrogen fixation in unicellular cyanobacteria, which may vary according to the genotype or the ecotype of the strains.Members of the unicellular cyanobacterial genus Cyanothece are diazotrophs that thrive in marine as well as terrestrial environments. This genus was originally grouped together with Synechococcus spp. but was later separated on the basis of distinct morphological and biochemical differences between the two genera (Komárek, 1976; Rippka and Cohen-Bazire, 1983). Some of the features that define the largely heterogeneous genus Cyanothece are oval to cylindrical cells, larger than 3 µm in size (they can be as large as 24 µm in diameter), radially arranged thylakoids, and a mucilaginous layer surrounding the cells (Komárek and Cepák, 1998; Porta et al., 2000; Liberton et al., 2011).It was recently demonstrated that Cyanothece sp. ATCC 51142, a member of the genus Cyanothece, has the unique ability to produce molecular hydrogen at exceptionally high rates under aerobic conditions (Bandyopadhyay et al., 2010). This striking observation was attributed to the nitrogenase enzyme system of Cyanothece sp. ATCC 51142. Our study also indicated that high rates of respiration in this strain might contribute to its nitrogenase-mediated aerobic hydrogen production. Glycerol was found to be an efficient source of reductants and energy for this process. In an effort to investigate if this atypical cyanobacterial trait was a characteristic of the genus Cyanothece, five additional Cyanothece spp. strains from different ecological habitats were sequenced to completion. The six strains display more than 90% identity in their 16S ribosomal RNA sequence but exhibit striking variability with respect to their genome sizes (with the largest genome being 7.8 Mb and the smallest being 4.4 Mb), the number of plasmids, and the percentage of pseudogenes (Bandyopadhyay et al., 2011). In addition, two of the strains possess linear chromosomal elements, features not known to occur in any other photosynthetic bacteria sequenced to date, which may impart niche-specific advantages to these strains. Analysis of the genome sequence of the Cyanothece spp. strains showed the presence of a nitrogenase gene cluster in all five strains, and preliminary analysis showed that four of the five strains were capable of aerobic nitrogen fixation and hydrogen production (Bandyopadhyay et al., 2011). In this study, we have focused on the patterns of nitrogen fixation and respiration in six different Cyanothece spp. strains in an effort to elucidate the underlying differences and similarities in these processes in unicellular diazotrophic strains with similar genotypic but varied ecological backgrounds. Our study reveals inherent differences in the regulation of these processes, which are likely controlled by strain-specific cellular signals. However, despite the differences in the patterns of nitrogenase activity, aerobic nitrogen fixation and hydrogen production was found to be a characteristic of this genus, with most members exhibiting nitrogenase-mediated hydrogen production at rates higher than any other wild-type cyanobacterial strain.     

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.     

Regulation of nitrogen fixation in Rhizobium sp.

Regulation of nitrogen fixation by ammonium and glutamate was examined in Rhizobium sp. 32H1 growing in defined liquid media. Whereas nitrogenase synthesis in Klebsiella pneunoniae is normally completely repressed during growth on NH4+, nitrogenase activity was detected in cultures of Rhizobium sp. grown with excess NH4+. However, an "ammonium effect" on activity was invariably observed in cultures grown on NH4+ as sole nitrogen source; the nitrogenase activity was, depending on conditions, 14 to 36% of that of comparable glutamate-grown cultures. Glutamate inhibited utilization of exogenous NH4+ and, in one of two procedures described, glutamate partially alleviated the ammonium effect on nitrogenase activity. NH4+, apparently produced from N2, was excreted into the culture medium when growth was initiated on glutamate, but not when NH4+ was thesole source of fixed nitrogen for growth. These findings are discussed in relation to nitrogen fixation by Rhizobium bacteroids.     

Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

Arginine and nitrogen mobilization in cyanobacteria

Cyanobacteria have evolved mechanisms to adapt to environmental stress and nutrient availability, including accumulation of storage compounds in inclusions and granules. As arginine is a key building block of cyanophycin, a dynamic nitrogen reservoir in many cyanobacteria, arginine metabolism plays a key role in cyanobacterial nitrogen storage and remobilization. Recently, an arginine dihydrolase AgrE/ArgZ was identified as a major arginine‐degrading enzyme in nondiazotrophic Synechocystis, which catalyzes the conversion of arginine into ornithine and ammonia. The N‐terminal domain of AgrE/ArgZ is responsible for arginine dihydrolase activity. Burnat et al. (2019) identified the arginine catabolic pathway in diazotrophic Anabaena, which starts with the reaction catalyzed by AgrE/ArgZ. Moreover, this study identified the C‐terminal domain of AgrE/ArgZ as an ornithine cyclodeaminase that catalyze the conversion of ornithine to proline. The results demonstrated that arginine is catabolized to generate glutamate by the concerted action of AgrE/ArgZ and bifunctional proline oxidase PutA in the vegetative cells of Anabaena. These findings expand our knowledge on nitrogen mobilization and redistribution in Anabaena under nitrogen‐fixation conditions. AgrE/ArgZ is widely present in many diazotrophic cyanobacteria and may be important for their contribution to marine nitrogen fixation. AgrE/ArgZ may have potential applications in metabolic engineering and biotechnology.     

Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown at constant growth rate in a chemostat with different molar ratios of sucrose to ammonium (C/N) in the influent media. Both compounds were consumed at essentially the same ratios as were present in the influent media. At low (C/N)-ratios, the cultures were ammonium-limited. At increased (C/N)-ratio ammonium-assimilating cultures additionally began to fix dinitrogen. The (C/N)-ratio at which nitrogenase activity became measurable, increased when the ambient oxygen concentration was increased. Immunoblotting revealed the appearance of nitrogenase proteins when the activity became detectable. Nitrogenase activity as determined either by acetylene reduction or by total nitrogen fixation gave constant relative activities of 1:3.8 (mol of N₂ fixed per mol of acetylene reduced) under all sets of conditions used in this investigation. In spite of the oxygen dependent variation of the (C/N)-ratio, nitrogenase became active when the ammonium supply was less than about 14 nmol of ammonium per g of protein. This suggests that oxygen was not directly involved in the onset of dinitrogen fixation.     

Unicellular, aerobic nitrogen-fixing cyanobacteria of the genus Cyanothece.

Two marine, unicellular aerobic nitrogen-fixing cyanobacteria, Cyanothece strain BH63 and Cyanothece strain BH68, were isolated from the intertidal sands of the Texas Gulf coast in enrichment conditions designed to favor rapid growth. By cell morphology, ultrastructure, a GC content of 40%, and aerobic nitrogen fixation ability, these strains were assigned to the genus Cyanothece. These strains can use molecular nitrogen as the sole nitrogen source and are capable of photoheterotrophic growth in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and glycerol. The strains demonstrated a doubling time of 10 to 14 h in the presence of nitrate and 16 to 20 h under nitrogen-fixing conditions. Rapid growth of nitrogen-fixing cultures can be obtained in continuous light even when the cultures are continuously shaken or bubbled with air. Under 12-h alternating light and dark cycles, the aerobic nitrogenase activity was confined to the dark phase. The typical rates of aerobic nitrogenase activity in Cyanothece strains BH63 and BH68 were 1,140 and 1,097 nmol of C2H2 reduced per mg (dry weight) per h, respectively, and nitrogenase activity was stimulated twofold by light. Ultrastructural observations revealed that numerous inclusion granules formed between the photosynthetic membranes in cells grown under nitrogen-fixing conditions. These Cyanothece strains posses many characteristics that make them particularly attractive for a detailed analysis of the interaction of nitrogen fixation and photosynthesis in an aerobic diazotroph.     

Oxygen relations of nitrogen fixation in cyanobacteria.

The enigmatic coexistence of O2-sensitive nitrogenase and O2-evolving photosynthesis in diazotrophic cyanobacteria has fascinated researchers for over two decades. Research efforts in the past 10 years have revealed a range of O2 sensitivity of nitrogenase in different strains of cyanobacteria and a variety of adaptations for the protection of nitrogenase from damage by both atmospheric and photosynthetic sources of O2. The most complex and apparently most efficient mechanisms for the protection of nitrogenase are incorporated in the heterocysts, the N2-fixing cells of cyanobacteria. Genetic studies indicate that the controls of heterocyst development and nitrogenase synthesis are closely interrelated and that the expression of N2 fixation (nif) genes is regulated by pO2.     

Effects of fertilization rate and crop rotation on diazotrophic cyanobacteria in paddy field

Application of chemical fertilizers at the recommended level (medium fertility) or lower stimulated growth of the diazotrophic cyanobacterial population and nitrogenase activity in a paddy field. High fertilizer levels proved to be inhibitory to nitrogen-fixing cyanobacteria indicating that indiscriminate use of chemical fertilizers for a longer period drastically disturbed the natural ecological balance. The rice–mustard–moong (RMM) crop rotation was observed to be more suitable for cyanobacterial nitrogen fixation than rice–wheat–maize rotation. The cropped plots had higher nitrogenase activity than fallow plots. The low fertility coupled with RMM rotation were found to be best suited for promoting nitrogen fixation by cyanobacteria to supply the rice plants. A top dressing of chemical nitrogenous fertilizer drastically suppressed the cyanobacterial nitrogenase activity (ARA) within 12 h; the magnitude of inhibition varied with respect to the cropping system. The inhibition was overcome by the 10th day and the ARA value reached the preapplication value or even higher in the case of low fertility and medium fertility level plots. A regression equation was established to predict nitrogen fixation in a given soil ecosystem.     

Low temperature delays timing and enhances the cost of nitrogen fixation in the unicellular cyanobacterium Cyanothece

Marine nitrogen-fixing cyanobacteria are largely confined to the tropical and subtropical ocean. It has been argued that their global biogeographical distribution reflects the physiologically feasible temperature range at which they can perform nitrogen fixation. In this study we refine this line of argumentation for the globally important group of unicellular diazotrophic cyanobacteria, and pose the following two hypotheses: (i) nitrogen fixation is limited by nitrogenase activity at low temperature and by oxygen diffusion at high temperature, which is manifested by a shift from strong to weak temperature dependence of nitrogenase activity, and (ii) high respiration rates are required to maintain very low levels of oxygen for nitrogenase, which results in enhanced respiratory cost per molecule of fixed nitrogen at low temperature. We tested these hypotheses in laboratory experiments with the unicellular cyanobacterium Cyanothece sp. {"type":"entrez-nucleotide","attrs":{"text":"BG043511","term_id":"12489990","term_text":"BG043511"}}BG043511. In line with the first hypothesis, the specific growth rate increased strongly with temperature from 18 to 30 °C, but leveled off at higher temperature under nitrogen-fixing conditions. As predicted by the second hypothesis, the respiratory cost of nitrogen fixation and also the cellular C:N ratio rose sharply at temperatures below 21 °C. In addition, we found that low temperature caused a strong delay in the onset of the nocturnal nitrogenase activity, which shortened the remaining nighttime available for nitrogen fixation. Together, these results point at a lower temperature limit for unicellular nitrogen-fixing cyanobacteria, which offers an explanation for their (sub)tropical distribution and suggests expansion of their biogeographical range by global warming.     

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other. Specific rates of dinitrogen fixation as well as of the regeneration of the cellular ATP pool were compared with specific rates of cellular respiration. With glucose-limited cultures, the rate of regeneration of the ATP pool and the rate of respiration varied in direct proportion to each other. This relationship, however, was dependent on the dissolved oxygen concentration. As compared to the phosphate-sufficient control, phosphate-limited cultures exhibited the same nitrogenase activity but significantly increased respiratory activities. Rates of ATP regeneration and of cellular respiration of phosphate-limited cultures did not fit into the relationship characteristic of glucose-limited cultures. However, a linear relationship between the rates of dinitrogen fixation and ATP regeneration was identified irrespective of the type of growth limitation and the dissolved oxygen concentration. The results suggest that the ATP supply rather than cellular oxygen consumption is of primary importance in keeping nitrogenase activity in aerobic cultures of A. vinelandii.     

Nitrogenase Inhibition in Nodules from Pea Plants Grown Under Salt Stress Occurs at the Physiological Level and can be Alleviated by B and Ca

In order to shed new light on the mechanisms of salt-mediated symbiotic N₂-fixation inhibition, the effect of salt stress (75 mM) on N₂-fixation in pea root nodules induced by R. leguminosarum was studied at the gene expression, protein production and enzymatic activity levels. Acetylene reduction assays for nitrogenase activity showed no activity in salt-stressed plants. To know whether salt inhibits N₂-fixing activity at a molecular or at a physiological level, expression of the nifH gene, encoding the nitrogenase reductase component of the nitrogenase enzyme was analyzed by RT-PCR analysis of total RNA extracted from nodulated roots. The nifH messenger RNA was present both in plants grown in the presence and absence of salt, although a reduction was observed in salt-stressed plants. Similar results were obtained for the immunodetection of the nitrogenase reductase protein in Western-blot assays, indicating that nitrogen fixation failed mainly at physiological level. Given that nutrient imbalance is a typical effect of salt stress in plants and that Fe is a prosthetic component of nitrogenase reductase and other proteins required by symbiotic N₂-fixation, as leghemoglobin, plants were analyzed for Fe contents by atomic absorption and the results confirmed that Fe levels were severely reduced in nodules developed in salt-stressed plants. In a previous papers (El-Hamdaoui et al., 2003b), we have shown that supplementing inoculated legumes with boron (B) and calcium (Ca) prevents nitrogen fixation decline under saline conditions stress. Analysis of salt-stressed nodules fed with extra B and Ca indicated that Fe content and nitrogenase activity was similar to that of non-stressed plants. These results indicate a linkage between Fe deprivation and salt-mediated failure of nitrogen fixation, which is prevented by B and Ca leading to increase of salt tolerance.     

Isolation and Characterization of NH+4-Excreting Mutants of Enterobacter gergoviae

NH4+-excreting mutants were isolated from Enterobacter gergoviae 57–7 wild type as methylamine resistant strains which were obtained by mutagenesis with a transposable element Tn5. The MG 61 mutants excreted 2 mmol/L of ammonium during a diazotrophic growth. The growth of MG 61 mutants were slower than the growth of wild types because of its excreting ammonium. MG 61 mutants expressed up to 86% of the fully depressed nitrogenase activity when grown in a medium containing 20 mmol/L ammonium. By contrast the ammonium grown cultures of wild type had no nitrogenase activity. In the presence of 5 mmol/L or 30 mmol/L of ammonium in the medium, the growth of MG 61 mutants was as same as CK and much slower than that of the wild types which means that the mutants could not utilize amonium very well in the medium. But MG 61 mutants could utilize glutamate as a sole nitrogen source. In the presence of nitrate (10 mmol/L) in the medium, MG 61 mutants grew slowly but excreted 7.8 mmol/L of ammonium.     

Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria

The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp. strain ATCC 51142 and the non-diazotrophic Synechocystis sp. strain PCC 6803. Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions. In Cyanothece sp. ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced. Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely. Knock-out mutants were constructed in Synechocystis sp. PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions. The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve. Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Delta cphA and Delta cphB mutants. These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp. ATCC 51142 and represents a key nitrogen reserve in these organisms. However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.     

‘Respiratory protection’ of the nitrogenase in dinitrogen-fixing cyanobacteria

Several filamentous and unicellular cyanobacteria were grown photoautotrophically with nitrate or dinitrogen as N-sources, and some respiratory properties of the cells or isolated plasma (CM) and thylakoid (ICM) membranes were compared. Specific cytochrome c oxidase activities in membranes from dinitrogen-fixing cells were between 10- and 50-times higher than those in membranes from nitrate-grown cells, ICM of heterocysts but CM of unicells being mainly responsible for the stimulation. Whole cell respiration (oxygen uptake) of diazotrophic unicells paralleled increased cytochrome oxidase activities of the isolated membranes. Mass spectrometric measurements of the uptake of isotopically labeled oxygen revealed that (low) light inhibited respiration of diazotrophic unicells to a much lesser degree than that of nitrate-grown cells which indicates the prevailing (respiratory) role of CM in the former. Normalized growth yields of diazotrophic unicells grown in continuous light were significantly higher than those of cells grown in a 12/12 hrs light/dark cycle. Mass spectrometry showed that overall nitrogen uptake by the former was higher than by the latter; in particular, and in marked contrast to the time course of nitrogenase activity (acetylene reduction) there was no appreciable nitrogen uptake or protein synthesis during dark periods; likewise, there was no 14-CO₂ fixation, nor chloropholl synthesis, nor cell division in the dark. By contrast, growth in continuous light gave sustained rates of nitrogen and carbon dioxide incorporation over the whole time range. Our results will be discussed in terms of respiratory protection as an essential strategy of keeping apart nitrogenase and oxygen, either atmospheric or photosynthetically produced within the same cell.     

Iron-Stimulated N(2) Fixation and Growth in Natural and Cultured Populations of the Planktonic Marine Cyanobacteria Trichodesmium spp

In light of recent proposals that iron (Fe) availability may play an important role in controlling oceanic primary production and nutrient flux, its regulatory impact on N(2) fixation and production dynamics was investigated in the widespread and biogeochemically important diazotrophic, planktonic cyanobacteria Trichodesmium spp. Fe additions, as FeCl(3) and EDTA-chelated FeCl(3), enhanced N(2) fixation (nitrogenase activity), photosynthesis (CO(2) fixation), and growth (chlorophyll a production) in both naturally occurring and cultured (on unenriched oligotrophic seawater) Trichodesmium populations. Maximum enhancement of these processes occurred under FeEDTA-amended conditions. On occasions, EDTA alone led to enhancement. No evidence for previously proposed molybdenum or phosphorus limitation was found. Our findings geographically extend support for Fe limitation of N(2) fixation and primary production to tropical and subtropical oligotrophic ocean waters often characterized by Trichodesmium blooms.     

联合固氮菌Enterobactergergoviae57—7泌铵突变株的分离和特性

经 Tn5转座子诱变从野生型菌株 ( Enterobacter gergoviae 5 7- 7)筛选到抗甲胺 ( 0 .3mol/L )的泌铵突变株 MG61 ,该突变株在固氮生长时能分泌铵 2 .0 mmol/L。由于泌铵 MG61的生长比野生型菌株慢 ,在培养基中含铵 ( 5 mmol/L或 30 mmol/L)条件下 ,MG61的生长速率与对照相同 ,而远比野生型菌株慢 ,表明 MG 61不能很好地利用铵。在含 2 0 mmol/L铵的培养基中 MG 61仍表达 86%的固氮活性 ,而野生型菌株完全丧失了固氮活性。在谷氨酸存在下 MG61的生长速率及固氮酶活性都比对照高。在硝酸盐存在下 MG61的生长速率与对照相同 ,但泌铵量达 7.8mmol/L。     

Cyanide assimilation in Rhizobium ORS 571: influence of the nitrogenase catalyzed hydrogen production on the efficiency of growth

When cyanide is gradually added to a nitrogenfixing culture, Rhizobium ORS 571 is capable of assimilating large amounts of cyanide using its nitrogenase. Under these conditions the molar growth yield on succinate (Y _succ) increases from 27 at the start of cyanide addition to 38 at the end. The respiratory chain of cells grown at a concentration of 7 mM cyanide is still very sensitive to cyanide. The increase in growth yield is explained by a decrease in hydrogen production by nitrogenase as soon as cyanide is assimilated. This is confirmed by calculating the influence of hydrogen production on Y _succ. Hydrogen production by nitrogenase has a greater influence on growth yields than the presence or absence of hydrogenase activity. At the end of cyanide addition when all cell nitrogen is synthesized from cyanide and no nitrogen fixation occurs, nitrogenase will be in a very oxidized state.