Effects of D-erythrose on nitrogenase activity in whole filaments of Anabaena sp. strain 7120.

D-Erythrose, which has been shown to enhance nitrogenase activity (acetylene reduction) by isolated heterocysts, was studied for its effects on nitrogenase activity and nitrite uptake by whole filaments of Anabaena sp. strain 7120. D-Erythrose had little effect on acetylene reduction in the light; however, at a concentration of 10 mM, it could restore 3'-(3,4-dichlorophenyl)-1',1'-dimethyl urea-inhibited or dark-limited levels to light-supported levels. Sucrose, glucose, or fructose did not exhibit similar effects. D-Erythrose had little effect on nitrite uptake, an indirect measure of nitrite reductase activity by nitrate-grown whole filaments. It was concluded that erythrose effects were mediated by heterocysts and were therefore specific for nitrogenase.     

Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.Abbreviations IgG Immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TEM transmission electron micrograph     

Developmental patterns related to nitrogen fixation in theNostoc-Gunnera magellanica Lam. symbiosis

Developmental patterns related to nitrogen fixation in the heterocystous cyanobacteriumNostoc harboured in distinct colonies along the stem ofGunnera magellanica Lam. plantlets were examined using successive plant sections. Pronounced morphological, physiological and biochemical alterations in the cyanobacterium were demonstrated. Close to the growing apex the cyanobacterial biomass, contained in smallGunnera cells, was low and consisted mostly of vegetative cells showing a high density of different storage structures except for cyanophycin granules. In contrast, both the total and specific nitrogenase activity and the relative nitrogenase protein level were at maximum within this part; while the frequency of heterocysts increased from zero to 30% within the same area. The nitrogenase protein was localized only in the heterocysts throughout the plant. Further down theGunnera stem there was a progressive increase in both the cyanobacterial biomass and the heterocyst frequency, which finally constituted about 60% of the cyanobacterial cell population. Throughout this part of the stem, cyanophycin granules were frequent in the vegetativeNostoc cells. At the base of the stem, degeneratedNostoc cells dominated and the nitrogenase activity was close to zero, although the nitrogenase protein remained. Degeneration of theNostoc cells and leaf shedding coincided. Both intact plants (approx. 20 mm in height) and plant stem sections (2 mm in length) showed substantial nitrogenase activity, although sectioning caused a 30% reduction in total nitrogenase activity.     

Isolation of cyanobacterial heterocysts with high and sustained dinitrogen-fixation capacity supported by endogenous reductants

A method is described for the preparation of cyanobacterial heterocysts with high nitrogen-fixation (acetylene-reduction) activity supported by endogenous reductants. The starting material was Anabaena variabilis ATCC 29413 grown in the light in the presence of fructose. Heterocysts produced from such cyanobacteria were more active than those from photoautotrophically-grown A. variabilis, presumably because higher reserves of carbohydrate were stored within the heterocysts. It proved important to avoid subjecting the cyanobacteria to low temperatures under aerobic conditions, as inhibition of respiration appeared to lead to inactivation of nitrogenase. Low temperatures were not harmful in the absence of O₂. A number of potential osmoregulators at various concentrations were tested for use in heterocyst isolation. The optimal concentration (0.2M sucrose) proved to be a compromise between adequate osmotic protection for isolated heterocysts and avoidance of inhibition of nitrogenase by high osmotic strength. Isolated heterocysts without added reductants such as H₂ had about half the nitrogen-fixation activity expected on the basis of intact filaments. H₂ did not increase the rate of acetylene reduction, suggesting that the supply of reductant from heterocyst metabolism did not limit nitrogen fixation under these conditions. Such heterocysts had linear rates of acetylene reduction for at least 2 h, and retained their full potential for at least 12 h when stored at 0°C under N₂.     

Survey of the Distribution of Different Types of Nitrogenases and Hydrogenases in Heterocyst-Forming Cyanobactera

As a first step toward developing the methodology for screening large numbers of heterocyst-forming freshwater cyanobacteria strains for the presence of various types of nitrogenases and hydrogenases, we surveyed the distribution of these genes and their activities in 14 strains from culture collections. The nitrogenase genes include nif1 encoding a Mo-type nitrogenase expressed in heterocysts, nif2 expressed in vegetative cells and heterocysts under anaerobic conditions, and vnf encoding a V-type nitrogenase expressed in heterocysts. Two methods proved to be valuable in surveying the distribution of nitrogenase types. The first method was Southern blot hybridization of DNA digested with two different endonucleases and hybridized with nifD1, nifD2, and vnfD probes. The second method was ethane formation from acetylene to detect the presence of active V-nitrogenase. We found that all 14 strains have nifD1 genes, and eight strains also have nifD2 genes. Four of the strains have vnfD genes, in addition to nifD2 genes. It is curious that three of these four strains had similar hybridization patterns with all of the nifD1, nifD2, and vnfD probes, suggesting that there could be some bias in strains used in the present study or in strains held in culture collections. This point will need to be assessed in the future. For surveying the distribution of hydrogenases, Southern blot hybridization was an effective method. All strains surveyed had hup genes, with the majority of them also having hox genes.     

Correlation between nitrogenase and glutamine synthetase expression in the cyanobacterium Anabaena cylindrical

Distribution pattern and levels of nitrogenase (EC 1.7.99.2) and glutamine synthetase (GS, EC 6.3.1.2) were studied in N₂-, NO₃^? and NH₄⁺ grown Anabaena cylindrica (CCAP 1403/2a) using immunogold electron microscopy. In N₂- and NO₃^? grown cultures, heterocysts were formed and nitrogenase activity was present. The nitrogenase antigen appeared within the heterocysts only and showed an even distribution. The level of nitrogenase protein in the heterocysts was identical with both nitrogen sources. In NO₃^? grown cells the 30% reduction in the nitrogenase activity was due to a corresponding decrease in the heterocyst frequency and not to a repressed nitrogenase synthesis. In NH₄^? grown cells, the nitrogenase activity was almost zero and new heterocysts were formed to a very low extent. The heterocysts found showed practically no nitrogenase protein throughout the cytoplasm, although some label occurred at the periphery of the heterocyst. This demonstrates that heterocyst differentiation and nitrogenase expression are not necessarily correlated and that while NH₄⁺ caused repression of both heterocyst and nitrogenase synthesis, NO₃^? caused inhibition of heterocyst differentiation only. The glutamine synthetase protein label was found throughout the vegetative cells and the heterocysts of all three cultures. The relative level of the GS antigen varied in the heterocysts depending on the nitrogen source, whereas the GS level was similar in all vegetative cells. In N₂- and NO₃⁺ grown cells, where nitrogenase was expressed, the GS level was ca 100% higher in the heterocysts compared to vegetative cells. In NH₄⁺ grown cells, where nitrogenase was repressed, the GS level was similar in the two cell types. The enhanced level of GS expressed in heterocysts of N₂ and NO₃^? grown cultures apparently is related to nitrogenase expression and has a role in assimilation of N₂derived ammonia.     

Regulation of nitrogenase levels in Anabaena sp. ATCC 33047 and other filamentous cyanobacteria

Incubation in the dark of photoautotrophically grown N₂-fixing heterocystous cyanobacteria leads to a loss of nitrogenase activity. Original levels of nitrogenase activity are rapidly regained upon re-illumination of the filaments, in a process dependent on de novo protein synthesis. Ammonia, acting indirectly through some of its metabolic derivatives, inhibits the light-promoted development of nitrogenase activity in filaments of Anabaena sp. ATCC 33047 and several other cyanobacteria containing mature heterocysts. The ammonia-mediated control system is also operative in N₂-fixing filaments in the absence of any added source of combined nitrogen, with the ammonia resulting from N₂-fixation already partially inhibiting full expression of nitrogenase. High nitrogenase levels, about two-fold higher than those in normal N₂-fixing Anabaena sp. ATCC 33047, are found in cell suspensions which have been treated with the glutamine synthetase inhibitor l-methionine-d,l-sulfoximine or subjected to nitrogen starvation. Filaments treated in either way are insensitive to the ammonia-promoted inhibition of nitrogenase development, although this insensitivity is only transitory for the nitrogen-starved filaments, which become ammonia-sensitive once they regain their normal nitrogen status.Abbreviations Chl chlorophyll - EDTA ethylenediaminetetraacetic acid - MSX l-methionine-d,l-sulfoximine     

Inhibition of nitrogenase by nitrite and nitric oxide in Rhodopseudomonas sphaeroides f. sp. denitrificans

In cells of Rhodopseudomonas sphaeroides f. sp. denitrificans nitrite and nitric oxide, the products of denitrification, inhibit activity of nitrogenase enzyme.Ferredoxin-linked CO₂ fixation, with H₂ as a reductant, was also inhibited by nitrite and NO in denitrifying cells.EPR spectroscopy of cell preparations treated with NO showed that it reacts with non-haem iron-sulphur proteins to form iron-nitrosyl complexes. Nitrite also reacts with these iron-sulphur proteins, but the formation of ironnitrosyl complexes was dependent on the presence of dithionite. Since nitrite is reduced to NO by dithionite it is likely that nitrogenase and CO₂ fixation reactions are inhibited not only by nitrite itself, but also by nitric oxide.Abbreviation DPPH 1,1-diphenyl-2-picrylhydrazyl     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.In the light, addition of NADP induced a lag in H₂- or NADH-supported acetylene reduction apparently by competing with nitrogenase for electrons at the reducing side of photosystem I. Time reversal of this inibition reflects a regulation of photosystem I-dependent nitrogenase activity by the NADPH/NADP ratio in the cell. This was directly demonstrated by differently adjusted NADPH/NADP ratios.NADPH donates electrons to nitrogenase in the dark and in the light, the light reaction being DBMIB-sensitive. NADPH-supported acetylene reduction was inhibited by NADP. This inhibition was not reversed with time, pointing to an involvement of ferredoxin: NADP oxidoreductase (EC 1.18.1.2) in this pathway. Apparently, in the dark, this enzyme is able to directly reduce ferredoxin, whereas in the light electrons from NADPH first have to pass through photosystem I before reducing ferredoxin, hence nitrogenase.Intermediates of glycolysis, like glucose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate supported nitrogenase activity in the dark, each with catalytic amounts of both NAD and NADP as equally effective cofactors.We conclude that in heterocysts electrons for nitrogen fixation are essentially supplied by dark reactions, mainly by glycolysis. NADH (and hydrogen) contribute electrons via photosystem I in the light, whereas the NADPH/NADP ratio regulates linear and cyclic electron flow at the reducing side of photosystem I to provide a ratio of ATP/electrons most effective for nitrogenase.Abbvreviations ATCC American Type Culture Collection - Diaminodurol (DAD) 2,3,5,6-tetramethyl-p-phenylenediamine dihydrochloride - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DNP-INT 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol - E Einstein (mol photons) - FNR ferredoxin - NADP oxidoreductase (EC 1.18.1.2) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Metronidazole 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole     

Electron donation to nitrogenase in heterocysts of cyanobacteria

Various electron donors were found to stimulate C₂H₂ reduction (N₂ fixation) by isolated heterocysts from Anabaena variabilis and Anabaena cylindrica. Intermediates of glycolysis and the tricarboxylic acid cycle as well as unphosphorylated sugars like glucose, fructose and erythrose were among these electron donors. The transfer of electrons from donors like H₂, NADH, glyoxylate and glycollate was strictly light-dependent, whereas others like NADPH or pyruvate plus coenzyme A supported C₂H₂ reduction also in the dark. In all cases, the overall activity was enhanced by light. The stimulation by light was more distinct with heterocysts from A. variabilis than with heterocysts from A. cylindrica.The present communication establishes that pyruvate supports C₂H₂ reduction by heterocysts from either A. variabilis or A. cylindrica with rates comparable to those with other electron donors. Pyruvate could, however, support C₂H₂ reduction only in the presence of coenzyme A, and the concentrations of both coenzyme A and pyruvate were crucial. A pyruvate-dependent reduction of ferredoxin by extracts from heterocysts was recorded spectrophotometrically. Glyoxylate, which is an inhibitor of thiamine pyrophosphate-dependent decarboxylations, inhibited pyruvate-dependent C₂H₂ reduction. This result supports the conclusion that pyruvate is metabolised by pyruvate: ferredoxin oxidoreductase in heterocysts. High concentrations of pyruvate and other electron donors inhibited C₂H₂ reduction which suggests that nitrogenase activity in heterocysts may be controlled by the availability of electron donors.Dedicated to Professor Norbert Pfennig, Konstanz, on the occasion of his 60th birthday     

Ultrastructural characterisation of cells specialised for nitrogen fixation in a non-heterocystous cyanobacterium,Trichodesmium spp.

Summary Trichodesmium is the first described example of a filamentous cyanobacterium without heterocysts that contains cells specialised for nitrogen fixation. The ultrastructure of cells with and without nitrogenase were compared using primarilyTrichodesmium tenue Wille, but alsoT. thiebautii Gomont andT. erythraeum Ehrenberg et Gomont. Immunohistochemistry demonstrated that the cytoplasm of certain cells was densely labelled with antibodies against Fe-protein (dinitrogenase reductase). Comparative TEM-image analysis revealed that these cells were also distinguished by a denser thylakoid network, dividing the vacuole-like space into smaller units. The nitrogenase-containing cells also exhibited less extensive gas vacuoles as well as fewer and smaller cyanophycin granules compared to cells which lacked nitrogenase. Carboxysomes were present in both cell types in equal proportion. Longitudinal sections showed that cells with nitrogenase were arranged adjacent to each other, and that groups of cells with and without nitrogenase may coexist in the same trichome. The correlation between modifications in ultrastructure and the presence of nitrogenase suggests a new type of cyanobacterial cell specialisation related to nitrogen fixation. The results obtained also question the systematic affiliation of the genusTrichodesmium.     

Glutamine synthetase: activity and localization in cyanobacteria of the cycadsCycas revoluta andZamia skinneri

Nitrogen-fixing cyanobacteria inhabit the zone between the inner and outer cortex of cycad coralloid roots. In the growing tip of such roots the cyanobacterial heterocyst frequency, nitrogenase activity (C₂H₂-reduction) and glutamine synthetase activity (both transferase and biosynthetic) were comparable to those found in freeliving cyanobacteria. The relative level of glutamine synthetase protein and its pattern of cellular/subcellular localization in heterocysts and vegetative cells were also similar to those of free-living cyanobacteria. However, there was a progressive decline in nitrogenase activity along the coralloid root with maximum reduction occurring in the regions farthest from the growing tip. A similar but less pronounced pattern was observed for glutamine synthetase activity. Distribution of glutamine synthetase protein in cyanobacteria in the first 2–3 mm of the root tip indicated a slight decrease in the heterocysts and vegetative cells. However, the overall level of cyanobacterial glutamine synthetase protein did not change because of a drastic increase in the numbers of heterocysts, which contain a proportionally higher level of glutamine synthetase than the vegetative cells.Abbreviation GS glutamine synthetase     

Relationship between nitrogen fixation and nitrate metabolism in the Nodularia strains M1 and M2

Nitrogen fixation and nitrate-reduction activities were determined in photoautotrophic cultures of two wild-type strains of cyanobacterium Nodularia, spp. M1 and M2. Air could support growth of the two strains at a similar rate in the presence or absence of exogenous nitrate, ammonium and/or bicarbonate. Nitrogenase activity in air-grown cultures varied with culture age, and totally disappeared after 6 h of darkness. Recovery took place upon culture re-illumination. Ammonium at a concentration of 1 mM resulted in the total disappearance of nitrogenase activity and of heterocysts. In contrast, 20 mM nitrate hardly affected nitrogenase activity and heterocyst formation after ten generations. Under the same conditions, either ammonium or nitrate completely abolished nitrogenase activity and heterocyst formation in Anabaena sp. PCC 7119, a typical heterocystous strain. The inefficiency of nitrate in inhibiting nitrogen fixation in Nodularia M1 and M2 seemed to be caused by a low nitrate-reductase activity, and not by an impairment of nitrate-uptake activity. On the other hand, the presence of nitrate was not required for uptake activity to be expressed in Nodularia.Abbreviation NR nitrate reductase We thank C. Fernández-Cabrera (Consejo Superior de Investigaciones Científicas, CSIC, Madrid, Spain) for technical assistance, and Dr. G. Pérez-Silva (CSIC) for his collaboration in the Anabaena NR assays. This work was supported by grants from Spanish CI-CyT (PB 87-0204 and PB 92-0497).     

Nitrogen fixation by Anabaena cylindrica

Summary Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO₂ fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.     

Immunochemical evidence that nitrogenase is restricted to the heterocysts in Anabaena cylindrica

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under anaerobic conditions was studied by immunoferritin labelling of the Fe-Mo protein (Component I). Differentiating cultures, incubated under an argon atmosphere, were treated with DCMU 12 h following initiation of induction. DCMU inhibited photosynthetic O₂ production, thus insuring strict anaerobic conditions, but had no effect on nitrogenase induction. Fe-Mo protein levels, as determined by rocket immunoelectrophoresis, increased 5-fold within 24h of DCMU treatment. Immunoferritin labelling of aldehyde fixed, ultrathin cryosections of anaerobically induced filaments showed that the Fe-Mo protein was restricted to the heterocyst. Ferritin labelling was shown to be specific by the following criteria: (a) substituting preimmune goat serum for the anti-Fe-Mo protein IgG prevented ferritin labelling; (b) ferritin-conjugated, non-homologous rabbit anti-goat IgG did not bind; (c) incubation of anti-Fe-Mo protein IgG treated sections with rabbit anti-goat IgG prior to the treatment with the ferritin label also prevented labelling. The results provide direct immunochemical evidence that nitrogenase is restricted to the heterocysts even under strictly anaerobic conditions.     

Induction of H2-Uptake and Nitrogenase Activities in the Cyanobacterium Anabaena variabilis ATCC 29413: Effects of Hydrogen and Organic Substrate

A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells of the cyanobacterium Anabaena variabilis was performed. The induction of heterocysts is followed by the induction of both in vivo hydrogen uptake and nitrogenase activities. Interestingly, a low but significant H₂-uptake [2–7 μmoles of H₂ · mg⁻¹ (Chl a) · h⁻¹] occurs in cultures with no heterocysts and with no nitrogenase activity. A slight stimulatory effect (30–40%) of H₂ on in vivo H₂-uptake was observed during the early stages of nitrogenase induction. However, exogenous H₂ does not further stimulate the induction of in vivo hydrogen uptake observed during heterocyst differentiation. Similarly, organic carbon (fructose) did not influence the induction of either in vivo hydrogen uptake or nitrogenase activities. Exogenous fructose supports higher in vivo hydrogen uptake and nitrogenase activities when the cells enter late exponential phase of growth. Received: 22 November 1995 / Accepted: 22 December 1995     

Carbon-nitrogen requirements for the expression of nitrogenase activity in cultured Parasponia-Rhizobium strain ANU 289

Nutritional factors controlling derepression of nitrogenase activity in Parasponia-Rhizobium strain ANU 289 were studied in stationary and agitated liquid cultures. Altering type and/or concentrations of the constituents of the derepression medium in respect of carbon and nitrogen sources influenced both derepression kinetics as well as the maximal level of activity. Hexose sugars and disaccharides stimulated nitrogenase activity three to six-fold compared to pentose sugars. Activity was also modulated by combining sugars with some organic acids such as succinate, fumarate and pyruvate but not with others (e.g. -ketoglutarate, malate, malonate). Of the range of nitrogen sources tested, either casamino acids (at 0.05%, but not at 0.1%), glutamate, proline or to a lesser extent histidine (each at 5 mM N) supported significant derepression of nitrogenase activity. Notably glutamine, urea, alanine, ammonium sulfate, nitrate, nitrite (each at 5 mM N) and yeast extract (0.05%) failed to derepress or support nitrogenase activity. Ammonium (5 mM) abolished established nitrogenase activity of rapidly agitated cultures within 15 h after addition. This inhibitory effect was alleviated by the addition of methionine sulfoximime (10 mM). Thus, in view of strong glutamine effects, ammonium repression appears to be mediated by glutamine and not by ammonium itself.Abbreviations HEPES [4-(2-hydroxyethyl)-1-piperazine-ethane; sulfonic acid] - MOPS [3-(N-morpholino) propane sulphonic acid] - MSX Methionine sulfoximine     

Heterocyst and akinete induction with altered pattern in Anabaena cylindrica,caused by neo-peptone

Neo-peptone B119 (Difco) was found to have a significant effect on differentiation of heterocysts and akinetes in Anabaena cylindrica. On adding neopeptone (0.4 g/l) to exponential phase culture of A. cylindrica, the following effects were observed (i) increased heterocyst frequency with altered heterocyst spacing and presence of double and multiple heterocysts after 24 h in cultures grown on N-free medium, (ii) induction of regular pattern of heterocysts after 48 h, in culture grown on medium supplemented with NH₄Cl, (iii) induction of pro-akinetes after 48 h in both N-free and ammonium-grown cultures. The higher concentrations of neo-peptone were lytic to A. cylindrica, and, its lytic and inductive effects could be decreased by acid hydrolysis or supplementation of NH₄Cl. Gel-filtration of neo-peptone showed that the inductive as well as the lytic effect was associated with some active factor(s) with molecular weight between 10,000–20,000. The retention of the inductive effect on autoclavation but its loss on trypsin digestion suggested that active factor(s) may be heat stable polypeptide(s). The heterocyst induction by active factor(s) decreased and akinete induction increased with increasing culture age. The pro-akinetes induced during exponential phase divided before maturation, while those induced during late exponential phase, could achieve full maturity. Growth and nitrogenase activity was unaffected while there was an increase in mean cell length on treatment of A. cylindrica with active factor(s) from neo-peptone, indicating that the effect may be mediated through cell division process(es).Abbreviations used N Nitrogen - chl chlorophyll     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

Isolation and characterisation of nitrate reductase mutants and regulation of nitrate reductase and nitrogenase in the cyanobacterium Nostoc muscorum

Summary Chlorate resistant mutants of the cyanobacterium Nostoc muscorum isolated after N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis were found to be defective/blocked in nitrate reductase (NR).The parent strain possessed active NR in the presence of nitrogen as nitrate and only basal levels of activity in ammonia and N-free grown cultures. Addition of ammonia suppressed the NR activity in the parent strain whereas addition of L-methionine DL-sulphoximine (MSX) restored NR activity. A similar repression by ammonia, glutamine and derepression with MSX were also observed for nitrogenase synthesis.One class of mutants lacked NR activity (nar ^-) whereas the specific activity of NR was low in another class of mutants (nar ^def). Unlike the parent, the mutants synthesized nitrogenase and differentiated heterocysts in the presence of nitrate nitrogen. Uptake studies of nitrite and ammonia in mutants revealed that they possessed both nitrite reductase and glutamine synthetases (GS) at low levels, and the same level respectively in comparison with the parent.