Feasibility of 55Fe Autoradiography as Performed on N2-Fixing Anabaena spp. Populations and Associated Bacteria

⁵⁵Fe emits low-energy X rays and Auger electrons by electron capture decay. Auger electrons are useful for autoradiographic examination of ⁵⁵Fe incorporation among microbial communities. Attainable resolution, in terms of silver grain deposition, is excellent and comparable to ³H. Two known Fe-demanding processes, photosynthetic CO₂ fixation and N₂ fixation, were examined by autoradiography of Anabaena populations. During photosynthetically active (illuminated) N₂-fixing periods, biological incorporation of ⁵⁵FeCl₃ by vegetative cells and heterocysts was evident. When N₂ fixation was suppressed by NH₄⁺ additions, heterocysts revealed no incorporation of ⁵⁵Fe. Conversely, when N₂-fixing Anabaena filaments were placed in darkness, ⁵⁵Fe incorporation decreased in vegetative cells, whereas heterocysts showed sustained rates of ⁵⁵Fe incorporation. Bacteria actively incorporated ⁵⁵Fe under both light and dark conditions. The chelated (by Na₂-ethylenediaminetetraacetate) form of ⁵⁵FeCl₃ was more readily incorporated than the nonchelated form. Furthermore, abiotic adsorption of ⁵⁵Fe to filters and nonliving particles proved lower when chelated ⁵⁵Fe was used in experiments. ⁵⁵Fe autoradiography is useful for observing the fate and cellular distribution of various forms of Fe among aquatic microbial communities.     

Adenine nucleotide levels in and nitrogen fixation by the cyanobacterium Anabaena sp. strain 7120.

Adenine nucleotide levels were determined in whole filaments of Anabaena sp. 7120 grown under different N2-fixing or non-N2-fixing conditions. These were compared with levels in isolated heterocysts, Rhodospirillum rubrum, and Azotobacter vinelandii. Adenine nucleotides in whole filaments of Anabaena sp. do not reflect the energetic expense of N2 fixation as they do in R. rubrum and A. vinelandii. However, adenine nucleotide levels in heterocysts were similar to the levels found in N2-fixing R. rubrum, i.e., an ATP:ADP ratio near 1 and an energy charge between 0.5 and 0.7. Nitrogenase activity was only 50% of optimal in permeabilized heterocysts at an exogenous ATP:ADP ratio of 3.33. Hydrogen, which increases acetylene reduction activity, also causes a transient increase (2 to 5 min) in the ATP:ADP ratio. Hydrogen has little effect on energy charge.     

14C-labeled metabolites in heterocysts and vegetative cells of Anabaena cylindrica filaments and their presumptive function as transport vehicles of organic carbon and nitrogen

To investigate the transport of primary metabolites in Anabaena cylindrica from vegetative cells into heterocysts, intact filaments were labeled with the heterocysts were separated from the vegetative cells after different time intervals, and the labeling patterns were determined. After a 20-s fixation time, a high percentage of labeling of alanine, glutamate and glutamine, and, to a lesser extent, glucose 6-phosphate was found in heterocysts as compared with whole filaments. The results can be explained if transport of alanine, glutamate, and sugars from vegetative cells into heterocysts is assumed. Alanine can serve as a precursor for reducing equivalents if it is oxidized to glutamine which flows back to the vegetative cells. This idea is supported by an experiment in which exogenous alanine is readily converted by isolated heterocysts to glutamate and glutamine under a N2-H2 atmosphere. The incorporation of [14C]carbonate in isolated heterocysts demonstrated the absence of the reductive pentose phosphate pathway; however, it revealed marked activity of an acid fixation reaction.     

Simultaneous occurrence of two different glyceraldehyde-3-phosphate dehydrogenases in heterocystous N(2)-fixing cyanobacteria

Enzyme activity determinations and Western and Northern blot analyses have shown the presence of two catalytically different glyceraldehyde-3-phosphate dehydrogenases (GAPDH) in both vegetative cells and heterocysts of several N(2)-fixing Anabaena strains: (a) the gap2-encoded NAD(P)-dependent GAPDH2 (EC 1.2.1.59), the enzyme involved in the photosynthetic carbon assimilation pathway, which is present at higher levels in vegetative cells, and (b) the gap3-encoded NAD-dependent GAPDH3 (EC 1.2.1.12), presumably involved in carbohydrate anabolism and catabolism, which is the predominant GAPDH in heterocysts. In contrast, the gap1-encoded GAPDH1, which is the other NAD-dependent cyanobacterial GAPDH, is virtually absent in both cell types. These findings are discussed in the context of carbon metabolism of heterocystous N(2)-fixing cyanobacteria.     

Localized Tetrazolium Reduction in Relation to N(2) Fixation, CO(2) Fixation, and H(2) Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N(2) fixation (acetylene reduction), CO(2) fixation, and H(2) utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, CO(2) fixation and H(2) utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N(2)-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.     

Molecular cloning and characterization of hetR genes from filamentous cyanobacteria

HetR, a serine type protease, plays an important role in heterocyst differentiation in filamentous cyanobacteria. We isolated and sequenced the hetR genes from different heterocystous and filamentous nonheterocystous cyanobacteria. The hetR gene in the heterocyst forming Anabaena variabilis ATCC 29413 FD was interrupted by interposon mutagenesis (mutant strain WSIII8). This mutant does not form heterocysts and shows no diazotrophic growth under aerobic conditions. However, under anaerobic N(2)-fixing conditions, the WSIII8 cells are able to grow, and high nitrogenase (Nif2) activity is detectable. Nif2 expression was demonstrated in each vegetative cell of the filament by immunolocalization 4 h after nitrogen step-down.     

FraH is required for reorganization of intracellular membranes during heterocyst differentiation in Anabaena sp. strain PCC 7120

In the filamentous, heterocyst-forming cyanobacteria, two different cell types, the CO(2)-fixing vegetative cells and the N(2)-fixing heterocysts, exchange nutrients and regulators for diazotrophic growth. In the model organism Anabaena sp. strain PCC 7120, inactivation of fraH produces filament fragmentation under conditions of combined nitrogen deprivation, releasing numerous isolated heterocysts. Transmission electron microscopy of samples prepared by either high-pressure cryo-fixation or chemical fixation showed that the heterocysts of a ΔfraH mutant lack the intracellular membrane system structured close to the heterocyst poles, known as the honeycomb, that is characteristic of wild-type heterocysts. Using a green fluorescent protein translational fusion to the carboxyl terminus of FraH (FraH-C-GFP), confocal microscopy showed spots of fluorescence located at the periphery of the vegetative cells in filaments grown in the presence of nitrate. After incubation in the absence of combined nitrogen, localization of FraH-C-GFP changed substantially, and the GFP fluorescence was conspicuously located at the cell poles in the heterocysts. Fluorescence microscopy and deconvolution of images showed that GFP fluorescence originated mainly from the region next to the cyanophycin plug present at the heterocyst poles. Intercellular transfer of the fluorescent tracers calcein (622 Da) and 5-carboxyfluorescein (374 Da) was either not impaired or only partially impaired in the ΔfraH mutant, suggesting that FraH is not important for intercellular molecular exchange. Location of FraH close to the honeycomb membrane structure and lack of such structure in the ΔfraH mutant suggest a role of FraH in reorganization of intracellular membranes, which may involve generation of new membranes, during heterocyst differentiation.     

Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-plex isobaric peptide tags

The filamentous cyanobacterium Nostoc sp. strain PCC 7120 is capable of fixing atmospheric nitrogen. The labile nature of the core process requires the terminal differentiation of vegetative cells to form heterocysts, specialized cells with altered cellular and metabolic infrastructure to mediate the N2-fixing process. We present an investigation targeting the cellular proteomic expression of the heterocysts compared to vegetative cells of a population cultured under N2-fixing conditions. New 8-plex iTRAQ reagents were used on enriched replicate heterocyst and vegetative cells, and replicate N2-fixing and non-N2-fixing filaments to achieve accurate measurements. With this approach, we successfully identified 506 proteins, where 402 had confident quantifications. Observations provided by purified heterocyst analysis enabled the elucidation of the dominant metabolic processes between the respective cell types, while emphasis on the filaments enabled an overall comparison. The level of analysis provided by this investigation presents various tools and knowledge that are important for future development of cyanobacterial biohydrogen production.     

Occurrence and localization of two distinct hydrogenases in the heterocystous cyanobacterium Anabaena sp. strain 7120

Two distinct types of hydrogenase occur in Anabaena 7120 and are distinguishable in whole filaments by the application of selective assay methods. A reversible hydrogenase occurs both in heterocysts and vegetative cells and can be selectively assayed by measuring H2 evolution from reduced methyl viologen. Activities in aerobically grown filaments were low but could be increased by 2 to 3 orders of magnitude by growing cells microaerobically. The presence of the reversible hydrogenase was independent of the N2-fixing properties of the organism, and activity did not respond to added H2 in the culture. Illumination was necessary during derepression of the reversible hydrogenase, and addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea increased the amount of enzyme that was synthesized. An uptake hydrogenase occurred only in heterocysts of aerobically grown filaments, but a small amount of activity also was present in the vegetative cells of filaments grown microaerobically with 20% H2. It was assayed selectively by measuring an oxyhydrogen reaction at atmospheric levels of O2. Additional uptake hydrogenase could be elicited by including H2 or by removing O2 from the sparging gas of a culture.     

Superoxide dismutase and catalase in the protection of the proton-donating systems of nitrogen fixation in the blue-green alga Anabaena cylindrica.

1. Superoxide dismutase activity was present in the heterocysts and vegetative cells of Anabaena cylindrica, but was always lower in the heterocysts. 2. No qualitative differences were found in the superoxide dismutase from the two cellular types. 3. Catalase activity was also present in both cellular types. 4. Most of the NADP reductase activity, as assayed with menadione or ferredoxin as electron acceptor, was localized within the heterocysts. 5. Studies on H2 consumption showed that most of the hydrogenase activity was associated with the heterocysts. 6. The results are discussed in terms of the postulate that superoxide dismutase and catalase are involved in the protection of the proton-donating systems participating in N2 fixation and H2 metabolism of heterocysts.     

Inhibition of cell division blocks the synthesis of the second nitrogenase (Nif2) in the cyanobacterium Anabaena variabilis

Anabaena variabilis ATCC 29413 belongs to the cyanobacteria that use a specific cell type, heterocysts, for fixation of atmospheric nitrogen under aerobic conditions. Nitrogen fixation under anaerobic conditions is catalyzed by a Mo-dependent nitrogenase (Nif2) that is expressed in the vegetative cells. We demonstrate here using immunolocalization/light microscopy (LM) that the synthesis of NifH2 is mainly initiated in dividing vegetative cells along the trichomes. Blocking cell division by cephalexin abolished nitrogenase synthesis under anaerobic conditions.     

Isolation and preliminary characterization of auxotrophs of a filamentous Cyanobacterium.

Auxotrophic mutants of the filamentous cyanobacterium Anabaena variabilis were isolated by a method in which, after mutagenesis and before penicllin enrichment, mutant and wild-type cells were separated by cavitation. Auxotrophs were identified by their inability to grow on minimal medium, and they were partially characterized by replica plating to media supplemented with single nutrients or specific groups of nutrients. Of the 83 auxotrophs isolated, 65 required an inorganic source of nitrogen for growth. In addition, auxotrophs were isolated that required methionine (six), uracil (two), adenine (one), biotin (two), and nicotinic acid (two). (The number of isolates of each type is indicated in parentheses.) The nutrient requirements of five auxotrophs appeared complex and were not determined. A large proportion of the mutants requiring inorgainic fixed nitrogen was altered in the differentiation of heterocysts. The following morphological aberrancies were observed: abnormally high and abnormally low frequencies of heterocysts; thick, uneven heterocyst envelopes; incompletely developed pore regions; very distinct pore regions; and protoplasts separated from the envelope of the heterocyst. Spontaneously occurring, N2-fixing, prototrophic revertants of mutants with aberrant heterocysts have been isolated at a frequency of 2 X 10(-8) to 4 X 10(-8) of the cells plated. That most such revertants produced morphologically normal heterocysts is consisten with the idea that heterocysts play an essential role in aerobic N2 fixation.     

四唑(Tetrazolium)对固氮鱼腥藻固氮和氢代谢的影响

固氮鱼腥藻(Anabaena azotica Ley)细胞能还原无色的TTC和NBT分别成为红色或蓝色的甲(月朁)(formazan)沉淀。异形胞还原TTC的速率高于营养细胞。前异形胞及异形胞附近的营养细胞对NBT的还原作用最强。而异形胞对NBT不起还原作用。无论在异形胞形成红色甲(月朁)或在营养细胞形成蓝色甲(月朁)后都抑制固氮酶活性。NBT甲(月朁)对固氮酶活性的抑制作用大于TTC甲(月朁),因为NBT氧化还原电位低于TTC。 TTC和NBT两者都明显地抑制固氮鱼腥藻完整细胞的放氢。因鱼腥藻的放氢是由固氮酶催化的结果。四唑抑制放氢推想是由于它截取了固氮酶催化系统中的电子的缘故。固氮微生物(包括蓝色细菌和根瘤菌)对四唑还原与吸氢酶之间有无相关是一个争论的问题。一些学者认为分离豆科植物体的一些根瘤菌株培养于含有TTC的琼脂培养基,如还原,便可证明这些根瘤菌株能氧化氢;换言之,应用TTC的还原可作为一些根瘤菌的菌落具有吸氢酶的验证。相反,我们发现固氮鱼腥藻还原TTC和NBT之后,都没有影响吸氢的能力。因此,我们推想固氮鱼腥藻对四唑之还原与吸氢酶是没有直接的关系。     

Localized Tetrazolium Reduction in Relation to N2 Fixation, CO2 Fixation, and H2 Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N₂ fixation (acetylene reduction), ¹⁴CO₂ fixation, and ³H₂ utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, ¹⁴CO₂ fixation and ³H₂ utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N₂-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.     

Cyanobacterial heterocysts: terminal pores proposed as sites of gas exchange

In many filamentous cyanobacteria, oxygenic photosynthesis is restricted to vegetative cells, whereas N(2) fixation is confined to microoxic heterocysts. The heterocyst has an envelope that provides a barrier to gas exchange: N(2) and O(2) diffuse into heterocysts at similar rates, which ensures that concentrations of N(2) are high enough to saturate N(2) fixation while respiration maintains O(2) at concentrations low enough to prevent nitrogenase inactivation. I propose that the main gas-diffusion pathway is through the terminal pores that connect heterocysts with vegetative cells. Transmembrane proteins would make the narrow pores permeable enough and they might provide a means of regulating the rate of gas exchange, increasing it by day, when N(2) fixation is most active, and decreasing it at night, minimizing O(2) entry. Comparisons are made with stomata, which regulate gas exchange in plants.     

Control of phycobiliprotein proteolysis and heterocyst differentiation in Anabaena.

Phycobiliprotein degradation can be initiated in cultures of the cyanobacterium Anabaena by removal of combined nitrogen from the medium. Certain strains of Anabaena differentiate cells specialized for aerobic nitrogen fixation (heterocysts) under such conditions. We describe here a procedure for the preparation of extracts from heterocysts or vegetative cells that contain an activity capable of degrading only the phycobiliproteins in a mixture of soluble Anabaena proteins in vitro. This activity increased under nitrogen starvation conditions or in ammonia-replete cultures treated with the glutamine synthetase inhibitor methionine sulfoximine. The increase in activity induced by nitrogen starvation was prevented by chloramphenicol or by carbon starvation. Under all these conditions, phycobiliprotein degradative activity assayed in vitro was correlated with the loss of phycobiliprotein absorbance in vivo. Finally, starvation of a met auxotroph of Anabaena for methionine (in the presence of ammonia) did not induce phycobiliprotein degradation in vivo or the increase in proteinase activity. Together with direct measurements of ppGpp, these results indicate that proteolysis in Anabaena is not controlled by compounds associated with the stringent response in Escherichia coli. Since the increase in proteinase activity appears to be regulated by the same variables that control heterocyst differentiation, the activity should provide a useful biochemical marker for the early events of differentiation.