Structural aspects of akinete germination in the cyanobacterium Anabaena variabilis

Electronmicroscopical investigations of light activated akinetes in different phases before outgrowth of the germinating cell showed two alterations in the akinete envelope, obviously in connection with the germination process. After induction of germination the akinetes show formation of an expanding more or less electron dense layer between the outer cell wall layer (outer membrane, L_IV) and the condensed part of the akinete coat (the transformed sheath of the vegetative cell). Between this new formed layer and the mentioned part of the akinete coat thick laminar layers are deposited which contain alternately electron dense and electron transparent strata. The expanding layer is assumed to be a mucous layer which acts as swelling body causing, after bursting of the layered shell, the expulsion of the germinating cell in the manner characteristic for Anabaena variabilis.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

The influence of light quality on akinete formation and germination in the toxic cyanobacterium Anabaena circinalis

Physiological control of akinete formation and subsequent germination is likely to be important in understanding and predicting how natural populations of cyanobacteria respond to their environment. While previous research has indicated nutrient limitation may be important in akinete formation new results presented here indicate that in the toxic and bloom-forming species Anabaena circinalis there was a profound effect of spectral quality. Under 40 μmol photons m^?2 s^?1 photosynthetically active irradiance (PAR) of predominately red irradiance akinete production was maximal at 2.1 × 10^?4 akinetes vegetative cell^?1 d^?1, some 3000 times greater than the 6.5 × 10^?8 akinetes vegetative cell^?1 d^?1 observed under equivalent PAR but predominately blue light. For cells grown under a range of predominantly red, white and green irradiance even short exposures to blue light reduced akinete formation rates by a factor of ten relative to controls, indicating that exposure to blue light inhibits akinete formation. Germination of akinetes was not influenced by the irradiance under which akinetes were formed: 88 ± 4.1% (mean ± 1 S.D.) of akinetes germinated with no evidence of an effect on germination success due to their production under predominately red, white or green irradiance (germination of akinetes produced under blue light was not tested). Spectral quality had a significant impact on both vegetative cell and germling growth rates. The results indicate a significant reduction in the cellular differentiation of A. circinalis vegetative cells into akinetes that is mediated by blue light. In an ecological context the production of akinetes will be greater in environments with less blue light; potentially including those with slower flow, more stratification, less vertical mixing and more turbidity. The resulting spatial pattern of akinete production is likely to influence the location of akinetes in sediments and the development of subsequent blooms from excysting germlings.     

The ultrastructure of Anabaena azollae in Azolla pinnata

The water fern Azolla pinnata R. Br. was collected in Northwestern India and its structure was examined using scanning and transmission electron microscopy. Emphasis was given to the symbiotic cyanobacterium. Anabaena azollae , and to its relationship to the hairs of the leaf cavities. The cyanobacterial filaments are loosely arranged and often adhere to the protruding hairs and the folded cell walls of the cavities. The vegetative cells show a typical bilayered cell wall. Thylakoids are few and evenly dispersed in mature vegetative cells and appear to lack phycobilisomes. Clusters of polyglucoside granules are distinguished between the thylakoids. Thylakoid membranes are often seen forming whirls and lattices. Polyhedral bodies (carboxysomes) appear particularly frequently in younger cells and in the proximity of polyphosphate bodies. Presence of structured granules, often positioned at both sides of the cross-wall of neighbouring vegetative cells, suggest a positive nitrogen balance. A high frequency of heterocysts is noted, while spores are not observed.
The outer cell wall of the unbranched, mostly two-celled, hairs shows frequent invaginations. The cytoplasma of the mature hair contains numerous organelles, and is penetrated by an electron transparent network with blebs and vesieles appearing. The exchange of metabolites between the symbiotic partners is discussed in relation to the structures noted.     

The Azolla, Anabaena azollae Relationship: I. Initial Characterization of the Association

Cultures of Azolla caroliniana Willd. free of the symbiotic blue-green alga, Anabaena azollae, were obtained by treatment of Azolla fronds with a regimen of antibiotics. These symbiontfree plants can be maintained only on medium containing a combined nitrogen source.     

Characterization of HetN, a protein involved in heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120

AtlA is a major cell-lytic enzyme called autolysin in Streptococcus mutans . In this study, we identified the atlg gene-encoding autolysin (Atlg), consisting of 863 residues from Streptococcus sobrinus 6715DP, and confirmed lytic activity of recombinant Atlg by zymography of S. sobrinus cells. An atlA -inactivated mutant was constructed in S. mutans Xc, and the atlg gene product was characterized by plasmid complementation. Microscopic analysis, saliva-induced aggregation assay and autolysis assay of static cultures in air revealed that the atlg gene product partially complemented the role of AtlA. Furthermore, the capability of biofilm formation of the atlA- deficient mutant cultivated in air was restored by plasmid comprising the atlg gene. These findings suggest that Atlg may be involved in cell separation and biofilm formation in S. sobrinus .     

Heterocyst differentiation and tryptophan metabolism in the cyanobacterium Anabaena sp. CA

Anabaena sp. CA does not synthesize heterocysts or express nitrogenase activity when grown with nitrate as the nitrogen source. Heterocysts and nitrogenase are induced in such cultures by various tryptophan analogs. The effect does not require inhibition of de novo protein synthesis in the culture. It is restricted to tryptophan analogs only, and, more specifically, to those which can be incorporated into proteins. dl-7-Azatryptophan was effective at triggering both heterocysts and nitrogenase when incubated in the culture for only 1–2 h, even though 6–7 h was required for heterocysts to fully mature and nitrogenase activity to be expressed. Chloramphenicol completely negated this effect, supporting the idea that the analogs are either incorporated into protein themselves or trigger the synthesis of proteins which initiate complete development of mature heterocysts. Using toluene-permeabilized cells, we have shown that anthranilate synthetase, the first key enzyme in tryptophan biosynthesis, has glutamine-dependent activity. This activity can be effectively feedback inhibited by the various tryptophan analogs at concentrations which are also effective in triggering heterocyst differentiation. These data provide firm evidence for a link between tryptophan biosynthesis, nitrogenase synthesis, heterocyst differentiation, and primary ammonia assimilation.     

Characterization of cytochrome c 6 from the cyanobacterium Anabaena PCC 7119

 A soluble monoheme c–type cytochrome c ₆ has been isolated from the cyanobacterium Anabaena PCC 7119. It is a basic protein, with a molecular mass of 9.7 kDa, which accepts electrons from Anabaena ferredoxin in the ferredoxin-NADP⁺reductase-dependent NADPH cytochrome c reductase activity assay. The turnover of the reaction has an optimum pH at 7.5. Flavodoxin can also replace ferredoxin in this assay, but with only 20% efficiency. Plastocyanin from Anabaena PCC 7119, as well as the c ₆ cytochromes from the green algae Chlorella fusca and Monoraphidium braunii are also shown to accept electrons from Anabaena ferredoxin. The reduction potential of cytochrome c ₆ at pH 6.7 was determined to be 338 mV and is pH dependent, with pK _a ^ox=8.4±0.1 and pK _a ^red≈9.5. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using visible, EPR and ¹H-NMR spectroscopies. The amino acid sequence and the visible and NMR spectroscopic data indicate that the heme iron has a methionine-histidine axial coordination in the pH range 5–11. However, the EPR data for the ferricytochrome are complex and show that in this pH range five distinct forms are present. Between pH 5 and 9 the spectrum is dominated by two rhombic species, with g–values at 2.94, 2.29, 1.43 and at 2.84, 2.34, 1.56, which interconvert with a pK _a of 8.4. The NMR data also show a main interconversion between two cytochrome forms at this pH, which coincides with that determined from the pH dependence of the reduction potential. Both these forms were associated with a methionine-histidine heme-iron coordination by correlation with the visible and NMR spectral data, although having crystal field parameters atypical for this type of coordination. Anabaena cytochrome c ₆ is one more example of a heme protein for which the widely used crystal field analysis of the EPR data (truth diagram) fails to unequivocally determine the type of heme-iron ligation. Received: 17 May 1996 / Accepted: 13 January 1997     

Identification of an akinete marker gene in Anabaena variabilis

Cyanobacteria that form akinetes as well as heterocysts present a rare opportunity to investigate the relationships between alternative differentiation processes and pattern formation processes in a single bacterium. Because no akinete marker gene has been identified, akinete formation has been little studied genetically. We report the first identification of an akinete marker gene.     

Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis.

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis.     

Pattern of akinete differentiation in the cyanobacteriumScytonema fritschii

The differentiation of akinetes inScytonema fritschii occurred adjacent to the newly developed heterocysts in late exponential phase. The filaments exhibited cell division leading to the formation of heterocysts, interspersed by the potential akinetes which could be identified by the accumulation of a large number of granules. Upon maturity, the akinetes acquired thick envelopes and were seen in elongated series interrupted by dead necridia which resulted from crumpling of the newly developed heterocysts. The formation of akinetes was accompanied by a change in color of cultures from blue-green to brown. Of the inorganic nitrogen sources tested, ammonium nitrate supported the formation of maximum percentage of akinetes. The incorporation of 7-azatryptophan and rifampicin in nitrate-free and nitrogen sources resulted in the production of heterocysts at a very high frequency in the late-exponential phase coinciding with akinete formation but the frequency of the latter was reduced. The activity of nitrogenase, nitrate reductase and glutamate-ammonia ligase was absent in mature akinetes. The absorption spectra of chlorophylla and phycobiliproteins revealed the presence of negligible amounts of the former white the latter were absent. The dry mass steadily increased during akinete differentiation with a concomitant decrease in C/N ratios.     

Effect of glutamine on growth and heterocyst differentiation in the cyanobacterium Anabaena variabilis.

Mutants of the cyanobacterium Anabaena variabilis that were capable of increased uptake of glutamine, as compared with that in the parental strains, were isolated. Growth of these mutants and their parental strains was measured in media containing N2, ammonia, or glutamine as a source of nitrogen. All strains grew well with any one of these sources of fixed nitrogen. Much of the glutamine taken up by the cells was converted to glutamate. The concentrations of glutamine, glutamate, arginine, ornithine, and citrulline in free amino acid pools in glutamine-grown cells were high compared with the concentrations of these amino acids in ammonia-grown or N2-grown cells. All strains capable of heterocyst differentiation, including a strain which produced nonfunctional heterocysts, grew and formed heterocysts in the presence of glutamine. However, nitrogenase activity was repressed in glutamine-grown cells. Glutamine may not be the molecule directly responsible for repression of the differentiation of heterocysts.     

Biodiversity of Anabaena azollae isolates from different Azolla cultures

The random amplified polymorphic DNA (RAPD) profile of A. azollae strains isolated from four different Azolla cultures was studied by using different primers. The objective of this study was to determine whether polymerase chain reaction (PCR) with different primers could differentiate the isolated A. azollae strains from one another. The primers amplified specific sequences of the isolates and generated fingerprinting pattern characteristic of each isolate. Clear polymorphism was noticed among all the strains which depends on the primer sequence.     

Characterization of genes for a second Mo-dependent nitrogenase in the cyanobacterium Anabaena variabilis.

Anabaena variabilis ATCC 29413 is a filamentous heterocystous cyanobacterium that fixes nitrogen under a variety of environmental conditions. Under aerobic growth conditions, nitrogen fixation depends upon differentiation of heterocysts and expression of either a Mo-dependent nitrogenase or a V-dependent nitrogenase in those specialized cells. Under anaerobic conditions, a second Mo-dependent nitrogenase gene cluster, nifII, was expressed in vegetative cells long before heterocysts formed. A strain carrying a mutant gene in the nifII cluster did not fix nitrogen under anaerobic conditions until after heterocysts differentiated. The nifII cluster was similar in organization to the nifI cluster that is expressed in heterocysts and that includes nifBSUHDKENXW as well as three open reading frames that are conserved in both cyanobacterial nif clusters.     

Sporulation in the filamentous cyanobacterium Anabaena cylindrica

Sporulation in the filamentous cyanobacterium Anabaena cylindrica involves the transformation of a vegetative cell into a thick-walled resistant structure. Because this process occurs at predictable loci in each filament and involves a significant increase in cell size, the course of sporulation in a culture can be quantitatively determined. Sporulation occurs during the late logarithmic phase of a culture, a time of slow but unbalanced growth. Under the conditions imployed here, sporulation is not a synchronous event either between or within filaments. The information in this paper provides an estimate of the rate of spore differentiation and supports the previous notion that in the formation of strings of more than one spore, a gradient of spore maturation exists.     

Transport of leucine in the cyanobacterium Anabaena variabilis

The amino acid leucine was transported by the cyanobacterium Anabaena variabilis. The K _m for transport was 10.8 M; the V _max was 8.7 nmoles min^–1 mg^–1 chlorophyll a. Transport of leucine was energy dependent: uptake of leucine was inhibited in the dark, and by DCMU and cyanide. Transport was neither dependent on nor enhanced by Na⁺. Prior growth of cells with leucine did not repress transport of [¹⁴C]-leucine. Alanine, glycine, valine, and methionine were strong competitive inhibitors of leucine uptake; serine, threonine, isoleucine, norleucine, and d-alanine competitively inhibited to a lesser degree. Other amino acids or amino acid analogues, including d-leucine, -aminoisobutyrate, and d-serine did not inhibit the transport of leucine.Abbreviations Chl a chlorophyll a - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - TES N-tris(hydroxymethyl)-2-aminoethane-sulfonic acid - TCA trichloroacetic acid - Tris N-tris(hydroxymethyl)aminoethane     

Regulation of urea-uptake in the cyanobacterium Anabaena doliolum

Abstract The utilization of urea was studied in the cyanobacterium Anabaena doliolum . The uptake of urea was unaltered in the presence of ammonium. The cells receiving ATP exogenously showed an induced level of urea-uptake as compared with the control cells. Urease inhibitor acetohydroxamic acid and hydroxyurea as well as glutamate analogue, MSO, did not affect the uptake of urea. These results suggest: (1) urea and ammonia have different uptake sites, (2) urea-uptake is an energy dependent process, and (3) during short-term experiments, urea uptake is not linked with the enzyme urease or the ammonium assimilating enzyme glutamine synthetase.